register interest

Professor Stefan Knapp

Research Area: Protein Science and Structural Biology
Technology Exchange: Drug discovery and Protein interaction
Keywords: Kinases, Phosphatases, Cancer, Drug design and Crystallography
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Kinases and phosphatases control a large number of cellular processes like metabolism, transcription, cell migration, events in cell cycle progression, cell communication, differentiation as well as apoptosis. These essential processes are not only controlled by phosphorylation levels but also by various protein interactions, activators and modulators present in kinases and phosphatases. Furthermore, the central role of phosphorylation dependent signalling in the control of a large number of cellular processes make kinases and phosphatases very attractive drug targets. In particular kinases have been selected as targets for therapeutic invention since about 80 kinases map to loci that are implicated in human diseases and 164 kinases map to amplicons that have been detected frequently in tumors. Our aim is to determine a large number of structures of representative family members of these important signaling molecules in order to understand their function, regulation and how they interact in signaling cascades.

Name Department Institution Country
Professor Benjamin Turk Department of Pharmacology Yale University School of Medicine United States
Professor Laurent Meijer ManRos Therapeutics France
Professor Andrew Hopkins NDM Strategic Oxford University, Henry Wellcome Building for Molecular Physiology United Kingdom
Professor Franz Bracher Department of Pharmacy Ludwig-Maximilians-Universität München Germany
Professor James E. Bradner Dana-Farber Cancer Institute Harvard Medical School United States
Professor Juerg Schwaller Deartment of Haematology Basel University Hospital Switzerland
Professor Nathanael S. Gray Dana Farber Cancer Institute Harvard Medical School United States
Professor Jonathan Morris School of Chemistry University of New South Wales Australia
Professor Andrew Holland Department of Molecular Biology & Genetics Johns Hopkins University School of Medicine United States
Professor Friedrich W. Herberg Biochemistry University of Kassel Germany
Professor Asko Uri Chemistry University of Tartu Estonia
Professor Giulio Superti-Furga Research Center for Molecular Medicine Austria
Professor David Bates University of Bristol United Kingdom
Professor Paul Fish UCL School of Pharmacy United Kingdom
Professor Cheryl Arrowsmith University of Toronto Canada
Dr Swen Hoelder Institute of Cancer Research United Kingdom
Professor Tatjana Stankovic University of Birmingham United Kingdom
Dr Alex Bullock Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Panagis Filippakopoulos Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Frank von Delft Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Xin Lu Oxford Ludwig Institute Oxford University, Old Road Campus Research Building United Kingdom
Dr Stephan Feller (RDM) Oxford University,
Professor Paul Brennan Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Dr Susanne Muller-Knapp Structural Genomics Consortium Oxford University, NDM Research Building United Kingdom
Professor Benedikt M Kessler Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Daniel Ebner Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Dr Stuart Conway Department of Chemistry University of Oxford United Kingdom
Gebel J, Luh LM, Coutandin D, Osterburg C, Löhr F, Schäfer B, Frombach AS, Sumyk M, Buchner L, Krojer T et al. 2016. Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization. Cell Death Differ, | Show Abstract | Read more

Members of the p53 tumor-suppressor family are expressed as multiple isoforms. Isoforms with an N-terminal transactivation domain are transcriptionally active, while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63α inhibits the tumor-suppressor function of TAp73β. This can in principle be due to blocking of the promoter or by direct interaction between both proteins. p63 and p73 can hetero-oligomerize through their tetramerization domains and a hetero-tetramer consisting of two p63 and two p73 molecules is thermodynamically more stable than both homo-tetramers. Here we show that cells expressing both p63 and p73 exist in mouse epidermis and hair follicle and that hetero-tetramer complexes can be detected by immunoprecipitation in differentiating keratinocytes. Through structure determination of the hetero-tetramer, we reveal why this hetero-tetramer is the thermodynamically preferred species. We have created mutants that exclusively form either hetero-tetramers or homo-tetramers, allowing to investigate the function of these p63/p73 hetero-tetramers. Using these tools, we show that inhibition of TAp73β in squamous cell carcinomas is due to promoter squelching and not direct interaction.Cell Death and Differentiation advance online publication, 7 October 2016; doi:10.1038/cdd.2016.83.

Popp TA, Tallant C, Rogers C, Fedorov O, Brennan PE, Müller S, Knapp S, Bracher F. 2016. Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors. J Med Chem, 59 (19), pp. 8889-8912. | Show Abstract | Read more

CBP (CREB (cAMP responsive element binding protein) binding protein (CREBBP)) and P300 (adenovirus E1A-associated 300 kDa protein) are two closely related histone acetyltransferases (HATs) that play a key role in the regulation of gene transcription. Both proteins contain a bromodomain flanking the HAT catalytic domain that is important for the targeting of CBP/P300 to chromatin and which offeres an opportunity for the development of protein-protein interaction inhibitors. Here we present the development of CBP/P300 bromodomain inhibitors with 2,3,4,5-tetrahydro-1,4-benzoxazepine backbone, an N-acetyl-lysine mimetic scaffold that led to the recent development of the chemical probe I-CBP112. We present comprehensive SAR of this inhibitor class as well as demonstration of cellular on target activity of the most potent and selective inhibitor TPOP146, which showed 134 nM affinity for CBP with excellent selectivity over other bromodomains.

Ma Y, Wang L, Neitzel LR, Loganathan S, Tang N, Qin L, Emily CE, Guo Y, Knapp S, Beauchamp RD et al. 2016. The MAPK pathway regulates intrinsic resistance to BET inhibitors in colorectal cancer. Clin Cancer Res, | Show Abstract | Read more

PURPOSE: The bromodomain and extra-terminal domain (BET) family proteins are epigenetic readers for acetylated histone marks. Emerging BET bromodomain inhibitors have exhibited antineoplastic activities in a wide range of human cancers through suppression of oncogenic transcription factors, including MYC. However, the preclinical activities of BET inhibitors in advanced solid cancers are moderate at best. To improve BET-targeted therapy, we interrogated mechanisms mediating resistance to BET inhibitors in colorectal cancer (CRC). EXPERIMENTAL DESIGN: Using a panel of molecularly defined CRC cell lines, we examined the impact of BET inhibition on cellular proliferation and survival as well as MYC activity. We further tested the ability of inhibitors targeting the RAF/MEK/ERK (MAPK) pathway to enhance MYC suppression and circumvent intrinsic resistance to BET inhibitors. Key findings were validated using genetic approaches. RESULTS: BET inhibitors as monotherapy moderately reduced CRC cell proliferation and MYC expression. Blockade of the MAPK pathway synergistically sensitized CRC cells to BET inhibitors, leading to potent apoptosis and MYC downregulation in vitro and in vivo. A combination of JQ1 and trametinib, but neither agent alone, induced significant regression of subcutaneous CRC xenografts. ` Conclusions: Our findings suggest that the MAPK pathway confers intrinsic resistance to BET inhibitors in CRC and propose an effective combination strategy for the treatment of CRC.

Najafova Z, Tirado-Magallanes R, Subramaniam M, Hossan T, Schmidt G, Nagarajan S, Baumgart SJ, Mishra VK, Bedi U, Hesse E et al. 2016. BRD4 localization to lineage-specific enhancers is associated with a distinct transcription factor repertoire. Nucleic Acids Res, pp. gkw826-gkw826. | Show Abstract | Read more

Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4.

Myrianthopoulos V, Gaboriaud-Kolar N, Tallant C, Hall ML, Grigoriou S, Brownlee PM, Fedorov O, Rogers C, Heidenreich D, Wanior M et al. 2016. Discovery and Optimization of a Selective Ligand for the Switch/Sucrose Nonfermenting-Related Bromodomains of Polybromo Protein-1 by the Use of Virtual Screening and Hydration Analysis. J Med Chem, 59 (19), pp. 8787-8803. | Show Abstract | Read more

Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein-ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5).

Miranda F, Mannion D, Liu S, Zheng Y, Mangala LS, Redondo C, Herrero-Gonzalez S, Xu R, Taylor C, Chedom DF et al. 2016. Salt-Inducible Kinase 2 Couples Ovarian Cancer Cell Metabolism with Survival at the Adipocyte-Rich Metastatic Niche. Cancer Cell, 30 (2), pp. 273-289. | Show Abstract | Read more

The adipocyte-rich microenvironment forms a niche for ovarian cancer metastasis, but the mechanisms driving this process are incompletely understood. Here we show that salt-inducible kinase 2 (SIK2) is overexpressed in adipocyte-rich metastatic deposits compared with ovarian primary lesions. Overexpression of SIK2 in ovarian cancer cells promotes abdominal metastasis while SIK2 depletion prevents metastasis in vivo. Importantly, adipocytes induce calcium-dependent activation and autophosphorylation of SIK2. Activated SIK2 plays a dual role in augmenting AMPK-induced phosphorylation of acetyl-CoA carboxylase and in activating the PI3K/AKT pathway through p85α-S154 phosphorylation. These findings identify SIK2 at the apex of the adipocyte-induced signaling cascades in cancer cells and make a compelling case for targeting SIK2 for therapy in ovarian cancer.

Chang AN, Mahajan P, Knapp S, Barton H, Sweeney HL, Kamm KE, Stull JT. 2016. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities. Proc Natl Acad Sci U S A, 113 (27), pp. E3824-E3833. | Show Abstract | Read more

The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties.

Sdelci S, Lardeau CH, Tallant C, Klepsch F, Klaiber B, Bennett J, Rathert P, Schuster M, Penz T, Fedorov O et al. 2016. Mapping the chemical chromatin reactivation landscape identifies BRD4-TAF1 cross-talk. Nat Chem Biol, 12 (7), pp. 504-510. | Show Abstract | Read more

Bromodomain-containing proteins of the BET family recognize histone lysine acetylation and mediate transcriptional activation of target genes such as the MYC oncogene. Pharmacological inhibitors of BET domains promise therapeutic benefits in a variety of cancers. We performed a high-diversity chemical compound screen for agents capable of modulating BRD4-dependent heterochromatization of a generic reporter in human cells. In addition to known and new compounds targeting BRD4, we identified small molecules that mimic BRD4 inhibition without direct engagement. One such compound was a potent inhibitor of the second bromodomain of TAF1. Using this inhibitor, we discovered that TAF1 synergizes with BRD4 to control proliferation of cancer cells, making TAF1 an attractive epigenetic target in cancers driven by MYC.

da Motta LL, Ledaki I, Purshouse K, Haider S, De Bastiani MA, Baban D, Morotti M, Steers G, Wigfield S, Bridges E et al. 2016. The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer. Oncogene, | Show Abstract | Read more

The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.Oncogene advance online publication, 13 June 2016; doi:10.1038/onc.2016.184.

Montenegro RC, Clark PG, Howarth A, Wan X, Ceroni A, Siejka P, Nunez-Alonso GA, Monteiro O, Rogers C, Gamble V et al. 2016. BET inhibition as a new strategy for the treatment of gastric cancer. Oncotarget, 7 (28), pp. 43997-44012. | Show Abstract | Read more

Gastric cancer is one of the most common malignancies and a leading cause of cancer death worldwide. The prognosis of stomach cancer is generally poor as this cancer is not very sensitive to commonly used chemotherapies. Epigenetic modifications play a key role in gastric cancer and contribute to the development and progression of this malignancy. In order to explore new treatment options in this target area we have screened a library of epigenetic inhibitors against gastric cancer cell lines and identified inhibitors for the BET family of bromodomains as potent inhibitors of gastric cancer cell proliferations. Here we show that both the pan-BET inhibitor (+)-JQ1 as well as a newly developed specific isoxazole inhibitor, PNZ5, showed potent inhibition of gastric cancer cell growth. Intriguingly, we found differences in the antiproliferative response between gastric cancer cells tested derived from Brazilian patients as compared to those from Asian patients, the latter being largely resistant to BET inhibition. As BET inhibitors are entering clinical trials these findings provide the first starting point for future therapies targeting gastric cancer.

Milhas S, Raux B, Betzi S, Derviaux C, Roche P, Restouin A, Basse MJ, Rebuffet E, Lugari A, Badol M et al. 2016. Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery. ACS Chem Biol, 11 (8), pp. 2140-2148. | Show Abstract | Read more

Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.

Lavogina D, Kestav K, Chaikuad A, Heroven C, Knapp S, Uri A. 2016. Co-crystal structures of the protein kinase haspin with bisubstrate inhibitors. Acta Crystallogr F Struct Biol Commun, 72 (Pt 5), pp. 339-345. | Show Abstract | Read more

Haspin is a mitotic protein kinase that is responsible for the phosphorylation of Thr3 of histone H3, thereby creating a recognition motif for docking of the chromosomal passenger complex that is crucial for the progression of cell division. Here, two high-resolution models of haspin with previously reported inhibitors consisting of an ATP analogue and a histone H3(1-7) peptide analogue are presented. The structures of the complexes confirm the bisubstrate character of the inhibitors by revealing the signature binding modes of the moieties targeting the ATP-binding site and the protein substrate-binding site of the kinase. This is the first structural model of a bisubstrate inhibitor targeting haspin. The presented structural data represent a model for the future development of more specific haspin inhibitors.

Sutherell CL, Tallant C, Monteiro OP, Yapp C, Fuchs JE, Fedorov O, Siejka P, Müller S, Knapp S, Brenton JD et al. 2016. Identification and Development of 2,3-Dihydropyrrolo[1,2-a]quinazolin-5(1H)-one Inhibitors Targeting Bromodomains within the Switch/Sucrose Nonfermenting Complex. J Med Chem, 59 (10), pp. 5095-5101. | Show Abstract | Read more

Bromodomain containing proteins PB1, SMARCA4, and SMARCA2 are important components of SWI/SNF chromatin remodeling complexes. We identified bromodomain inhibitors that target these proteins and display unusual binding modes involving water displacement from the KAc binding site. The best compound binds the fifth bromodomain of PB1 with a KD of 124 nM, SMARCA2B and SMARCA4 with KD values of 262 and 417 nM, respectively, and displays excellent selectivity over bromodomains other than PB1, SMARCA2, and SMARCA4.

Gerstenberger BS, Trzupek JD, Tallant C, Fedorov O, Filippakopoulos P, Brennan PE, Fedele V, Martin S, Picaud S, Rogers C et al. 2016. Identification of a Chemical Probe for Family VIII Bromodomains through Optimization of a Fragment Hit. J Med Chem, 59 (10), pp. 4800-4811. | Show Abstract | Read more

The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.

Esvan YJ, Zeinyeh W, Boibessot T, Nauton L, Théry V, Knapp S, Chaikuad A, Loaëc N, Meijer L, Anizon F et al. 2016. Discovery of pyrido[3,4-g]quinazoline derivatives as CMGC family protein kinase inhibitors: Design, synthesis, inhibitory potency and X-ray co-crystal structure. Eur J Med Chem, 118 pp. 170-177. | Show Abstract | Read more

The design and synthesis of new pyrido[3,4-g]quinazoline derivatives is described as well as their protein kinase inhibitory potencies toward five CMGC family members (CDK5, CK1, GSK3, CLK1 and DYRK1A). The interest for this original tricyclic heteroaromatic scaffold as modulators of CLK1/DYRK1A activity was validated by nanomolar potencies (compounds 12 and 13). CLK1 co-crystal structures with two inhibitors revealed the binding mode of these compounds within the ATP-binding pocket.

Coutandin D, Osterburg C, Srivastav RK, Sumyk M, Kehrloesser S, Gebel J, Tuppi M, Hannewald J, Schäfer B, Salah E et al. 2016. Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level. Elife, 5 (MARCH2016), | Show Abstract | Read more

Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.

Mathea S, Abdul Azeez KR, Salah E, Tallant C, Wolfreys F, Konietzny R, Fischer R, Lou HJ, Brennan PE, Schnapp G et al. 2016. Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib. ACS Chem Biol, 11 (6), pp. 1595-1602. | Show Abstract | Read more

The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here, we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The cocrystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions -1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs.

Martin LJ, Koegl M, Bader G, Cockcroft XL, Fedorov O, Fiegen D, Gerstberger T, Hofmann MH, Hohmann AF, Kessler D et al. 2016. Structure-Based Design of an in Vivo Active Selective BRD9 Inhibitor. J Med Chem, 59 (10), pp. 4462-4475. | Show Abstract | Read more

Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.

Ghari F, Quirke AM, Munro S, Kawalkowska J, Picaud S, McGouran J, Subramanian V, Muth A, Williams R, Kessler B et al. 2016. Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response. Sci Adv, 2 (2), pp. e1501257. | Show Abstract | Read more

Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.

Sorrell FJ, Szklarz M, Abdul Azeez KR, Elkins JM, Knapp S. 2016. Family-wide Structural Analysis of Human Numb-Associated Protein Kinases. Structure, 24 (3), pp. 401-411. | Show Abstract | Read more

The highly diverse Numb-associated kinase (NAK) family has been linked to broad cellular functions including receptor-mediated endocytosis, Notch pathway modulation, osteoblast differentiation, and dendrite morphogenesis. Consequently, NAK kinases play a key role in a diverse range of diseases from Parkinson's and prostate cancer to HIV. Due to the plasticity of this kinase family, NAK kinases are often inhibited by approved or investigational drugs and have been associated with side effects, but they are also potential drug targets. The presence of cysteine residues in some NAK family members provides the possibility for selective targeting via covalent inhibition. Here we report the first high-resolution structures of kinases AAK1 and BIKE in complex with two drug candidates. The presented data allow a comprehensive structural characterization of the NAK kinase family and provide the basis for rational design of selective NAK inhibitors.

Kumar R, Li DQ, Müller S, Knapp S. 2016. Epigenomic regulation of oncogenesis by chromatin remodeling. Oncogene, 35 (34), pp. 4423-4436. | Show Abstract | Read more

Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein-protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy.

Zuercher WJ, Elkins JM, Knapp S. 2016. The Intersection of Structural and Chemical Biology - An Essential Synergy. Cell Chem Biol, 23 (1), pp. 173-182. | Show Abstract | Read more

The continual improvement in our ability to generate high resolution structural models of biological molecules has stimulated and supported innovative chemical biology projects that target increasingly challenging ligand interaction sites. In this review we outline some of the recent developments in chemical biology and rational ligand design and show selected examples that illustrate the synergy between these research areas.

Chaikuad A, Lang S, Brennan PE, Temperini C, Fedorov O, Hollander J, Nachane R, Abell C, Müller S, Siegal G, Knapp S. 2016. Structure-Based Identification of Inhibitory Fragments Targeting the p300/CBP-Associated Factor Bromodomain. J Med Chem, 59 (4), pp. 1648-1653. | Show Abstract | Read more

The P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.

Raux B, Voitovich Y, Derviaux C, Lugari A, Rebuffet E, Milhas S, Priet S, Roux T, Trinquet E, Guillemot JC et al. 2016. Exploring Selective Inhibition of the First Bromodomain of the Human Bromodomain and Extra-terminal Domain (BET) Proteins. J Med Chem, 59 (4), pp. 1634-1641. | Show Abstract | Read more

A midthroughput screening follow-up program targeting the first bromodomain of the human BRD4 protein, BRD4(BD1), identified an acetylated-mimic xanthine derivative inhibitor. This compound binds with an affinity in the low micromolar range yet exerts suitable unexpected selectivity in vitro against the other members of the bromodomain and extra-terminal domain (BET) family. A structure-based program pinpointed a role of the ZA loop, paving the way for the development of potent and selective BET-BRDi probes.

Petersen LK, Blakskjær P, Chaikuad A, Christensen AB, Dietvorst J, Holmkvist J, Knapp S, Kořínek M, Larsen LK, Pedersen AE et al. 2016. Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library Med. Chem. Commun., 7 (7), pp. 1332-1339. | Show Abstract | Read more

This journal is © The Royal Society of Chemistry 2016.A highly specific and potent (7 nM cellular IC50) inhibitor of p38α kinase was identified directly from a 12.6 million membered DNA-encoded small molecule library. This was achieved using the high fidelity yoctoReactor technology (yR) for preparing the DNA-encoded library, and a homogeneous screening technique-the binder trap enrichment technology (BTE). Although structurally atypical to other kinase blockers, this inhibitor was found by X-ray crystallography to interact with the ATP binding site and provide strong distortion of the P-loop. Remarkably, it assumed an alternative binding mode as it lacks key features of known kinase inhibitors such as typical hinge binding motifs. Interestingly, the inhibitor bound assuming a canonical type-II ('DFG-out') binding mode by forming hinge hydrogen bonds with the backbone, showed excellent shape complementarity, and formed a number of specific polar interactions. Moreover, the crystal structure showed, that although buried in the p38α active site, the original DNA attachment point of the compound was accessible through a channel created by the distorted P-loop conformation. This study demonstrates the usability of DNA-encoded library technologies for identifying novel chemical matter with alternative binding modes to provide a good starting point for drug development.

Lori L, Pasquo A, Lori C, Petrosino M, Chiaraluce R, Tallant C, Knapp S, Consalvi V. 2016. Effect of BET Missense Mutations on Bromodomain Function, Inhibitor Binding and Stability. PLoS One, 11 (7), pp. e0159180. | Show Abstract | Read more

Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin structure. Acetylated lysine residues are specifically recognized by bromodomains, small protein interaction modules that read these modification in a sequence and acetylation dependent way regulating the recruitment of transcriptional regulators and chromatin remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains are highly druggable protein interaction domains resulting in the development of a large number of bromodomain inhibitors. BET bromodomain inhibitors received a lot of attention in the oncology field resulting in the rapid translation of early BET bromodomain inhibitors into clinical studies. Here we investigated the effects of mutations present as polymorphism or found in cancer on BET bromodomain function and stability and the influence of these mutants on inhibitor binding. We found that most BET missense mutations localize to peripheral residues in the two terminal helices. Crystal structures showed that the three dimensional structure is not compromised by these mutations but mutations located in close proximity to the acetyl-lysine binding site modulate acetyl-lysine and inhibitor binding. Most mutations affect significantly protein stability and tertiary structure in solution, suggesting new interactions and an alternative network of protein-protein interconnection as a consequence of single amino acid substitution. To our knowledge this is the first report studying the effect of mutations on bromodomain function and inhibitor binding.

Elkins JM, Fedele V, Szklarz M, Abdul Azeez KR, Salah E, Mikolajczyk J, Romanov S, Sepetov N, Huang XP, Roth BL et al. 2016. Comprehensive characterization of the Published Kinase Inhibitor Set. Nat Biotechnol, 34 (1), pp. 95-103. | Show Abstract | Read more

Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome.

Picaud S, Fedorov O, Thanasopoulou A, Leonards K, Jones K, Meier J, Olzscha H, Monteiro O, Martin S, Philpott M et al. 2015. Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy. Cancer Res, 75 (23), pp. 5106-5119. | Show Abstract | Read more

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers.

Knapp S. 2016. Emerging Target Families: Intractable Targets. Handb Exp Pharmacol, 232 pp. 43-58. | Show Abstract | Read more

The druggability of a target is defined by the likelihood of a certain target binding site to be amendable to functional modulation by a small molecule in vivo. Thus, druggability depends on the ability of the developed small molecule to reach the target site, the properties of the ligand binding pocket and our ability to develop chemical matter that efficiently interact with the drug binding site of interest. Historically enzymes have been the main drug targets because the inhibition of their activity can be easily assayed and catalytic centres are often attractive drug binding sites. However, despite considerable effort, a number of classical enzyme families have not been successfully targeted. More recently protein-protein interactions received considerable attention and several clinical inhibitors have now been developed. Despite the considerable progress made expanding target space, a large number of targets with a very strong rationale for targeting remain intractable. In the following chapter I will summarize progress made in developing inhibitors for challenging drug binding sites and emerging target families.

Müller S, Chaikuad A, Gray NS, Knapp S. 2015. The ins and outs of selective kinase inhibitor development. Nat Chem Biol, 11 (11), pp. 818-821. | Read more

Fedorov O, Castex J, Tallant C, Owen DR, Martin S, Aldeghi M, Monteiro O, Filippakopoulos P, Picaud S, Trzupek JD et al. 2015. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance. Sci Adv, 1 (10), pp. e1500723. | Show Abstract | Read more

Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes.

Darby RA, Unsworth A, Knapp S, Kerr ID, Callaghan R. 2015. Overcoming ABCG2-mediated drug resistance with imidazo-[1,2-b]-pyridazine-based Pim1 kinase inhibitors. Cancer Chemother Pharmacol, 76 (4), pp. 853-864. | Show Abstract | Read more

PURPOSE: Multidrug efflux pumps such as ABCG2 confer drug resistance to a number of cancer types, leading to poor prognosis and outcome. To date, the strategy of directly inhibiting multidrug efflux pumps in order to overcome drug resistance in cancer has been unsuccessful. An alternative strategy is to target proteins involved in the regulation of multidrug efflux pump activity or expression. Pim1 kinase has been demonstrated to phosphorylate ABCG2, promote its oligomerisation and contribute to its ability to confer drug resistance. METHODS: In the present manuscript, imidazo-pyridazine-based inhibitors of Pim1 were examined for their ability to overcome ABCG2-mediated drug resistance. Drug efficacy was measured as a cytotoxic response or an effect on transport by ABCG2. Protein expression patterns were assessed using western immuno-blotting. RESULTS: The two Pim1 inhibitors increased the potency of flavopiridol, mitoxantrone, topotecan and doxorubicin, specifically in ABCG2-expressing cells. This effect was associated with an increase in the cellular accumulation of [(3)H]-mitoxantrone, suggesting direct impairment of the transporter. However, prolonged pre-incubation with the studied inhibitors greatly enhanced the effect on mitoxantrone accumulation. The inhibitors caused a significant time-dependent reduction in the expression of ABCG2 in the resistant cells, an effect that would improve drug efficacy. CONCLUSION: Consequently, it appears that the Pim1 inhibitors display a dual-mode effect on ABCG2-expressing cancer cells. This may provide a powerful new strategy in overcoming drug resistance by targeting proteins that regulate expression of efflux pumps.

Morozumi Y, Boussouar F, Tan M, Chaikuad A, Jamshidikia M, Colak G, He H, Nie L, Petosa C, de Dieuleveult M et al. 2016. Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells. J Mol Cell Biol, 8 (4), pp. 349-362. | Show Abstract | Read more

Although the conserved AAA ATPase and bromodomain factor, ATAD2, has been described as a transcriptional co-activator upregulated in many cancers, its function remains poorly understood. Here, using a combination of ChIP-seq, ChIP-proteomics, and RNA-seq experiments in embryonic stem cells where Atad2 is normally highly expressed, we found that Atad2 is an abundant nucleosome-bound protein present on active genes, associated with chromatin remodelling, DNA replication, and DNA repair factors. A structural analysis of its bromodomain and subsequent investigations demonstrate that histone acetylation guides ATAD2 to chromatin, resulting in an overall increase of chromatin accessibility and histone dynamics, which is required for the proper activity of the highly expressed gene fraction of the genome. While in exponentially growing cells Atad2 appears dispensable for cell growth, in differentiating ES cells Atad2 becomes critical in sustaining specific gene expression programmes, controlling proliferation and differentiation. Altogether, this work defines Atad2 as a facilitator of general chromatin-templated activities such as transcription.

Rouka E, Simister PC, Janning M, Kumbrink J, Konstantinou T, Muniz JR, Joshi D, O'Reilly N, Volkmer R, Ritter B et al. 2015. Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3). J Biol Chem, 290 (42), pp. 25275-25292. | Show Abstract | Read more

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3.

Hammitzsch A, Tallant C, Fedorov O, O'Mahony A, Brennan PE, Hay DA, Martinez FO, Al-Mossawi MH, de Wit J, Vecellio M et al. 2015. CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses. Proc Natl Acad Sci U S A, 112 (34), pp. 10768-10773. | Show Abstract | Read more

Th17 responses are critical to a variety of human autoimmune diseases, and therapeutic targeting with monoclonal antibodies against IL-17 and IL-23 has shown considerable promise. Here, we report data to support selective bromodomain blockade of the transcriptional coactivators CBP (CREB binding protein) and p300 as an alternative approach to inhibit human Th17 responses. We show that CBP30 has marked molecular specificity for the bromodomains of CBP and p300, compared with 43 other bromodomains. In unbiased cellular testing on a diverse panel of cultured primary human cells, CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines. CBP30 also inhibited IL-17A secretion by Th17 cells from healthy donors and patients with ankylosing spondylitis and psoriatic arthritis. Transcriptional profiling of human T cells after CBP30 treatment showed a much more restricted effect on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials.

Chaikuad A, Knapp S, von Delft F. 2015. Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens. Acta Crystallogr D Biol Crystallogr, 71 (Pt 8), pp. 1627-1639. | Show Abstract | Read more

The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth. The lack of rational approaches towards the selection of successful chemical space and representative combinations has led to significant overlapping conditions, which are currently present in a multitude of commercially available crystallization screens. Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth. Four newly defined smears were classified by molecular-weight groups and enabled the preservation of specific properties related to different polymer sizes. These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives. The efficiency of the smear-based screens was evaluated on more than 220 diverse recombinant human proteins, which overall revealed a good initial crystallization success rate of nearly 50%. In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals. The defined smears therefore offer an alternative approach towards PEG sampling, which will benefit the design of crystallization screens sampling a wide chemical space of this key precipitant.

Gato-Cañas M, Martinez de Morentin X, Blanco-Luquin I, Fernandez-Irigoyen J, Zudaire I, Liechtenstein T, Arasanz H, Lozano T, Casares N, Chaikuad A et al. 2015. A core of kinase-regulated interactomes defines the neoplastic MDSC lineage. Oncotarget, 6 (29), pp. 27160-27175. | Show Abstract | Read more

Myeloid-derived suppressor cells (MDSCs) differentiate from bone marrow precursors, expand in cancer-bearing hosts and accelerate tumor progression. MDSCs have become attractive therapeutic targets, as their elimination strongly enhances anti-neoplastic treatments. Here, immature myeloid dendritic cells (DCs), MDSCs modeling tumor-infiltrating subsets or modeling non-cancerous (NC)-MDSCs were compared by in-depth quantitative proteomics. We found that neoplastic MDSCs differentially expressed a core of kinases which controlled lineage-specific (PI3K-AKT and SRC kinases) and cancer-induced (ERK and PKC kinases) protein interaction networks (interactomes). These kinases contributed to some extent to myeloid differentiation. However, only AKT and ERK specifically drove MDSC differentiation from myeloid precursors. Interfering with AKT and ERK with selective small molecule inhibitors or shRNAs selectively hampered MDSC differentiation and viability. Thus, we provide compelling evidence that MDSCs constitute a distinct myeloid lineage distinguished by a "kinase signature" and well-defined interactomes. Our results define new opportunities for the development of anti-cancer treatments targeting these tumor-promoting immune cells.

Huston A, Arrowsmith CH, Knapp S, Schapira M. 2015. Probing the epigenome. Nat Chem Biol, 11 (8), pp. 542-545. | Show Abstract | Read more

© 2015 Nature America, Inc. All rights reserved.Epigenetic chemical probes are having a strong impact in biological discovery and target validation. Systematic coverage of emerging epigenetic target classes with these potent, selective, cell-active chemical tools will profoundly influence understanding of the human biology and pathology of chromatintemplated mechanisms.

Arrowsmith CH, Audia JE, Austin C, Baell J, Bennett J, Blagg J, Bountra C, Brennan PE, Brown PJ, Bunnage ME et al. 2015. The promise and peril of chemical probes. Nat Chem Biol, 11 (8), pp. 536-541. | Show Abstract | Read more

© 2015 Nature America, Inc. All rights reserved.Chemical probes are powerful reagents with increasing impacts on biomedical research. However, probes of poor quality or that are used incorrectly generate misleading results. To help address these shortcomings, we will create a community-driven wiki resource to improve quality and convey current best practice.

Alexander LT, Möbitz H, Drueckes P, Savitsky P, Fedorov O, Elkins JM, Deane CM, Cowan-Jacob SW, Knapp S. 2015. Type II Inhibitors Targeting CDK2. ACS Chem Biol, 10 (9), pp. 2116-2125. | Show Abstract | Read more

Kinases can switch between active and inactive conformations of the ATP/Mg(2+) binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in-out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target.

Hay DA, Rogers CM, Fedorov O, Tallant C, Martin S, Monteiro OP, Mueller S, Knapp S, Schofield CJ, Brennan PE. 2015. Design and synthesis of potent and selective inhibitors of BRD7 and BRD9 bromodomains MEDCHEMCOMM, 6 (7), pp. 1381-1386. | Show Abstract | Read more

© The Royal Society of Chemistry.Emerging evidence suggests bromodomain-containing proteins 7 and 9 (BRD7 and BRD9) have roles in the regulation of human transcription and disease including cancer. We describe potent and selective inhibitors of the BRD7 and BRD9 bromodomains intended for use as tools to elucidate the biological roles of BRD7 and BRD9 in healthy and diseased cells.

Harrington L, Alexander LT, Knapp S, Bayley H. 2015. Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor Angewandte Chemie - International Edition, 54 (28), pp. 8154-8159. | Show Abstract | Read more

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Protein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label-free determination of inhibition constants. The procedure circumvents many sources of bias or false-positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips.

Harrington L, Alexander LT, Knapp S, Bayley H. 2015. Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor. Angew Chem Int Ed Engl, 54 (28), pp. 8154-8159. | Show Abstract | Read more

Protein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label-free determination of inhibition constants. The procedure circumvents many sources of bias or false-positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips.

Clark PG, Vieira LC, Tallant C, Fedorov O, Singleton DC, Rogers CM, Monteiro OP, Bennett JM, Baronio R, Müller S et al. 2015. LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor. Angew Chem Int Ed Engl, 54 (21), pp. 6217-6221. | Show Abstract | Read more

The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam LP99 is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains. Development of LP99 from a fragment hit was expedited through balancing structure-based inhibitor design and biophysical characterization against tractable chemical synthesis: Complexity-building nitro-Mannich/lactamization cascade processes allowed for early structure-activity relationship studies whereas an enantioselective organocatalytic nitro-Mannich reaction enabled the synthesis of the lead scaffold in enantioenriched form and on scale. This epigenetic probe was shown to inhibit the association of BRD7 and BRD9 to acetylated histones in vitro and in cells. Moreover, LP99 was used to demonstrate that BRD7/9 plays a role in regulating pro-inflammatory cytokine secretion.

Clark PG, Vieira LC, Tallant C, Fedorov O, Singleton DC, Rogers CM, Monteiro OP, Bennett JM, Baronio R, Müller S et al. 2015. LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor. Angew Chem Weinheim Bergstr Ger, 127 (21), pp. 6315-6319. | Show Abstract | Read more

The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam LP99 is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains. Development of LP99 from a fragment hit was expedited through balancing structure-based inhibitor design and biophysical characterization against tractable chemical synthesis: Complexity-building nitro-Mannich/lactamization cascade processes allowed for early structure-activity relationship studies whereas an enantioselective organocatalytic nitro-Mannich reaction enabled the synthesis of the lead scaffold in enantioenriched form and on scale. This epigenetic probe was shown to inhibit the association of BRD7 and BRD9 to acetylated histones in vitro and in cells. Moreover, LP99 was used to demonstrate that BRD7/9 plays a role in regulating pro-inflammatory cytokine secretion.

Bennett J, Fedorov O, Tallant C, Monteiro O, Meier J, Gamble V, Savitsky P, Nunez-Alonso GA, Haendler B, Rogers C et al. 2016. Discovery of a Chemical Tool Inhibitor Targeting the Bromodomains of TRIM24 and BRPF. J Med Chem, 59 (4), pp. 1642-1647. | Show Abstract | Read more

TRIM24 is a transcriptional regulator as well as an E3 ubiquitin ligase. It is overexpressed in diverse tumors, and high expression levels have been linked to poor prognosis in breast cancer patients. TRIM24 contains a PHD/bromodomain offering the opportunity to develop protein interaction inhibitors that target this protein interaction module. Here we identified potent acetyl-lysine mimetic benzimidazolones TRIM24 bromodomain inhibitors. The best compound of this series is a selective BRPF1B/TRIM24 dual inhibitor that bound with a KD of 137 and 222 nM, respectively, but exerted good selectivity over other bromodomains. Cellular activity of the inhibitor was demonstrated using FRAP assays as well as cell viability data.

Martin E, Knapp S, Engh RA, Moebitz H, Varin T, Roux B, Meiler J, Berdini V, Baumann A, Vieth M. 2015. Perspective on computational and structural aspects of kinase discovery from IPK2014. Biochim Biophys Acta, 1854 (10 Pt B), pp. 1595-1604. | Show Abstract | Read more

Recent advances in understanding the activity and selectivity of kinase inhibitors and their relationships to protein structure are presented. Conformational selection in kinases is studied from empirical, data-driven and simulation approaches. Ligand binding and its affinity are, in many cases, determined by the predetermined active and inactive conformation of kinases. Binding affinity and selectivity predictions highlight the current state of the art and advances in computational chemistry as it applies to kinase inhibitor discovery. Kinome wide inhibitor profiling and cell panel profiling lead to a better understanding of selectivity and allow for target validation and patient tailoring hypotheses. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Kovackova S, Chang L, Bekerman E, Neveu G, Barouch-Bentov R, Chaikuad A, Heroven C, Šála M, De Jonghe S, Knapp S et al. 2015. Selective Inhibitors of Cyclin G Associated Kinase (GAK) as Anti-Hepatitis C Agents. J Med Chem, 58 (8), pp. 3393-3410. | Show Abstract | Read more

Cyclin G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Cocrystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV life cycle (i.e., viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer, and Parkinson's disease).

Chen P, Chaikuad A, Bamborough P, Bantscheff M, Bountra C, Chung CW, Fedorov O, Grandi P, Jung D, Lesniak R et al. 2016. Discovery and Characterization of GSK2801, a Selective Chemical Probe for the Bromodomains BAZ2A and BAZ2B. J Med Chem, 59 (4), pp. 1410-1424. | Show Abstract | Read more

Bromodomains are acetyl-lysine specific protein interaction domains that have recently emerged as a new target class for the development of inhibitors that modulate gene transcription. The two closely related bromodomain containing proteins BAZ2A and BAZ2B constitute the central scaffolding protein of the nucleolar remodeling complex (NoRC) that regulates the expression of noncoding RNAs. However, BAZ2 bromodomains have low predicted druggability and so far no selective inhibitors have been published. Here we report the development of GSK2801, a potent, selective and cell active acetyl-lysine competitive inhibitor of BAZ2A and BAZ2B bromodomains as well as the inactive control compound GSK8573. GSK2801 binds to BAZ2 bromodomains with dissociation constants (KD) of 136 and 257 nM for BAZ2B and BAZ2A, respectively. Crystal structures demonstrated a canonical acetyl-lysine competitive binding mode. Cellular activity was demonstrated using fluorescent recovery after photobleaching (FRAP) monitoring displacement of GFP-BAZ2A from acetylated chromatin. A pharmacokinetic study in mice showed that GSK2801 had reasonable in vivo exposure after oral dosing, with modest clearance and reasonable plasma stability. Thus, GSK2801 represents a versatile tool compound for cellular and in vivo studies to understand the role of BAZ2 bromodomains in chromatin biology.

Falke H, Chaikuad A, Becker A, Loaëc N, Lozach O, Abu Jhaisha S, Becker W, Jones PG, Preu L, Baumann K et al. 2015. 10-iodo-11H-indolo[3,2-c]quinoline-6-carboxylic acids are selective inhibitors of DYRK1A. J Med Chem, 58 (7), pp. 3131-3143. | Show Abstract | Read more

The protein kinase DYRK1A has been suggested to act as one of the intracellular regulators contributing to neurological alterations found in individuals with Down syndrome. For an assessment of the role of DYRK1A, selective synthetic inhibitors are valuable pharmacological tools. However, the DYRK1A inhibitors described in the literature so far either are not sufficiently selective or have not been tested against closely related kinases from the DYRK and the CLK protein kinase families. The aim of this study was the identification of DYRK1A inhibitors exhibiting selectivity versus the structurally and functionally closely related DYRK and CLK isoforms. Structure modification of the screening hit 11H-indolo[3,2-c]quinoline-6-carboxylic acid revealed structure-activity relationships for kinase inhibition and enabled the design of 10-iodo-substituted derivatives as very potent DYRK1A inhibitors with considerable selectivity against CLKs. X-ray structure determination of three 11H-indolo[3,2-c]quinoline-6-carboxylic acids cocrystallized with DYRK1A confirmed the predicted binding mode within the ATP binding site.

Edwards AM, Arrowsmith CH, Bountra C, Bunnage ME, Feldmann M, Knight JC, Patel DD, Prinos P, Taylor MD, Sundström M, SGC Open Source Target-Discovery Partnership. 2015. Preclinical target validation using patient-derived cells. Nat Rev Drug Discov, 14 (3), pp. 149-150. | Show Abstract | Read more

The Structural Genomics Consortium (SGC) and its clinical, industry and disease-foundation partners are launching open-source preclinical translational medicine studies.

Drouin L, McGrath S, Vidler LR, Chaikuad A, Monteiro O, Tallant C, Philpott M, Rogers C, Fedorov O, Liu M et al. 2015. Structure enabled design of BAZ2-ICR, a chemical probe targeting the bromodomains of BAZ2A and BAZ2B. J Med Chem, 58 (5), pp. 2553-2559. | Show Abstract | Read more

The bromodomain containing proteins BAZ2A/B play essential roles in chromatin remodeling and regulation of noncoding RNAs. We present the structure based discovery of a potent, selective, and cell active inhibitor 13 (BAZ2-ICR) of the BAZ2A/B bromodomains through rapid optimization of a weakly potent starting point. A key feature of the presented inhibitors is an intramolecular aromatic stacking interaction that efficiently occupies the shallow bromodomain pockets. 13 represents an excellent chemical probe for functional studies of the BAZ2 bromodomains in vitro and in vivo.

Martin E, Knapp S, Engh RA, Moebitz H, Varin T, Roux B, Meiler J, Berdini V, Baumann A, Vieth M. 2015. Perspective on computational and structural aspects of kinase discovery from IPK2014 Biochimica et Biophysica Acta - Proteins and Proteomics, 1854 (10), pp. 1595-1604. | Show Abstract | Read more

© 2015 Elsevier B.V. All rights reserved.Recent advances in understanding the activity and selectivity of kinase inhibitors and their relationships to protein structure are presented. Conformational selection in kinases is studied from empirical, data-driven and simulation approaches. Ligand binding and its affinity are, in many cases, determined by the predetermined active and inactive conformation of kinases. Binding affinity and selectivity predictions highlight the current state of the art and advances in computational chemistry as it applies to kinase inhibitor discovery. Kinome wide inhibitor profiling and cell panel profiling lead to a better understanding of selectivity and allow for target validation and patient tailoring hypotheses. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Kestav K, Lavogina D, Raidaru G, Chaikuad A, Knapp S, Uri A. 2015. Bisubstrate inhibitor approach for targeting mitotic kinase Haspin. Bioconjug Chem, 26 (2), pp. 225-234. | Show Abstract | Read more

During the past decade, the basophilic atypical kinase Haspin has emerged as a key player in mitosis responsible for phosphorylation of Thr3 residue of histone H3. Here, we report the construction of conjugates comprising an aromatic fragment targeted to the ATP-site of Haspin and a peptide mimicking the N-terminus of histone H3. The combination of effective solid phase synthesis procedures and a high throughput binding/displacement assay with fluorescence anisotropy readout afforded the development of inhibitors with remarkable subnanomolar affinity toward Haspin. The selectivity profiles of novel conjugates were established by affinity studies with a model basophilic kinase (catalytic subunit of cAMP-dependent protein kinase) and by a commercial 1-point inhibition assay with 43 protein kinases.

Homan KT, Larimore KM, Elkins JM, Szklarz M, Knapp S, Tesmer JJ. 2015. Identification and structure-function analysis of subfamily selective G protein-coupled receptor kinase inhibitors. ACS Chem Biol, 10 (1), pp. 310-319. | Show Abstract | Read more

Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson's disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

Ekambaram R, Manoharan GB, Enkvist E, Ligi K, Knapp S, Uri A. 2015. PIM kinase-responsive microsecond-lifetime photoluminescent probes based on selenium-containing heteroaromatic tricycle RSC ADVANCES, 5 (117), pp. 96750-96757. | Show Abstract | Read more

© The Royal Society of Chemistry.A new structural fragment was synthesized for construction of protein binding-responsive photoluminescent probes. In complex with protein kinases of the PIM family, bisubstrate inhibitors containing benzo[4,5]seleno[3,2-d]pyrimidin-4-one moiety revealed microsecond-lifetime phosphorescence emission after pulse excitation with near-UV radiation. The phosphorescence signal was substantially (more than 50-fold) amplified by a covalently bound fluorescent dye (PromoFluor-555 or PromoFluor-647) whose absorption spectrum well overlapped with the phosphorescence emission spectrum of the selenium-containing heteroaromatic tricycle. The developed organic small-molecule long-lifetime photoluminescence probes possess subnanomolar affinity towards kinases of the PIM family and reveal especially strong emission signal with PIM-2 isozyme. The developed probes have potential to be used for monitoring of activity of PIM kinases for diagnosis of cancer.

Harrington L, Alexander LT, Knapp S, Bayley H. 2015. Inside Cover: Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor (Angew. Chem. Int. Ed. 28/2015) Angewandte Chemie International Edition, 54 (28), pp. 8002-8002. | Read more

Horne GA, Stewart HJ, Dickson J, Knapp S, Ramsahoye B, Chevassut T. 2015. Nanog requires BRD4 to maintain murine embryonic stem cell pluripotency and is suppressed by bromodomain inhibitor JQ1 together with Lefty1. Stem Cells Dev, 24 (7), pp. 879-891. | Show Abstract | Read more

Embryonic stem cells (ESCs) are maintained in an undifferentiated state through expression of the core transcriptional factors Nanog, Oct4, and Sox2. However, the epigenetic regulation of pluripotency is poorly understood. Differentiation of ESCs is accompanied by a global reduction of panacetylation of histones H3 and H4 suggesting that histone acetylation plays an important role in maintenance of ESC pluripotency. Acetylated lysine residues on histones are read by members of the bromodomain family that includes BET (bromodomain and extraterminal domain) proteins for which highly potent and selective inhibitors have been developed. In this study we demonstrate that the pan-BET bromodomain inhibitor JQ1 induces rapid spontaneous differentiation of murine ESCs by inducing marked transcriptional downregulation of Nanog as well as the stemness markers Lefty1 and Lefty2, but not Myc, often used as a marker of BET inhibitor activity in cancer. We show that the effects of JQ1 are recapitulated by knockdown of the BET family member BRD4 implicating this protein in Nanog regulation. These data are also supported by chromatin immunoprecipitation experiments which confirm BRD4 binding at the Nanog promoter that is known to require acetylation by the histone acetyltransferase MOF for transcriptional activity. In further support of our findings, we show that JQ1 antagonizes the stem cell-promoting effects of the histone deacetylase inhibitors sodium butyrate and valproic acid. Our data suggest that BRD4 is critical for the maintenance of ESC pluripotency and that this occurs primarily through the maintenance of Nanog expression.

Toi M, Pillai MR, Gupta S, Badwe R, Carmo-Fonseca M, Costa L, Chow LWC, Knapp S, Kumar R. 2015. The global cancer genomics consortium’s symposium: New era of molecular medicine and epigenetic cancer medicine - Cross section of genomics and epigenetics Genes and Cancer, 6 (1-2), pp. 1-8. | Show Abstract

© 2015, Impact Journals LLC. All rights reserved.The Global Cancer Genomics Consortium (GCGC) colleagues continue to function together as an interactive multidisciplinary team of cancer biologists and oncologists with interests in genomics and building a bidirectional bridge between cancer clinics and laboratories while taking advantage of shared resources among its member scientists. The GCGC includes member scientists from six institutions in Lisbon, United Kingdom, Japan, India and United States, and was formed in December 2010 for a period of five years. Driven by valuable lessons learned from the previous symposiums, the fourth GCGC Symposium focused on a cross section of genomic and epigenetic cancer medicine and it’s for this reason we chose the conference theme - New Era of Molecular Medicine and Epigenetic Cancer Medicine: Cross Section of Genomics and Epigenetics. This year’s symposium was co-organized by the Organization for Oncology and Translational Research (OOTR) at the Shiran Hall, Kyoto University, Kyoto, Japan, from November 14 and 15, 2014. The symposium attracted around 80 participants from 14 countries, and counted with 23 invited platform speakers. Scientific sessions included eight platform sessions and one poster session, and three plenary lectures. The symposium focused on cancer stem cells and self-renewal, cancer transcriptome, tumor heterogeneity, tumor biology, breast cancer genomics, targeted therapeutics and personalized medicine. The issues of cancer stem cells and tumor heterogeneity were echoed in most of the scientific presentations. The meeting concluded with an oral presentation by the best poster awardee and closing remarks by meeting co-chairs.

Aldeghi M, Heifetz A, Bodkin MJ, Knapp S, Biggin PC. 2016. Accurate calculation of the absolute free energy of binding for drug molecules. Chem Sci, 7 (1), pp. 207-218. | Show Abstract | Read more

Accurate prediction of binding affinities has been a central goal of computational chemistry for decades, yet remains elusive. Despite good progress, the required accuracy for use in a drug-discovery context has not been consistently achieved for drug-like molecules. Here, we perform absolute free energy calculations based on a thermodynamic cycle for a set of diverse inhibitors binding to bromodomain-containing protein 4 (BRD4) and demonstrate that a mean absolute error of 0.6 kcal mol(-1) can be achieved. We also show a similar level of accuracy (1.0 kcal mol(-1)) can be achieved in pseudo prospective approach. Bromodomains are epigenetic mark readers that recognize acetylation motifs and regulate gene transcription, and are currently being investigated as therapeutic targets for cancer and inflammation. The unprecedented accuracy offers the exciting prospect that the binding free energy of drug-like compounds can be predicted for pharmacologically relevant targets.

Tallant C, Valentini E, Fedorov O, Overvoorde L, Ferguson FM, Filippakopoulos P, Svergun DI, Knapp S, Ciulli A. 2015. Molecular basis of histone tail recognition by human TIP5 PHD finger and bromodomain of the chromatin remodeling complex NoRC. Structure, 23 (1), pp. 80-92. | Show Abstract | Read more

Binding of the chromatin remodeling complex NoRC to RNA complementary to the rDNA promoter mediates transcriptional repression. TIP5, the largest subunit of NoRC, is involved in recruitment to rDNA by interactions with promoter-bound TTF-I, pRNA, and acetylation of H4K16. TIP5 domains that recognize posttranslational modifications on histones are essential for recruitment of NoRC to chromatin, but how these reader modules recognize site-specific histone tails has remained elusive. Here, we report crystal structures of PHD zinc finger and bromodomains from human TIP5 and BAZ2B in free form and bound to H3 and/or H4 histones. PHD finger functions as an independent structural module in recognizing unmodified H3 histone tails, and the bromodomain prefers H3 and H4 acetylation marks followed by a key basic residue, KacXXR. Further low-resolution analyses of PHD-bromodomain modules provide molecular insights into their trans histone tail recognition, required for nucleosome recruitment and transcriptional repression of the NoRC complex.

Wang J, Knapp S, Pyne NJ, Pyne S, Elkins JM. 2014. Crystal Structure of Sphingosine Kinase 1 with PF-543. ACS Med Chem Lett, 5 (12), pp. 1329-1333. | Show Abstract | Read more

The most potent inhibitor of Sphingosine Kinase 1 (SPHK1) so far identified is PF-543. The crystal structure of SPHK1 in complex with inhibitor PF-543 to 1.8 Å resolution reveals the inhibitor bound in a bent conformation analogous to that expected of a bound sphingosine substrate but with a rotated head group. The structural data presented will aid in the design of SPHK1 and SPHK2 inhibitors with improved properties.

Thanasopoulou A, Dumrese K, Picaud S, Fedorov O, Knapp S, Schwaller J. 2014. Targeting Aberrant Self-Renewal of Leukemic Cells with a Novel CBP/p300 Bromodomain Inhibitor BLOOD, 124 (21),

Chaikuad A, Petros AM, Fedorov O, Xu J, Knapp S. 2014. Structure-based approaches towards identification of fragments for the low-druggability ATAD2 bromodomain MEDCHEMCOMM, 5 (12), pp. 1843-1848. | Show Abstract | Read more

© The Royal Society of Chemistry.The transcriptional co-regulator ATAD2 is a prognostic marker for patient survival in many cancers. ATAD2 harbours a bromodomain which may offer an opportunity for pharmacological intervention, but its shallow, polar binding surface makes the development of inhibitors challenging. Here we optimized crystal transfer/soaking conditions enabling crystallographic fragment screening. We describe nine crystal structures of fragments including thymidine, a novel acetyl-lysine mimetic ligand and the evaluation of the binding properties of the identified fragments using NMR chemical shift perturbation experiments. The presented binding modes offer chemical starting points for the development of more potent ATAD2 inhibitors. This journal is

Abdul Azeez KR, Knapp S, Fernandes JM, Klussmann E, Elkins JM. 2014. The crystal structure of the RhoA-AKAP-Lbc DH-PH domain complex. Biochem J, 464 (2), pp. 231-239. | Show Abstract | Read more

The RhoGEF (Rho GTPase guanine-nucleotide-exchange factor) domain of AKAP-Lbc (A-kinase-anchoring protein-Lbc, also known as AKAP13) catalyses nucleotide exchange on RhoA and is involved in the development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA-GDP and the AKAP-Lbc RhoGEF [DH (Dbl-homologous)-PH (pleckstrin homology)] domain to 2.1 Å (1 Å = 0.1 nm) resolution. The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF. Nucleotide-exchange assays comparing the activity of the DH-PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained by the RhoA-AKAP-Lbc structure. Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA. Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

Quinn ER, Ciceri P, Mueller-Knapp S, O'Mahony A, Fedorov O, Filippakopoulos P, Hunt JP, Lasater EA, Pallares G, Picaud S et al. 2014. Dual kinase/bromodomain inhibitors for rationally designed polypharmacology CANCER RESEARCH, 74 (19), pp. 5387-5387. | Read more

Chaikuad A, Tacconi EM, Zimmer J, Liang Y, Gray NS, Tarsounas M, Knapp S. 2014. A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics. Nat Chem Biol, 10 (10), pp. 853-860. | Show Abstract | Read more

Activation of the ERK pathway is a hallmark of cancer, and targeting of upstream signaling partners led to the development of approved drugs. Recently, SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induces a so-far-unknown binding pocket that accommodates the piperazine-phenyl-pyrimidine decoration. This new binding pocket was created by an inactive conformation of the phosphate-binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed two canonical but distinct type I binding modes. Notably, the new binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell-based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity.

Knapp S, Sundström M. 2014. Recently targeted kinases and their inhibitors-the path to clinical trials. Curr Opin Pharmacol, 17 (1), pp. 58-63. | Show Abstract | Read more

Protein kinases have emerged as one of the most important drug target families for the treatment of cancer. To date, 28 inhibitors with reported activity versus one or multiple kinases have been approved for clinical use. However, the majority of new clinical trials are focused on new subindications using already approved kinase inhibitors or target well validated kinase targets with novel inhibitors. In contrast, relatively few clinical trials have been initiated using specific inhibitors that inhibit novel kinase targets, despite significant validation efforts in the public domain. Analysis of the target validation history of first in class kinase inhibitors revealed a long delay between initial disease association and development of inhibitors. As part of this analysis, we have investigated which first in class inhibitor that entered phase I clinical trials over the last five years and also considered which research approaches that were used to validate them.

Knapp S, Sundstrom M. 2014. Recently targeted kinases and their inhibitors - the path to clinical trials CURRENT OPINION IN PHARMACOLOGY, 17 pp. 58-63. | Read more

Nagarajan S, Hossan T, Alawi M, Najafova Z, Indenbirken D, Bedi U, Taipaleenmäki H, Ben-Batalla I, Scheller M, Loges S et al. 2014. Bromodomain Protein BRD4 Is Required for Estrogen Receptor-Dependent Enhancer Activation and Gene Transcription Cell Reports, 8 (2), pp. 460-469. | Show Abstract | Read more

The estrogen receptor α (ERα) controls cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to control gene transcription. A deeper understanding of these transcriptional mechanisms may uncover therapeutic targets for ERα-dependent cancers. We show that BRD4 regulates ERα-induced gene expression by affecting elongation-associated phosphorylationof RNA polymerase II (RNAPII) and histone H2Bmonoubiquitination. Consistently, BRD4 activity isrequired for proliferation of ER+ breast and endometrial cancer cells and uterine growth in mice. Genome-wide studies revealed an enrichment of BRD4 on transcriptional start sites of active genes and a requirement of BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we demonstrate that BRD4 occupancy on distal EREs enriched for H3K27ac is required for recruitment and elongation of RNAPII on EREs and the production of ERα-dependent enhancer RNAs. These results uncover BRD4 as a central regulator of ERα function and potential therapeutic target. © 2014 The Authors.

Nagarajan S, Hossan T, Alawi M, Najafova Z, Indenbirken D, Bedi U, Taipaleenmäki H, Ben-Batalla I, Scheller M, Loges S et al. 2014. Bromodomain protein BRD4 is required for estrogen receptor-dependent enhancer activation and gene transcription. Cell Rep, 8 (2), pp. 460-469. | Show Abstract | Read more

The estrogen receptor α (ERα) controls cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to control gene transcription. A deeper understanding of these transcriptional mechanisms may uncover therapeutic targets for ERα-dependent cancers. We show that BRD4 regulates ERα-induced gene expression by affecting elongation-associated phosphorylation of RNA polymerase II (RNAPII) and histone H2B monoubiquitination. Consistently, BRD4 activity is required for proliferation of ER(+) breast and endometrial cancer cells and uterine growth in mice. Genome-wide studies revealed an enrichment of BRD4 on transcriptional start sites of active genes and a requirement of BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we demonstrate that BRD4 occupancy on distal EREs enriched for H3K27ac is required for recruitment and elongation of RNAPII on EREs and the production of ERα-dependent enhancer RNAs. These results uncover BRD4 as a central regulator of ERα function and potential therapeutic target.

Zhao Z, Wu H, Wang L, Liu Y, Knapp S, Liu Q, Gray NS. 2014. Exploration of type II binding mode: A privileged approach for kinase inhibitor focused drug discovery? ACS Chem Biol, 9 (6), pp. 1230-1241. | Show Abstract | Read more

The ATP site of kinases displays remarkable conformational flexibility when accommodating chemically diverse small molecule inhibitors. The so-called activation segment, whose conformation controls catalytic activity and access to the substrate binding pocket, can undergo a large conformational change with the active state assuming a 'DFG-in' and an inactive state assuming a 'DFG-out' conformation. Compounds that preferentially bind to the DFG-out conformation are typically called 'type II' inhibitors in contrast to 'type I' inhibitors that bind to the DFG-in conformation. This review surveys the large number of type II inhibitors that have been developed and provides an analysis of their crystallographically determined binding modes. Using a small library of type II inhibitors, we demonstrate that more than 200 kinases can be targeted, suggesting that type II inhibitors may not be intrinsically more selective than type I inhibitors.

Hay DA, Fedorov O, Martin S, Singleton DC, Tallant C, Wells C, Picaud S, Philpott M, Monteiro OP, Rogers CM et al. 2014. Discovery and optimization of small-molecule ligands for the CBP/p300 bromodomains. J Am Chem Soc, 136 (26), pp. 9308-9319. | Show Abstract | Read more

Small-molecule inhibitors that target bromodomains outside of the bromodomain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromodomain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benzimidazole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromodomain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (Kd = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromodomains as therapeutic targets.

Rooney TP, Filippakopoulos P, Fedorov O, Picaud S, Cortopassi WA, Hay DA, Martin S, Tumber A, Rogers CM, Philpott M et al. 2014. A series of potent CREBBP bromodomain ligands reveals an induced-fit pocket stabilized by a cation-π interaction. Angew Chem Int Ed Engl, 53 (24), pp. 6126-6130. | Show Abstract | Read more

The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein-bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced-fit pocket forming a cation-π interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells.

Filippakopoulos P, Knapp S. 2014. Targeting bromodomains: epigenetic readers of lysine acetylation. Nat Rev Drug Discov, 13 (5), pp. 337-356. | Show Abstract | Read more

Lysine acetylation is a key mechanism that regulates chromatin structure; aberrant acetylation levels have been linked to the development of several diseases. Acetyl-lysine modifications create docking sites for bromodomains, which are small interaction modules found on diverse proteins, some of which have a key role in the acetylation-dependent assembly of transcriptional regulator complexes. These complexes can then initiate transcriptional programmes that result in phenotypic changes. The recent discovery of potent and highly specific inhibitors for the BET (bromodomain and extra-terminal) family of bromodomains has stimulated intensive research activity in diverse therapeutic areas, particularly in oncology, where BET proteins regulate the expression of key oncogenes and anti-apoptotic proteins. In addition, targeting BET bromodomains could hold potential for the treatment of inflammation and viral infection. Here, we highlight recent progress in the development of bromodomain inhibitors, and their potential applications in drug discovery.

Maiolica A, de Medina-Redondo M, Schoof EM, Chaikuad A, Villa F, Gatti M, Jeganathan S, Lou HJ, Novy K, Hauri S et al. 2014. Modulation of the chromatin phosphoproteome by the Haspin protein kinase. Mol Cell Proteomics, 13 (7), pp. 1724-1740. | Show Abstract | Read more

Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr(3) of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg(2) and Lys(4) adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys(4) of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser(137) of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser(137) resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser(137) might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.

Brady DC, Crowe MS, Turski ML, Hobbs GA, Yao X, Chaikuad A, Knapp S, Xiao K, Campbell SL, Thiele DJ, Counter CM. 2014. Copper is required for oncogenic BRAF signalling and tumorigenesis. Nature, 509 (7501), pp. 492-496. | Show Abstract | Read more

The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

Huber KV, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A, Jemth AS, Göktürk C, Sanjiv K, Strömberg K et al. 2014. Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature, 508 (7495), pp. 222-227. | Show Abstract | Read more

Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.

Cowan-Jacob SW, Jahnke W, Knapp S. 2014. Novel approaches for targeting kinases: allosteric inhibition, allosteric activation and pseudokinases. Future Med Chem, 6 (5), pp. 541-561. | Show Abstract | Read more

Protein kinases are involved in many essential cellular processes and their deregulation can lead to a variety of diseases, including cancer. The pharmaceutical industry has invested heavily in the identification of kinase inhibitors to modulate these disease-promoting pathways, resulting in several successful drugs. However, the field is challenging as it is difficult to identify novel selective inhibitors with good pharmacokinetic/pharmacodynamic properties. In addition, resistance to kinase inhibitor treatment frequently arises. The identification of non-ATP site targeting ('allosteric') inhibitors, the identification of kinase activators and the expansion of kinase target space to include the less studied members of the family, including atypical- and pseudo-kinases, are potential avenues to overcome these challenges. In this perspective, the opportunities and challenges of following these approaches and others will be discussed.

Guetzoyan L, Ingham RJ, Nikbin N, Rossignol J, Wolling M, Baumert M, Burgess-Brown NA, Strain-Damerell CM, Shrestha L, Brennan PE et al. 2014. Machine-assisted synthesis of modulators of the histone reader BRD9 using flow methods of chemistry and frontal affinity chromatography MEDCHEMCOMM, 5 (4), pp. 540-546. | Show Abstract | Read more

A combination of conventional organic synthesis, remotely monitored flow synthesis and bioassay platforms, were used for the evaluation of novel inhibitors targeting bromodomains outside the well-studied bromodomain and extra terminal (BET) family, here exemplified by activity measurements on the bromodomain of BRD9 protein, a component of some tissue-specific SWi/SNF chromatin remodelling complexes. The Frontal Affinity Chromatography combined with Mass Spectrometry (FAC-MS) method proved to be reliable and results correlated well with an independent thermal shift assay. © 2014 the Partner Organisations.

Ciceri P, Müller S, O'Mahony A, Fedorov O, Filippakopoulos P, Hunt JP, Lasater EA, Pallares G, Picaud S, Wells C et al. 2014. Dual kinase-bromodomain inhibitors for rationally designed polypharmacology. Nat Chem Biol, 10 (4), pp. 305-312. | Show Abstract | Read more

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has, however, necessitated the application of combination therapies, which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, which are clinical PLK1 and JAK2-FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors.

Van Ameijde J, Overvoorde J, Knapp S, Den Hertog J, Ruijtenbeek R, Liskamp RM. 2014. A new way to interrogate PTPs Assay and Drug Development Technologies, 12 (2), pp. 105-106. | Read more

Mueller S, Knapp S. 2014. Discovery of BET bromodomain inhibitors and their role in target validation MEDCHEMCOMM, 5 (3), pp. 288-296. | Show Abstract | Read more

Bromodomains (BRDs) are protein interaction modules that selectively recognize ε-N-acetylated lysine residues. BRDs are present in diverse proteins that play key functions in chromatin organization and regulation of gene transcription. Aberrant transcription is a hallmark of many diseases in particular cancer and inflammation. The complexity of molecular processes regulating gene transcription identified transcriptional regulators as interesting targets for the development of specific chemical tool molecules (chemical probes) that help to understand the molecular mechanisms of transcription and to explore the potential of BRD mediated interactions as sites for pharmaceutical intervention. Recently a number of highly specific inhibitors have been developed against the BET (bromo and extra terminal) family of bromodomains. The availability of selective BRD inhibitors had a significant impact on the validation of bromodomain-containing proteins as targets for drug development and for our understanding of the biological roles of these proteins. In this review we will summarize the discovery of BET bromodomain inhibitors and their roles in target validation. © 2014 The Royal Society of Chemistry.

Liu S, Knapp S, Ahmed AA. 2014. The structural basis of PI3K cancer mutations: from mechanism to therapy. Cancer Res, 74 (3), pp. 641-646. | Show Abstract | Read more

While genetic alteration in the p85α-p110α (PI3K) complex represents one of the most frequent driver mutations in cancer, the wild-type complex is also required for driving cancer progression through mutations in related pathways. Understanding the mechanistic basis of the function of the phosphoinositide 3-kinase (PI3K) is essential for designing optimal therapeutic targeting strategies. Recent structural data of the p85α/p110α complex unraveled key insights into the molecular mechanisms of the activation of the complex and provided plausible explanations for the well-established biochemical data on p85/p110 dimer regulation. A wealth of biochemical and biologic information supported by recent genetic findings provides a strong basis for additional p110-independent function of p85α in the regulation of cell survival. In this article, we review the structural, biochemical, and biologic mechanisms through which p85α regulates the cancer cell life cycle with an emphasis on the recently discovered genetic alterations in cancer. As cancer progression is dependent on multiple biologic processes, targeting key drivers such as the PI3K may be required for efficacious therapy of heterogeneous tumors typically present in patients with late-stage disease.

Selner NG, Luechapanichkul R, Chen X, Neel BG, Zhang ZY, Knapp S, Bell CE, Pei D. 2014. Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases. Biochemistry, 53 (2), pp. 397-412. | Show Abstract | Read more

The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >10(5)-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >10(5)-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3-18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A cocrystal structure of PTP1B bound with a nephrin pY(1193) peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities, and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed.

Chaikuad A, Keates T, Vincke C, Kaufholz M, Zenn M, Zimmermann B, Gutiérrez C, Zhang RG, Hatzos-Skintges C, Joachimiak A et al. 2014. Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies. Biochem J, 459 (1), pp. 59-69. | Show Abstract | Read more

GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent 'off-target' of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.

Wilbek TS, Skovgaard T, Sorrell FJ, Knapp S, Berthelsen J, Strømgaard K. 2015. Identification and characterization of a small-molecule inhibitor of death-associated protein kinase 1. Chembiochem, 16 (1), pp. 59-63. | Read more

Hammitzsch A, de Wit J, Ridley A, Al-Mossawi MH, Knapp S, Bowness P. 2014. BROMODOMAIN INHIBITORS REDUCE TH17-TYPE RESPONSES IN SPONDYLOARTHRITIS IN VITRO CLINICAL AND EXPERIMENTAL RHEUMATOLOGY, 32 (5), pp. 810-810.

Philpott M, Rogers CM, Yapp C, Wells C, Lambert JP, Strain-Damerell C, Burgess-Brown NA, Gingras AC, Knapp S, Müller S. 2014. Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching. Epigenetics Chromatin, 7 (1), pp. 14. | Show Abstract | Read more

BACKGROUND: Acetylation of lysine residues in histone tails plays an important role in the regulation of gene transcription. Bromdomains are the readers of acetylated histone marks, and, consequently, bromodomain-containing proteins have a variety of chromatin-related functions. Moreover, they are increasingly being recognised as important mediators of a wide range of diseases. The first potent and selective bromodomain inhibitors are beginning to be described, but the diverse or unknown functions of bromodomain-containing proteins present challenges to systematically demonstrating cellular efficacy and selectivity for these inhibitors. Here we assess the viability of fluorescence recovery after photobleaching (FRAP) assays as a target agnostic method for the direct visualisation of an on-target effect of bromodomain inhibitors in living cells. RESULTS: Mutation of a conserved asparagine crucial for binding to acetylated lysines in the bromodomains of BRD3, BRD4 and TRIM24 all resulted in reduction of FRAP recovery times, indicating loss of or significantly reduced binding to acetylated chromatin, as did the addition of known inhibitors. Significant differences between wild type and bromodomain mutants for ATAD2, BAZ2A, BRD1, BRD7, GCN5L2, SMARCA2 and ZMYND11 required the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to amplify the binding contribution of the bromodomain. Under these conditions, known inhibitors decreased FRAP recovery times back to mutant control levels. Mutation of the bromodomain did not alter FRAP recovery times for full-length CREBBP, even in the presence of SAHA, indicating that other domains are primarily responsible for anchoring CREBBP to chromatin. However, FRAP assays with multimerised CREBBP bromodomains resulted in a good assay to assess the efficacy of bromodomain inhibitors to this target. The bromodomain and extraterminal protein inhibitor PFI-1 was inactive against other bromodomain targets, demonstrating the specificity of the method. CONCLUSIONS: Viable FRAP assays were established for 11 representative bromodomain-containing proteins that broadly cover the bromodomain phylogenetic tree. Addition of SAHA can overcome weak binding to chromatin, and the use of tandem bromodomain constructs can eliminate masking effects of other chromatin binding domains. Together, these results demonstrate that FRAP assays offer a potentially pan-bromodomain method for generating cell-based assays, allowing the testing of compounds with respect to cell permeability, on-target efficacy and selectivity.

Rudolf AF, Skovgaard T, Knapp S, Jensen LJ, Berthelsen J. 2014. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination. PLoS One, 9 (6), pp. e98800. | Show Abstract | Read more

Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening conditions. Furthermore a marked improvement in the correlation was found by using kinase constructs containing the catalytic domain in presence of additional domains or subunits.

Ekambaram R, Enkvist E, Manoharan GB, Ugandi M, Kasari M, Viht K, Knapp S, Issinger OG, Uri A. 2014. Benzoselenadiazole-based responsive long-lifetime photoluminescent probes for protein kinases. Chem Commun (Camb), 50 (31), pp. 4096-4098. | Show Abstract | Read more

Benzoselenadiazole-containing inhibitors of protein kinases were constructed and their capability to emit phosphorescence in the kinase-bound state was established. Labelling of the inhibitors with a red fluorescent dye led to sensitive responsive photoluminescent probes for protein kinase CK2 that emitted red light with a long (microsecond-scale) decay time upon excitation of the probes with a pulse of near-UV light.

Costa L, Casimiro S, Gupta S, Knapp S, Pillai MR, Toi M, Badwe R, Carmo-Fonseca M, Kumar R. 2014. The global cancer genomics consortium’s third annual symposium: From oncogenomics to cancer care Genes and Cancer, 5 (3-4), pp. 64-70. | Show Abstract

© 2014, Impact Journals LLC. All Rights Reserved.The Global Cancer Genomics Consortium (GCGC) is a cohesive network of oncologists, cancer biologists and structural and genomic experts residing in six institutions from Portugal, United Kingdom, Japan, India, and United States. The team is using its combined resources and infrastructures to address carefully selected, shared, burning questions in cancer medicine. The Third Annual Symposium was organized by the Institute of Molecular Medicine, Lisbon Medical School, Lisbon, Portugal, from September 18 to 20, 2013. To highlight the benefits and limitations of recent advances in cancer genomics, the meeting focused on how to better translate our gains in oncogenomics to cancer patients while engaging our younger colleagues in cancer medicine at-large. Over two hundreds participants actively discussed some of the most recent advances in the areas cancer genomics, transcriptomics and cancer system biology and how to best apply such knowledge to cancer therapeutics, biomarkers discovery and drug development, and an essential role played by bio-banking throughout the process. In brief, the GCGC symposium provided a platform for students and translational cancer researchers to share their excitement and worries as we are beginning to translate the gains in oncogenomics to a better cancer patient treatment

Chen C, Ha BH, Thévenin AF, Lou HJ, Zhang R, Yip KY, Peterson JR, Gerstein M, Kim PM, Filippakopoulos P et al. 2014. Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity. Mol Cell, 53 (1), pp. 140-147. | Show Abstract | Read more

Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the "DFG+1" residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.

Fedorov O, Lingard H, Wells C, Monteiro OP, Picaud S, Keates T, Yapp C, Philpott M, Martin SJ, Felletar I et al. 2014. [1,2,4]triazolo[4,3-a]phthalazines: inhibitors of diverse bromodomains. J Med Chem, 57 (2), pp. 462-476. | Show Abstract | Read more

Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]triazolo[4,3-a]phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology.

Ferguson FM, Fedorov O, Chaikuad A, Philpott M, Muniz JR, Felletar I, von Delft F, Heightman T, Knapp S, Abell C, Ciulli A. 2013. Targeting low-druggability bromodomains: fragment based screening and inhibitor design against the BAZ2B bromodomain. J Med Chem, 56 (24), pp. 10183-10187. | Show Abstract | Read more

Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable.

van Ameijde J, Overvoorde J, Knapp S, den Hertog J, Ruijtenbeek R, Liskamp RM. 2014. A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates. Anal Biochem, 448 (1), pp. 9-13. | Show Abstract | Read more

A versatile assay for protein tyrosine phosphatases (PTP) employing 3-nitrophosphotyrosine containing peptidic substrates is described. These therapeutically important phosphatases feature in signal transduction pathways. The assay involves spectrophotometric detection of 3-nitrotyrosine production from 3-nitrophosphotyrosine containing peptidic substrates, which are accepted by many PTPs. Compared to conventional chromogenic phosphate derivatives, the more realistic peptidic substrates allow evaluating substrate specificity. The assay's applicability is demonstrated by determining kinetic parameters for several PTP-substrate combinations and inhibitor evaluation, as well as detection of PTP activity in lysates. The convenient new assay may assist further adoption of PTPs in drug development.

Deng X, Elkins JM, Zhang J, Yang Q, Erazo T, Gomez N, Choi HG, Wang J, Dzamko N, Lee JD et al. 2013. Structural determinants for ERK5 (MAPK7) and leucine rich repeat kinase 2 activities of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones. Eur J Med Chem, 70 pp. 758-767. | Show Abstract | Read more

The benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one core was discovered as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor scaffold, previously. Further structure-activity relationship studies of this scaffold led to the discovery of ERK5-IN-1 (26) as the most selective and potent ERK5 inhibitor reported to date. 26 potently inhibits ERK5 biochemically with an IC₅₀ of 0.162 ± 0.006 μM and in cells with a cellular EC₅₀ for inhibiting epidermal growth factor induced ERK5 autophosphorylation of 0.09 ± 0.03 μM. Furthermore, 26 displays excellent selectivity over other kinases with a KINOMEscan selectivity score (S₁₀) of 0.007, and exhibits exceptional bioavailability (F%) of 90% in mice. 26 will serve as a valuable tool compound to investigate the ERK5 signaling pathway and as a starting point for developing an ERK5 directed therapeutic agent.

Picaud S, Wells C, Felletar I, Brotherton D, Martin S, Savitsky P, Diez-Dacal B, Philpott M, Bountra C, Lingard H et al. 2013. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proc Natl Acad Sci U S A, 110 (49), pp. 19754-19759. | Show Abstract | Read more

Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.

Schäfer G, Milić J, Eldahshan A, Götz F, Zühlke K, Schillinger C, Kreuchwig A, Elkins JM, Abdul Azeez KR, Oder A et al. 2013. Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions. Angew Chem Int Ed Engl, 52 (46), pp. 12187-12191. | Show Abstract | Read more

A good fit: Interactions between A-kinase anchoring proteins (AKAPs) and protein kinaseA (PKA) play key roles in a plethora of physiologically relevant processes whose dysregulation causes or is associated with diseases such as heart failure. Terpyridines have been developed as α-helix mimetics for the inhibition of such interactions and are the first biologically active, nonpeptidic compounds that block the AKAP binding site of PKA. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Fisher K, Gee F, Wang S, Xue F, Knapp S, Philpott M, Wells C, Rodriguez M, Snoek LB, Kammenga J, Poulin GB. 2013. Maintenance of muscle myosin levels in adult C. elegans requires both the double bromodomain protein BET-1 and sumoylation. Biol Open, 2 (12), pp. 1354-1363. | Show Abstract | Read more

Attenuation of RAS-mediated signalling is a conserved process essential to control cell proliferation, differentiation, and apoptosis. Cooperative interactions between histone modifications such as acetylation, methylation and sumoylation are crucial for proper attenuation in C. elegans, implying that the proteins recognising these histone modifications could also play an important role in attenuation of RAS-mediated signalling. We sought to systematically identify these proteins and found BET-1. BET-1 is a conserved double bromodomain protein that recognises acetyl-lysines on histone tails and maintains the stable fate of various lineages. Unexpectedly, adults lacking both BET-1 and SUMO-1 are depleted of muscle myosin, an essential component of myofibrils. We also show that this muscle myosin depletion does not occur in all animals at a specific time, but rather that the penetrance of the phenotype increases with age. To gain mechanistic insights into this process, we sought to delay the occurrence of the muscle myosin depletion phenotype and found that it requires caspase activity and MEK-dependent signalling. We also performed transcription profiling on these mutants and found an up-regulation of the FGF receptor, egl-15, a tyrosine kinase receptor acting upstream of MEK. Consistent with a MEK requirement, we could delay the muscle phenotype by systemic or hypodermal knock down of egl-15. Thus, this work uncovered a caspase- and MEK-dependent mechanism that acts specifically on ageing adults to maintain the appropriate net level of muscle myosin.

Harrington L, Cheley S, Alexander LT, Knapp S, Bayley H. 2013. Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate. Proc Natl Acad Sci U S A, 110 (47), pp. E4417-E4426. | Show Abstract | Read more

In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-molecule detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (~10(7) to 10(8) M(-1) · s(-1)), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.

Knapp S, Weinmann H. 2013. Small-molecule modulators for epigenetics targets ChemMedChem, 8 (11), pp. 1885-1891. | Read more

van Ameijde J, Overvoorde J, Knapp S, den Hertog J, Ruijtenbeek R, Liskamp RMJ. 2013. Real-Time Monitoring of the Dephosphorylating Activity of Protein Tyrosine Phosphatases Using Microarrays with 3-Nitrophosphotyrosine Substrates CHEMPLUSCHEM, 78 (11), pp. 1349-1357. | Show Abstract | Read more

Phosphatases and kinases regulate the crucial phosphorylation post-translational modification. In spite of their similarly important role in many diseases and therapeutic potential, phosphatases have received arguably less attention. One reason for this is a scarcity of high-throughput phosphatase assays. Herein, a new real-time, dynamic protein tyrosine phosphatase (PTP) substrate microarray assay measuring product formation is described. PTP substrates comprising a novel 3-nitrophosphotyrosine residue are immobilized in discrete spots. After reaction catalyzed by a PTP a 3-nitrotyrosine residue is formed that can be detected by specific, sequence-independent antibodies. The resulting microarray was successfully evaluated with a panel of recombinant PTPs and cell lysates, which afforded results comparable to data from other assays. Its parallel nature, convenience, and low sample requirements facilitate investigation of the therapeutically relevant PTP enzyme family. Keeping it real: The activity of important protein tyrosine phosphatases has been monitored in real time in parallel with a novel substrate microarray through formation of 3-nitrotyrosine (see figure). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Knapp S, Weinmann H. 2013. Small-molecule modulators for epigenetics targets. ChemMedChem, 8 (11), pp. 1885-1891. | Show Abstract | Read more

A capital conference: Influencing epigenetic mechanisms may be highly relevant for future therapies of various diseases such as cancer, inflammation, and metabolic disorders. Leading experts in the field gathered in Berlin on June 5-6, 2013 at a Bayer HealthCare Life Science Workshop to share recent success stories and to discuss future trends.

Vidler LR, Filippakopoulos P, Fedorov O, Picaud S, Martin S, Tomsett M, Woodward H, Brown N, Knapp S, Hoelder S. 2013. Discovery of novel small-molecule inhibitors of BRD4 using structure-based virtual screening. J Med Chem, 56 (20), pp. 8073-8088. | Show Abstract | Read more

Bromodomains (BRDs) are epigenetic readers that recognize acetylated-lysine (KAc) on proteins and are implicated in a number of diseases. We describe a virtual screening approach to identify BRD inhibitors. Key elements of this approach are the extensive design and use of substructure queries to compile a set of commercially available compounds featuring novel putative KAc mimetics and docking this set for final compound selection. We describe the validation of this approach by applying it to the first BRD of BRD4. The selection and testing of 143 compounds lead to the discovery of six novel hits, including four unprecedented KAc mimetics. We solved the crystal structure of four hits, determined their binding mode, and improved their potency through synthesis and the purchase of derivatives. This work provides a validated virtual screening approach that is applicable to other BRDs and describes novel KAc mimetics that can be further explored to design more potent inhibitors.

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De Nicola GF, Martin ED, Chaikuad A, Bassi R, Clark J, Martino L, Verma S, Sicard P, Tata R, Atkinson RA et al. 2013. Mechanism and consequence of the autoactivation of p38α mitogen-activated protein kinase promoted by TAB1 Nature Structural and Molecular Biology, 20 (10), pp. 1182-1192. | Show Abstract | Read more

p38α mitogen-activated protein kinase (p38α) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFβ-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical-genetic approaches and coexpression in mammalian, bacterial and cell-free systems revealed that mouse p38α autophosphorylation occurs in cis by direct interaction with TAB1(371-416). In isolated rat cardiac myocytes and perfused mouse hearts, TAT-TAB1(371-416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38α C-terminal kinase lobe. TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis. Interference with p38α recognition by TAB1 abolishes its cardiac toxicity. Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity. © 2013 Nature America, Inc. All rights reserved.

Canning P, Cooper CD, Krojer T, Murray JW, Pike AC, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V et al. 2013. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J Biol Chem, 288 (39), pp. 28304. | Read more

Gammons MV, Fedorov O, Ivison D, Du C, Clark T, Hopkins C, Hagiwara M, Dick AD, Cox R, Harper SJ et al. 2013. Topical antiangiogenic SRPK1 inhibitors reduce choroidal neovascularization in rodent models of exudative AMD. Invest Ophthalmol Vis Sci, 54 (9), pp. 6052-6062. | Show Abstract | Read more

PURPOSE: Exudative AMD (wet AMD) is treated by monthly injection into the eye of anti-VEGF proteins. VEGF is alternatively spliced to produce numerous isoforms that differ in angiogenic activity. Serine-rich protein kinase-1 (SRPK1) has been identified as a regulator of pro-angiogenic VEGF splicing by phosphorylating serine-rich splicing factor-1 (SRSF1), which binds to VEGF pre-mRNA. We tested the hypothesis that topical (eye drop) SRPK1-selective inhibitors could be generated that reduce pro-angiogenic isoforms, and prevent choroidal neovascularization in vivo. METHODS: Novel inhibitors were tested for SRPK inhibition in vitro, pro-angiogenic VEGF production in RPE cells by PCR and ELISA, and for inhibition of choroidal neovascularisation in mice and rats. RESULTS: A novel disubstituted furan inhibitor was selective for the SRPK family of kinases and reduced expression of pro-angiogenic but not antiangiogenic VEGF isoforms. This inhibitor and previously identified SRPK inhibitors significantly reduced choroidal neovascularisation in vivo. Topical administration of SRPK inhibitors dose-dependently blocked CNV with an EC50 of 9 μM. CONCLUSIONS: These results indicate that novel SRPK1 selective inhibitors could be a potentially novel topical (eye drop) therapeutic for wet AMD.

Knapp S. 2013. Testis specific gene expression drives disease progression and Rituximab resistance in lymphoma. EMBO Mol Med, 5 (8), pp. 1149-1150. | Read more

Da Costa D, Agathanggelou A, Perry T, Weston V, Petermann E, Zlatanou A, Oldreive C, Wei W, Stewart G, Longman J et al. 2013. BET inhibition as a single or combined therapeutic approach in primary paediatric B-precursor acute lymphoblastic leukaemia. Blood Cancer J, 3 (7), pp. e126. | Show Abstract | Read more

Paediatric B-precursor ALL is a highly curable disease, however, treatment resistance in some patients and the long-term toxic effects of current therapies pose the need for more targeted therapeutic approaches. We addressed the cytotoxic effect of JQ1, a highly selective inhibitor against the transcriptional regulators, bromodomain and extra-terminal (BET) family of proteins, in paediatric ALL. We showed a potent in vitro cytotoxic response of a panel of primary ALL to JQ1, independent of their prognostic features but dependent on high MYC expression and coupled with transcriptional downregulation of multiple pro-survival pathways. In agreement with earlier studies, JQ1 induced cell cycle arrest. Here we show that BET inhibition also reduced c-Myc protein stability and suppressed progression of DNA replication forks in ALL cells. Consistent with c-Myc depletion and downregulation of pro-survival pathways JQ1 sensitised primary ALL samples to the classic ALL therapeutic agent dexamethasone. Finally, we demonstrated that JQ1 reduces ALL growth in ALL xenograft models, both as a single agent and in combination with dexamethasone. We conclude that targeting BET proteins should be considered as a new therapeutic strategy for the treatment of paediatric ALL and particularly those cases that exhibit suboptimal responses to standard treatment.

Lori C, Lantella A, Pasquo A, Alexander LT, Knapp S, Chiaraluce R, Consalvi V. 2013. Effect of single amino acid substitution observed in cancer on Pim-1 kinase thermodynamic stability and structure. PLoS One, 8 (6), pp. e64824. | Show Abstract | Read more

Pim-1 kinase, a serine/threonine protein kinase encoded by the pim proto-oncogene, is involved in several signalling pathways such as the regulation of cell cycle progression and apoptosis. Many cancer types show high expression levels of Pim kinases and particularly Pim-1 has been linked to the initiation and progression of the malignant phenotype. In several cancer tissues somatic Pim-1 mutants have been identified. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of Pim-1 kinase. We expressed and purified some of the mutants of Pim-1 kinase that are expressed in cancer tissues and reported in the single nucleotide polymorphisms database. The point mutations in the variants significantly affect the conformation of the native state of Pim-1. All the mutants, expressed as soluble recombinant proteins, show a decreased thermal and thermodynamic stability and a lower activation energy values for kinase activity. The decreased stability accompanied by an increased flexibility suggests that Pim-1 variants may be involved in a wider network of protein interactions. All mutants bound ATP and ATP mimetic inhibitors with comparable IC50 values suggesting that the studied Pim-1 kinase mutants can be efficiently targeted with inhibitors developed for the wild type protein.

Ekambaram R, Enkvist E, Vaasa A, Kasari M, Raidaru G, Knapp S, Uri A. 2013. Selective bisubstrate inhibitors with sub-nanomolar affinity for protein kinase Pim-1. ChemMedChem, 8 (6), pp. 909-913. | Show Abstract | Read more

Potent and selective: The unique nature of the ATP binding pocket structure of Pim family protein kinases (PKs) was used for the development of bisubstrate inhibitors and a fluorescent probe with sub-nanomolar affinity. Conjugates of arginine-rich peptides with two ATP mimetic scaffolds were synthesized and tested as inhibitors of Pim-1. Against a panel of 124 protein kinases, a novel ARC-PIM conjugate selectively inhibited PKs of the Pim family.

Elkins JM, Wang J, Deng X, Pattison MJ, Arthur JS, Erazo T, Gomez N, Lizcano JM, Gray NS, Knapp S. 2013. X-ray crystal structure of ERK5 (MAPK7) in complex with a specific inhibitor. J Med Chem, 56 (11), pp. 4413-4421. | Show Abstract | Read more

The protein kinase ERK5 (MAPK7) is an emerging drug target for a variety of indications, in particular for cancer where it plays a key role mediating cell proliferation, survival, epithelial-mesenchymal transition, and angiogenesis. To date, no three-dimensional structure has been published that would allow rational design of inhibitors. To address this, we determined the X-ray crystal structure of the human ERK5 kinase domain in complex with a highly specific benzo[e]pyrimido[5,4-b]diazepine-6(11H)-one inhibitor. The structure reveals that specific residue differences in the ATP-binding site, compared to the related ERKs p38s and JNKs, allow for the development of ERK5-specific inhibitors. The selectivity of previously observed ERK5 inhibitors can also be rationalized using this structure, which provides a template for future development of inhibitors with potential for treatment of disease.

Lu M, Breyssens H, Salter V, Zhong S, Hu Y, Baer C, Ratnayaka I, Sullivan A, Brown NR, Endicott J et al. 2013. Restoring p53 function in human melanoma cells by inhibiting MDM2 and cyclin B1/CDK1-phosphorylated nuclear iASPP. Cancer Cell, 23 (5), pp. 618-633. | Show Abstract | Read more

Nearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.

Soundararajan M, Roos AK, Savitsky P, Filippakopoulos P, Kettenbach AN, Olsen JV, Gerber SA, Eswaran J, Knapp S, Elkins JM. 2013. Structures of Down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition. Structure, 21 (6), pp. 986-996. | Show Abstract | Read more

Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK subfamily: DYRK1A with an ATP-mimetic inhibitor and consensus peptide, and DYRK2 including NAPA and DH (DYRK homology) box regions. The current activation model suggests that DYRKs are Ser/Thr kinases that only autophosphorylate the second tyrosine of the activation loop YxY motif during protein translation. The structures explain the roles of this tyrosine and of the DH box in DYRK activation and provide a structural model for DYRK substrate recognition. Phosphorylation of a library of naturally occurring peptides identified substrate motifs that lack proline in the P+1 position, suggesting that DYRK1A is not a strictly proline-directed kinase. Our data also show that DYRK1A wild-type and Y321F mutant retain tyrosine autophosphorylation activity.

Picaud S, Da Costa D, Thanasopoulou A, Filippakopoulos P, Fish PV, Philpott M, Fedorov O, Brennan P, Bunnage ME, Owen DR et al. 2013. PFI-1, a highly selective protein interaction inhibitor, targeting BET Bromodomains. Cancer Res, 73 (11), pp. 3336-3346. | Show Abstract | Read more

Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycle progression. BET proteins are recruited on transcriptionally active chromatin via their two N-terminal bromodomains (BRD), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional downregulation of a number of oncogenes, providing a novel pharmacologic strategy for the treatment of cancer. Here, we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1, which efficiently blocks the interaction of BET BRDs with acetylated histone tails. Cocrystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell-cycle arrest, downregulation of MYC expression, as well as induction of apoptosis and induces differentiation of primary leukemic blasts. Intriguingly, cells exposed to PFI-1 showed significant downregulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10, providing an alternative strategy for the specific inhibition of this well-established oncology target.

Cited:

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Cheng Z, Gong Y, Ma Y, Lu K, Lu X, Pierce LA, Thompson RC, Muller S, Knapp S, Wang J. 2013. Inhibition of BET Bromodomain Targets Genetically Diverse Glioblastoma CLINICAL CANCER RESEARCH, 19 (7), pp. 1748-1759. | Read more

Hewings DS, Fedorov O, Filippakopoulos P, Martin S, Picaud S, Tumber A, Wells C, Olcina MM, Freeman K, Gill A et al. 2013. Optimization of 3,5-dimethylisoxazole derivatives as potent bromodomain ligands. J Med Chem, 56 (8), pp. 3217-3227. | Show Abstract | Read more

The bromodomain protein module, which binds to acetylated lysine, is emerging as an important epigenetic therapeutic target. We report the structure-guided optimization of 3,5-dimethylisoxazole derivatives to develop potent inhibitors of the BET (bromodomain and extra terminal domain) bromodomain family with good ligand efficiency. X-ray crystal structures of the most potent compounds reveal key interactions required for high affinity at BRD4(1). Cellular studies demonstrate that the phenol and acetate derivatives of the lead compounds showed strong antiproliferative effects on MV4;11 acute myeloid leukemia cells, as shown for other BET bromodomain inhibitors and genetic BRD4 knockdown, whereas the reported compounds showed no general cytotoxicity in other cancer cell lines tested.

Kumar R, Horvath A, Mazumder R, Toi M, Sato F, Pillai MR, Costa L, Carmo-Fonseca M, Knapp S, Dutt A et al. 2013. The Global Cancer Genomics Consortium's Second Annual Symposium: Genomics Medicine in Cancer Research Genes & Cancer, 4 (5-6), pp. 196-200. | Show Abstract | Read more

The Second Annual Symposium of the Global Cancer Genomics Consortium (GCGC) was held at the Tata Memorial Center in Mumbai, India, from November 19 to 20, 2012. Founded in late 2010, the GCGC aims to provide a platform for highly productive, collaborative efforts on next-generation cancer research through bridging the latest scientific and technology developments with clinical oncology challenges. This year's presenters brought together highly innovative interdisciplinary views and strategies to meet major challenges in cancer research. The symposium featured 3 major themes: OMICS approaches toward the identification of cancer molecular drivers, single-cell analysis in cancer, and clinical and translational genomics. Each theme was represented in presentations of new findings, with an obvious implication in cross-disciplinary components of OMICs and an overwhelming participation by students. In summary, the GCGC symposium provided a discussion and congregation of the latest advances in basic and translational cancer research and offered the participants with a highly cooperative network environment for future collaboration. © The Author(s) 2013.

Yu W, Smil D, Li F, Tempel W, Fedorov O, Nguyen KT, Bolshan Y, Al-Awar R, Knapp S, Arrowsmith CH et al. 2013. Bromo-deaza-SAH: a potent and selective DOT1L inhibitor. Bioorg Med Chem, 21 (7), pp. 1787-1794. | Show Abstract | Read more

Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia.

Canning P, Cooper CD, Krojer T, Murray JW, Pike AC, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V et al. 2013. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J Biol Chem, 288 (11), pp. 7803-7814. | Show Abstract | Read more

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.

Myrianthopoulos V, Kritsanida M, Gaboriaud-Kolar N, Magiatis P, Ferandin Y, Durieu E, Lozach O, Cappel D, Soundararajan M, Filippakopoulos P et al. 2013. Novel Inverse Binding Mode of Indirubin Derivatives Yields Improved Selectivity for DYRK Kinases. ACS Med Chem Lett, 4 (1), pp. 22-26. | Show Abstract | Read more

DYRK kinases are involved in alternative pre-mRNA splicing as well as in neuropathological states such as Alzheimer's disease and Down syndrome. In this study, we present the design, synthesis, and biological evaluation of indirubins as DYRK inhibitors with enhanced selectivity. Modifications of the bis-indole included polar or acidic functionalities at positions 5' and 6' and a bromine or a trifluoromethyl group at position 7, affording analogues that possess high activity and pronounced specificity. Compound 6i carrying a 5'-carboxylate moiety demonstrated the best inhibitory profile. A novel inverse binding mode, which forms the basis for the improved selectivity, was suggested by molecular modeling and confirmed by determining the crystal structure of DYRK2 in complex with 6i. Structure-activity relationships were further established, including a thermodynamic analysis of binding site water molecules, offering a structural explanation for the selective DYRK inhibition.

Knapp S. 2013. 3D Structure and Physiological Regulation of PAKs pp. 137-148. | Read more

Newman RH, Hu J, Rho HS, Xie Z, Woodard C, Neiswinger J, Cooper C, Shirley M, Clark HM, Hu S et al. 2013. Construction of human activity-based phosphorylation networks. Mol Syst Biol, 9 (1), pp. 655. | Show Abstract | Read more

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.

Knapp S, Arruda P, Blagg J, Burley S, Drewry DH, Edwards A, Fabbro D, Gillespie P, Gray NS, Kuster B et al. 2013. A public-private partnership to unlock the untargeted kinome. Nat Chem Biol, 9 (1), pp. 3-6. | Read more

Mehellou Y, Alessi DR, Macartney TJ, Szklarz M, Knapp S, Elkins JM. 2013. Structural insights into the activation of MST3 by MO25. Biochem Biophys Res Commun, 431 (3), pp. 604-609. | Show Abstract | Read more

The MO25 scaffolding protein operates as critical regulator of a number of STE20 family protein kinases (e.g. MST and SPAK isoforms) as well as pseudokinases (e.g. STRAD isoforms that play a critical role in activating the LKB1 tumour suppressor). To better understand how MO25 interacts and stimulates the activity of STE20 protein kinases, we determined the crystal structure of MST3 catalytic domain (residues 19-289) in complex with full length MO25β. The structure reveals an intricate web of interactions between MST3 and MO25β that function to stabilise the kinase domain in a closed, active, conformation even in the absence of ATP or an ATP-mimetic inhibitor. The binding mode of MO25β is reminiscent of the mechanism by which MO25α interacts with the pseudokinase STRADα. In particular we identified interface residues Tyr223 of MO25β and Glu58 and Ile71 of MST3 that when mutated prevent activation of MST3 by MO25β. These data provide molecular understanding of the mechanism by which MO25 isoforms regulates the activity of STE20 family protein kinases.

Hay D, Fedorov O, Filippakopoulos P, Martin S, Philpott M, Picaud S, Hewings DS, Uttakar S, Heightman TD, Conway SJ et al. 2013. The design and synthesis of 5- and 6-isoxazolylbenzimidazoles as selective inhibitors of the BET bromodomains. Medchemcomm, 4 (1), pp. 140-144. | Show Abstract | Read more

Simple 1-substituted 5- and 6-isoxazolyl-benzimidazoles have been shown to be potent inhibitors of the BET bromodomains with selectivity over the related bromodomain of CBP. The reported inhibitors were prepared from simple starting materials in two steps followed by separation of the regioisomers or regioselectively in three steps.

Fish PV, Filippakopoulos P, Bish G, Brennan PE, Bunnage ME, Cook AS, Federov O, Gerstenberger BS, Jones H, Knapp S et al. 2012. Identification of a chemical probe for bromo and extra C-terminal bromodomain inhibition through optimization of a fragment-derived hit. J Med Chem, 55 (22), pp. 9831-9837. | Show Abstract | Read more

The posttranslational modification of chromatin through acetylation at selected histone lysine residues is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The significance of this subset of the epigenetic code is interrogated and interpreted by an acetyllysine-specific protein-protein interaction with bromodomain reader modules. Selective inhibition of the bromo and extra C-terminal domain (BET) family of bromodomains with a small molecule is feasible, and this may represent an opportunity for disease intervention through the recently disclosed antiproliferative and anti-inflammatory properties of such inhibitors. Herein, we describe the discovery and structure-activity relationship (SAR) of a novel, small-molecule chemical probe for BET family inhibition that was identified through the application of structure-based fragment assessment and optimization techniques. This has yielded a potent, selective compound with cell-based activity (PFI-1) that may further add to the understanding of BET family function within the bromodomains.

De Antoni A, Maffini S, Knapp S, Musacchio A, Santaguida S. 2012. A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B. J Cell Biol, 199 (2), pp. 269-284. | Show Abstract | Read more

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B.

Hewings DS, Rooney TP, Jennings LE, Hay DA, Schofield CJ, Brennan PE, Knapp S, Conway SJ. 2012. Progress in the development and application of small molecule inhibitors of bromodomain-acetyl-lysine interactions. J Med Chem, 55 (22), pp. 9393-9413. | Show Abstract | Read more

Bromodomains, protein modules that recognize and bind to acetylated lysine, are emerging as important components of cellular machinery. These acetyl-lysine (KAc) "reader" domains are part of the write-read-erase concept that has been linked with the transfer of epigenetic information. By reading KAc marks on histones, bromodomains mediate protein-protein interactions between a diverse array of partners. There has been intense activity in developing potent and selective small molecule probes that disrupt the interaction between a given bromodomain and KAc. Rapid success has been achieved with the BET family of bromodomains, and a number of potent and selective probes have been reported. These compounds have enabled linking of the BET bromodomains with diseases, including cancer and inflammation, suggesting that bromodomains are druggable targets. Herein, we review the biology of the bromodomains and discuss the SAR for the existing small molecule probes. The biology that has been enabled by these compounds is summarized.

Tahtouh T, Elkins JM, Filippakopoulos P, Soundararajan M, Burgy G, Durieu E, Cochet C, Schmid RS, Lo DC, Delhommel F et al. 2012. Selectivity, cocrystal structures, and neuroprotective properties of leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid leucettamine B. J Med Chem, 55 (21), pp. 9312-9330. | Show Abstract | Read more

DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are implicated in the onset and development of Alzheimer's disease and Down syndrome. The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3β. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimer's disease.

Elkins JM, Santaguida S, Musacchio A, Knapp S. 2012. Crystal structure of human aurora B in complex with INCENP and VX-680. J Med Chem, 55 (17), pp. 7841-7848. | Show Abstract | Read more

We present the structure of the human Aurora B kinase domain in complex with the C-terminal Aurora-binding region of human INCENP and the Aurora kinase inhibitor VX-680. The structure unexpectedly reveals a dimeric arrangement of the Aurora B:INCENP complex, which was confirmed to exist in solution by analytical ultracentrifugation. The dimerization involves a domain swap of the activation loop, resulting in a different conformation of the DFG motif as compared to that seen in other kinase complexes with VX-680. The binding of INCENP differs significantly from that seen in the Xenopus laevis Aurora B:INCENP complex currently used as a model for structure-based design for this important oncology target.

Chaikuad A, Alfano I, Kerr G, Sanvitale CE, Boergermann JH, Triffitt JT, von Delft F, Knapp S, Knaus P, Bullock AN. 2012. Structure of the bone morphogenetic protein receptor ALK2 and implications for fibrodysplasia ossificans progressiva. J Biol Chem, 287 (44), pp. 36990-36998. | Show Abstract | Read more

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.

Prudent R, Vassal-Stermann E, Nguyen CH, Pillet C, Martinez A, Prunier C, Barette C, Soleilhac E, Filhol O, Beghin A et al. 2012. Pharmacological inhibition of LIM kinase stabilizes microtubules and inhibits neoplastic growth. Cancer Res, 72 (17), pp. 4429-4439. | Show Abstract | Read more

The emergence of tumor resistance to conventional microtubule-targeting drugs restricts their clinical use. Using a cell-based assay that recognizes microtubule polymerization status to screen for chemicals that interact with regulators of microtubule dynamics, we identified Pyr1, a cell permeable inhibitor of LIM kinase, which is the enzyme that phosphorylates and inactivates the actin-depolymerizing factor cofilin. Pyr1 reversibly stabilized microtubules, blocked actin microfilament dynamics, inhibited cell motility in vitro and showed anticancer properties in vivo, in the absence of major side effects. Pyr1 inhibition of LIM kinase caused a microtubule-stabilizing effect, which was independent of any direct effects on the actin cytoskeleton. In addition, Pyr1 retained its activity in multidrug-resistant cancer cells that were resistant to conventional microtubule-targeting agents. Our findings suggest that LIM kinase functions as a signaling node that controls both actin and microtubule dynamics. LIM kinase may therefore represent a targetable enzyme for cancer treatment.

Matzuk MM, McKeown MR, Filippakopoulos P, Li Q, Ma L, Agno JE, Lemieux ME, Picaud S, Yu RN, Qi J et al. 2012. Small-molecule inhibition of BRDT for male contraception. Cell, 150 (4), pp. 673-684. | Show Abstract | Read more

A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.

Hellwig S, Miduturu CV, Kanda S, Zhang J, Filippakopoulos P, Salah E, Deng X, Choi HG, Zhou W, Hur W et al. 2012. Small-molecule inhibitors of the c-Fes protein-tyrosine kinase. Chem Biol, 19 (4), pp. 529-540. | Show Abstract | Read more

The c-Fes protein-tyrosine kinase modulates cellular signaling pathways governing differentiation, the innate immune response, and vasculogenesis. Here, we report the identification of types I and II kinase inhibitors with potent activity against c-Fes both in vitro and in cell-based assays. One of the most potent inhibitors is the previously described anaplastic lymphoma kinase inhibitor TAE684. The crystal structure of TAE684 in complex with the c-Fes SH2-kinase domain showed excellent shape complementarity with the ATP-binding pocket and a key role for the gatekeeper methionine in the inhibitory mechanism. TAE684 and two pyrazolopyrimidines with nanomolar potency against c-Fes in vitro were used to establish a role for this kinase in osteoclastogenesis, illustrating the value of these inhibitors as tool compounds to probe the diverse biological functions associated with this unique kinase.

Filippakopoulos P, Picaud S, Mangos M, Keates T, Lambert JP, Barsyte-Lovejoy D, Felletar I, Volkmer R, Müller S, Pawson T et al. 2012. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell, 149 (1), pp. 214-231. | Show Abstract | Read more

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.

Adams CJ, Pike AC, Maniam S, Sharpe TD, Coutts AS, Knapp S, La Thangue NB, Bullock AN. 2012. The p53 cofactor Strap exhibits an unexpected TPR motif and oligonucleotide-binding (OB)-fold structure. Proc Natl Acad Sci U S A, 109 (10), pp. 3778-3783. | Show Abstract | Read more

Activation of p53 target genes for tumor suppression depends on the stress-specific regulation of transcriptional coactivator complexes. Strap (stress-responsive activator of p300) is activated upon DNA damage by ataxia telangiectasia mutated (ATM) and Chk2 kinases and is a key regulator of the p53 response. In addition to antagonizing Mdm2, Strap facilitates the recruitment of p53 coactivators, including JMY and p300. Strap is a predicted TPR-repeat protein, but shows only limited sequence identity with any protein of known structure. To address this and to elucidate the molecular mechanism of Strap activity we determined the crystal structure of the full-length protein at 2.05 Å resolution. The structure of Strap reveals an atypical six tetratricopeptide repeat (TPR) protein that also contains an unexpected oligonucleotide/oligosaccharide-binding (OB)-fold domain. This previously unseen domain organization provides an extended superhelical scaffold allowing for protein-protein as well as protein-DNA interaction. We show that both of the TPR and OB-fold domains localize to the chromatin of p53 target genes and exhibit intrinsic regulatory activity necessary for the Strap-dependent p53 response.

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Filippakopoulos P, Picaud S, Fedorov O, Keller M, Wrobel M, Morgenstern O, Bracher F, Knapp S. 2012. Benzodiazepines and benzotriazepines as protein interaction inhibitors targeting bromodomains of the BET family Bioorganic and Medicinal Chemistry, 20 (6), pp. 1878-1886. | Show Abstract | Read more

Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective protein interaction inhibitors of bromodomain and extra-terminal (BET) proteins, a family of transcriptional co-regulators that play a key role in cancer cell survival and proliferation, but the requirements for high affinity interaction of this compound class with bromodomains has not been described. Here we provide insight into the structure-activity relationship (SAR) and selectivity of this versatile scaffold. In addition, using high resolution crystal structures we compared the binding mode of a series of benzodiazepine (BzD) and related triazolo-benzotriazepines (BzT) derivatives including clinically approved drugs such as alprazolam and midazolam. Our analysis revealed the importance of the 1-methyl triazolo ring system for BET binding and suggests modifications for the development of further high affinity bromodomain inhibitors. © 2011 Elsevier Ltd. All rights reserved.

Zhang W, Prakash C, Sum C, Gong Y, Li Y, Kwok JJ, Thiessen N, Pettersson S, Jones SJ, Knapp S et al. 2012. Bromodomain-containing protein 4 (BRD4) regulates RNA polymerase II serine 2 phosphorylation in human CD4+ T cells. J Biol Chem, 287 (51), pp. 43137-43155. | Show Abstract | Read more

Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser-2-phosphorylated Pol II (Pol II Ser-2) at both enhancers and promoters of active genes. Disruption of bromodomain-histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser-2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited diacetylated H4 (lysine 5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells.

Vidler LR, Brown N, Knapp S, Hoelder S. 2012. Druggability analysis and structural classification of bromodomain acetyl-lysine binding sites. J Med Chem, 55 (17), pp. 7346-7359. | Show Abstract | Read more

Bromodomains are readers of the epigenetic code that specifically bind acetyl-lysine containing recognition sites on proteins. Recently the BET family of bromodomains has been demonstrated to be druggable through the discovery of potent inhibitors, sparking an interest in protein-protein interaction inhibitors that directly target gene transcription. Here, we assess the druggability of diverse members of the bromodomain family using SiteMap and show that there are significant differences in predicted druggability. Furthermore, we trace these differences in druggability back to unique amino acid signatures in the bromodomain acetyl-lysine binding sites. These signatures were then used to generate a new classification of the bromodomain family, visualized as a classification tree. This represents the first analysis of this type for the bromodomain family and can prove useful in the discovery of inhibitors, particularly for anticipating screening hit rates, identifying inhibitors that can be explored for lead hopping approaches, and selecting proteins for selectivity screening.

Meiby E, Knapp S, Elkins JM, Ohlson S. 2012. Fragment screening of cyclin G-associated kinase by weak affinity chromatography. Anal Bioanal Chem, 404 (8), pp. 2417-2425. | Show Abstract | Read more

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

Fedorov O, Niesen FH, Knapp S. 2012. Kinase inhibitor selectivity profiling using differential scanning fluorimetry. Methods Mol Biol, 795 pp. 109-118. | Show Abstract | Read more

Fast, robust, and inexpensive screening methods are the heart of drug discovery processes. Moreover, it is useful to have access to several established assay formats, for validation purposes. If a targeted protein is an enzyme, the logical and widely used approach is the direct measurement of the effect of the added ligands on its activity. A variety of enzymatic assay formats have been successfully applied for inhibitor screening of protein kinases. However, enzymatic assays require an active enzyme with a known substrate and often time-consuming assay optimization. Several alternative approaches have been recently developed that detect binding of ligands to proteins. This chapter overviews and provides the experimental protocol of the successful application of differential scanning fluorimetry (DSF) in our laboratory for fast and robust screening of medium-sized (<10,000) inhibitor libraries. DSF monitors the thermal stabilization of the native protein structure upon ligand binding. It allows selectivity profiling of any protein kinase without prior knowledge of either substrate or activity of the kinase under investigation. Comparative studies revealed that generated data is highly reproducible and correlates well with the results from other ligand binding methodologies, direct binding constants as well as enzymatic assays.

Brault L, Menter T, Obermann EC, Knapp S, Thommen S, Schwaller J, Tzankov A. 2012. PIM kinases are progression markers and emerging therapeutic targets in diffuse large B-cell lymphoma. Br J Cancer, 107 (3), pp. 491-500. | Show Abstract | Read more

BACKGROUND: PIM serine/threonine kinases are often highly expressed in haematological malignancies. We have shown that PIM inhibitors reduced the survival and migration of leukaemic cells. Here, we investigated PIM kinases in diffuse large B-cell lymphoma (DLBCL) biopsy samples and DLBCL cell lines. METHODS: Immunohistochemical staining for PIM kinases and CXCR4 was performed on tissue microarrays from a cohort of 101 DLBCL cases, and the effects of PIM inhibitors on the survival and migration of DLBCL cell lines were determined. RESULTS: PIM1 expression significantly correlated with the activation of signal transducer and activator of transcription (STAT) 3 and 5, P-glycoprotein expression, CXCR4-S339 phosphorylation, and cell proliferation. Whereas most cases exhibited cytoplasmic or cytoplasmic and nuclear PIM1 and PIM2 expression, 12 cases (10 of the non-germinal centre DLBCL type) expressed PIM1 predominately in the nucleus. Interestingly, nuclear expression of PIM1 significantly correlated with disease stage. Exposure of DLBCL cell lines to PIM inhibitors modestly impaired cellular proliferation and CXCR4-mediated migration. CONCLUSION: This work demonstrates that PIM expression in DLBCL is associated with activation of the JAK/STAT signalling pathway and with the proliferative activity. The correlation of nuclear PIM1 expression with disease stage and the modest response to small-molecule inhibitors suggests that PIM kinases are progression markers rather than primary therapeutic targets in DLBCL.

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Brault L, Menter T, Obermann EC, Knapp S, Thommen S, Schwaller J, Tzankov A. 2012. PIM kinases are progression markers and emerging therapeutic targets in diffuse large B-cell lymphoma British Journal of Cancer, 107 (3), pp. 491-500. | Show Abstract | Read more

Background: PIM serine/threonine kinases are often highly expressed in haematological malignancies. We have shown that PIM inhibitors reduced the survival and migration of leukaemic cells. Here, we investigated PIM kinases in diffuse large B-cell lymphoma (DLBCL) biopsy samples and DLBCL cell lines.Methods:Immunohistochemical staining for PIM kinases and CXCR4 was performed on tissue microarrays from a cohort of 101 DLBCL cases, and the effects of PIM inhibitors on the survival and migration of DLBCL cell lines were determined.Results:PIM1 expression significantly correlated with the activation of signal transducer and activator of transcription (STAT) 3 and 5, P-glycoprotein expression, CXCR4-S339 phosphorylation, and cell proliferation. Whereas most cases exhibited cytoplasmic or cytoplasmic and nuclear PIM1 and PIM2 expression, 12 cases (10 of the non-germinal centre DLBCL type) expressed PIM1 predominately in the nucleus. Interestingly, nuclear expression of PIM1 significantly correlated with disease stage. Exposure of DLBCL cell lines to PIM inhibitors modestly impaired cellular proliferation and CXCR4-mediated migration.Conclusion:This work demonstrates that PIM expression in DLBCL is associated with activation of the JAK/STAT signalling pathway and with the proliferative activity. The correlation of nuclear PIM1 expression with disease stage and the modest response to small-molecule inhibitors suggests that PIM kinases are progression markers rather than primary therapeutic targets in DLBCL. © 2012 Cancer Research UK All rights reserved.

Kettenbach AN, Wang T, Faherty BK, Madden DR, Knapp S, Bailey-Kellogg C, Gerber SA. 2012. Rapid determination of multiple linear kinase substrate motifs by mass spectrometry. Chem Biol, 19 (5), pp. 608-618. | Show Abstract | Read more

Kinase-substrate recognition depends on the chemical properties of the phosphorylatable residue as well as the surrounding linear sequence motif. Detailed knowledge of these characteristics increases the confidence of linking identified phosphorylation sites to kinases, predicting phosphorylation sites, and designing optimal peptide substrates. Here, we present a mass spectrometry-based approach for determining linear kinase substrate motifs by elaborating the positional and chemical preference of the kinase for a phosphorylatable residue using libraries of naturally-occurring peptides that are amenable to peptide identification by commonly used proteomics platforms. We applied this approach to a structurally and functionally diverse set of purified kinases, which recapitulated their previously described substrate motifs and discovered additional ones, including preferences of certain kinases for phosphorylatable residues adjacent to peptide termini. Furthermore, we identify specific and distinguishable motif elements for the four members of the polo-like kinase (Plk) family and verify members of these motif elements for Plk1 in vivo.

Pasquo A, Consalvi V, Knapp S, Alfano I, Ardini M, Stefanini S, Chiaraluce R. 2012. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations. PLoS One, 7 (2), pp. e32555. | Show Abstract | Read more

Protein tyrosine phosphatase ρ (PTPρ) belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

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Chaikuad A, Alfano I, Kerr G, Sanvitale CE, Boergermann JH, Triffitt JT, Von Delft F, Knapp S, Knaus P, Bullock AN. 2012. Structure of the bone morphogenetic protein receptor ALK2 and implications for fibrodysplasia ossificans progressiva Journal of Biological Chemistry, 287 (44), pp. 36990-36998. | Show Abstract | Read more

Background: Mutations in the ALK2 kinase cause extraskeletal bone formation. Results: We solved the structure of ALK2 in complex with the inhibitor FKBP12. Conclusion: Disease mutations break critical interactions that stabilize the inactive ALK2-FKBP12 complex leading to kinase activation. Significance: We offer an explanation for the effects of mutation and a structural template for the design of small molecule inhibitors. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Filippakopoulos P, Knapp S. 2012. The bromodomain interaction module. FEBS Lett, 586 (17), pp. 2692-2704. | Show Abstract | Read more

ε-N-acetylation of lysine residues (K(ac)) is one of the most abundant post-translation modifications (PTMs) in the human proteome. In the nucleus, acetylation of histones has been linked to transcriptional activation of genes but the functional consequences of most acetylation events and proteins recruited to these sites remains largely unknown. Bromodomains (BRDs) are small helical interaction modules that specifically recognize acetylation sites in proteins. BRDs have recently emerged as interesting targets for the development of specific protein interaction inhibitors, enabling a novel exiting strategy for the development of new therapies. This review provides an overview over sequence requirements of BRDs, known substrates and the structural mechanisms of specific K(ac) recognition.

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Filippakopoulos P, Knapp S. 2012. The bromodomain interaction module FEBS Letters, 586 (17), pp. 2692-2704. | Show Abstract | Read more

-N-acetylation of lysine residues (K ac) is one of the most abundant post-translation modifications (PTMs) in the human proteome. In the nucleus, acetylation of histones has been linked to transcriptional activation of genes but the functional consequences of most acetylation events and proteins recruited to these sites remains largely unknown. Bromodomains (BRDs) are small helical interaction modules that specifically recognize acetylation sites in proteins. BRDs have recently emerged as interesting targets for the development of specific protein interaction inhibitors, enabling a novel exiting strategy for the development of new therapies. This review provides an overview over sequence requirements of BRDs, known substrates and the structural mechanisms of specific K ac recognition. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Global Cancer Genomics Consortium. 2012. The Global Cancer Genomics Consortium: interfacing genomics and cancer medicine. Cancer Res, 72 (15), pp. 3720-3724. | Show Abstract | Read more

The Global Cancer Genomics Consortium (GCGC) is an international collaborative platform that amalgamates cancer biologists, cutting-edge genomics, and high-throughput expertise with medical oncologists and surgical oncologists; they address the most important translational questions that are central to cancer research and treatment. The annual GCGC symposium was held at the Advanced Centre for Treatment Research and Education in Cancer, Mumbai, India, from November 9 to 11, 2011. The symposium showcased international next-generation sequencing efforts that explore cancer-specific transcriptomic changes, single-nucleotide polymorphism, and copy number variations in various types of cancers, as well as the structural genomics approach to develop new therapeutic targets and chemical probes. From the spectrum of studies presented at the symposium, it is evident that the translation of emerging cancer genomics knowledge into clinical applications can only be achieved through the integration of multidisciplinary expertise. In summary, the GCGC symposium provided practical knowledge on structural and cancer genomics approaches, as well as an exclusive platform for focused cancer genomics endeavors.

Elkins JM, Knapp S. 2012. The structure of the full-length tetrameric PKA regulatory RIIβ complex reveals the mechanism of allosteric PKA activation. Sci Signal, 5 (224), pp. pe21. | Show Abstract | Read more

The catalytic activity of protein kinases is usually tightly controlled by posttranslational modifications and diverse sets of regulatory proteins. Protein kinases are highly dynamic enzymes, and structures of kinases in various activation states and costructures with regulatory proteins have provided critical insights into the complex regulatory mechanisms of this large and diverse protein family. The crystal structure of protein kinase A (PKA) provided a reference model for our understanding of kinase catalytic function. Now, more than two decades later, the high-resolution model of a full-length tetrameric PKA holoenzyme has been published, revealing the structural mechanisms underlying allosteric PKA activation.

Brennan P, Filippakopoulos P, Knapp S. 2012. The therapeutic potential of acetyl-lysine and methyl-lysine effector domains Drug Discovery Today: Therapeutic Strategies,

Brennan P, Filippakopoulos P, Knapp S. 2012. The therapeutic potential of acetyl-lysine and methyl-lysine effector domains Drug Discovery Today: Therapeutic Strategies, 9 (2-3), pp. e101-e110. | Show Abstract | Read more

Epigenetic reader domains are protein interaction modules that selectively recognize common post-translational modification on histones and other nuclear proteins such as ε-N-acetylated lysine or methyllysine/arginine residues. Interactions mediated by these effector domains result in the recruitment of gene specific transcriptional regulators. This review focusses on reader domains that recognize acetylated and methylated lysine and arginine residues. Bromodomains selectively recognize acetylated lysines residues and inhibitors have recently emerged as promising lead compounds for the treatment of cancer and inflammatory diseases, acting by specifically repressing expression of oncogenes and pro-inflammatory cytokines. Initial inhibitors have also been reported for methyllysine binding domains. Here we review recent development of this emerging target area. © 2012 Elsevier Ltd. All rights reserved.

Huber K, Brault L, Fedorov O, Gasser C, Filippakopoulos P, Bullock AN, Fabbro D, Trappe J, Schwaller J, Knapp S, Bracher F. 2012. 7,8-dichloro-1-oxo-β-carbolines as a versatile scaffold for the development of potent and selective kinase inhibitors with unusual binding modes. J Med Chem, 55 (1), pp. 403-413. | Show Abstract | Read more

Development of both potent and selective kinase inhibitors is a challenging task in modern drug discovery. The innate promiscuity of kinase inhibitors largely results from ATP-mimetic binding to the kinase hinge region. We present a novel class of substituted 7,8-dichloro-1-oxo-β-carbolines based on the distinct structural features of the alkaloid bauerine C whose kinase inhibitory activity does not rely on canonical ATP-mimetic hinge interactions. Intriguingly, cocrystal structures revealed an unexpected inverted binding mode and the presence of halogen bonds with kinase backbone residues. The compounds exhibit excellent selectivity over a comprehensive panel of human protein kinases while inhibiting selected kinases such as the oncogenic PIM1 at low nanomolar concentrations. Together, our biochemical and structural data suggest that this scaffold may serve as a valuable template for the design and development of specific inhibitors of various kinases including the PIM family of kinases, CLKs, DAPK3 (ZIPK), BMP2K (BIKE), and others.

Filippakopoulos P, Picaud S, Fedorov O, Keller M, Wrobel M, Morgenstern O, Bracher F, Knapp S. 2012. Benzodiazepines and benzotriazepines as protein interaction inhibitors targeting bromodomains of the BET family. Bioorg Med Chem, 20 (6), pp. 1878-1886. | Show Abstract | Read more

Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective protein interaction inhibitors of bromodomain and extra-terminal (BET) proteins, a family of transcriptional co-regulators that play a key role in cancer cell survival and proliferation, but the requirements for high affinity interaction of this compound class with bromodomains has not been described. Here we provide insight into the structure-activity relationship (SAR) and selectivity of this versatile scaffold. In addition, using high resolution crystal structures we compared the binding mode of a series of benzodiazepine (BzD) and related triazolo-benzotriazepines (BzT) derivatives including clinically approved drugs such as alprazolam and midazolam. Our analysis revealed the importance of the 1-methyl triazolo ring system for BET binding and suggests modifications for the development of further high affinity bromodomain inhibitors.

Shrestha A, Hamilton G, O'Neill E, Knapp S, Elkins JM. 2012. Analysis of conditions affecting auto-phosphorylation of human kinases during expression in bacteria. Protein Expr Purif, 81 (1), pp. 136-143. | Show Abstract | Read more

Bacterial over-expression of kinases is often associated with high levels of auto-phosphorylation resulting in heterogeneous recombinant protein preparations or sometimes in insoluble protein. Here we present expression systems for nine kinases in Escherichia coli and, for the most heavily phosphorylated, the characterisation of factors affecting auto-phosphorylation. Experiments showed that the level of auto-phosphorylation was proportional to the rate of expression. Comparison of phosphorylation states following in vitro phosphorylation with phosphorylation states following expression in E. coli showed that the non-physiological 'hyper-phosphorylation' was occurring at sites that would require local unfolding to be accessible to a kinase active site. In contrast, auto-phosphorylation on unphosphorylated kinases that had been expressed in bacteria overexpressing λ-phosphatase was only observed on distinct exposed sites. Remarkably, the Ser/Thr kinase PLK4 auto-phosphorylated on a tyrosine residue (Tyr177) located in the activation segment. The results give support to a mechanism in which auto-phosphorylation occurs before or during protein folding. In addition, the expression systems and protocols presented will be a valuable resource to the research community.

Muller S, Filippakopoulos P, Knapp S. 2011. Bromodomains as therapeutic targets. Expert Rev Mol Med, 13 pp. e29. | Show Abstract | Read more

Acetylation of lysine residues is a post-translational modification with broad relevance to cellular signalling and disease biology. Enzymes that 'write' (histone acetyltransferases, HATs) and 'erase' (histone deacetylases, HDACs) acetylation sites are an area of extensive research in current drug development, but very few potent inhibitors that modulate the 'reading process' mediated by acetyl lysines have been described. The principal readers of ɛ-N-acetyl lysine (K(ac)) marks are bromodomains (BRDs), which are a diverse family of evolutionary conserved protein-interaction modules. The conserved BRD fold contains a deep, largely hydrophobic acetyl lysine binding site, which represents an attractive pocket for the development of small, pharmaceutically active molecules. Proteins that contain BRDs have been implicated in the development of a large variety of diseases. Recently, two highly potent and selective inhibitors that target BRDs of the BET (bromodomains and extra-terminal) family provided compelling data supporting targeting of these BRDs in inflammation and in an aggressive type of squamous cell carcinoma. It is likely that BRDs will emerge alongside HATs and HDACs as interesting targets for drug development for the large number of diseases that are caused by aberrant acetylation of lysine residues.

Hewings DS, Wang M, Philpott M, Fedorov O, Uttarkar S, Filippakopoulos P, Picaud S, Vuppusetty C, Marsden B, Knapp S et al. 2011. 3,5-dimethylisoxazoles act as acetyl-lysine-mimetic bromodomain ligands. J Med Chem, 54 (19), pp. 6761-6770. | Show Abstract | Read more

Histone-lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone-bromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound 4d, which has IC(50) values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains.

Philpott M, Yang J, Tumber T, Fedorov O, Uttarkar S, Filippakopoulos P, Picaud S, Keates T, Felletar I, Ciulli A et al. 2011. Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery. Mol Biosyst, 7 (10), pp. 2899-2908. | Show Abstract | Read more

Histone lysine acetylation is a key component of epigenetic regulation of gene transcription. Bromodomains, found in histone acetyl transferases and other chromatin-associated proteins, bind selectively to acetylated lysines, acting as "readers" of the histone code, and have recently been shown to contain a druggable binding pocket. Here we report the development of high-throughput assays that quantify the binding of bromodomains to acetylated histone peptides. We have used these assays to screen for histone binding partners of as yet uncharacterized bromodomains, adding to current knowledge of the histone code and expanding the repertoire of assays for chemical probe discovery. We have also demonstrated that these assays can be used to detect small molecule binding from the very weak to the nanomolar range. This assay methodology is thereby anticipated to provide the basis both for broader interactome profiling and for small molecule inhibitor discovery.

Miduturu CV, Deng X, Kwiatkowski N, Yang W, Brault L, Filippakopoulos P, Chung E, Yang Q, Schwaller J, Knapp S et al. 2011. High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors. Chem Biol, 18 (7), pp. 868-879. | Show Abstract | Read more

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.

Debdab M, Carreaux F, Renault S, Soundararajan M, Fedorov O, Filippakopoulos P, Lozach O, Babault L, Tahtouh T, Baratte B et al. 2011. Leucettines, a class of potent inhibitors of cdc2-like kinases and dual specificity, tyrosine phosphorylation regulated kinases derived from the marine sponge leucettamine B: modulation of alternative pre-RNA splicing. J Med Chem, 54 (12), pp. 4172-4186. | Show Abstract | Read more

We here report on the synthesis, optimization, and biological characterization of leucettines, a family of kinase inhibitors derived from the marine sponge leucettamine B. Stepwise synthesis of analogues starting from the natural structure, guided by activity testing on eight purified kinases, led to highly potent inhibitors of CLKs and DYRKs, two families of kinases involved in alternative pre-mRNA splicing and Alzheimer's disease/Down syndrome. Leucettine L41 was cocrystallized with CLK3. It interacts with key residues located within the ATP-binding pocket of the kinase. Leucettine L41 inhibits the phosphorylation of serine/arginine-rich proteins (SRp), a family of proteins regulating pre-RNA splicing. Indeed leucettine L41 was demonstrated to modulate alternative pre-mRNA splicing, in a cell-based reporting system. Leucettines should be further explored as pharmacological tools to study and modulate pre-RNA splicing. Leucettines may also be investigated as potential therapeutic drugs in Alzheimer's disease (AD) and in diseases involving abnormal pre-mRNA splicing.

Lopez-Ramos M, Prudent R, Moucadel V, Sautel CF, Barette C, Lafanechere L, Mouawad L, Grierson D, Schmidt F, Florent J-C et al. 2011. New potent dual inhibitors of CK2 and Pim kinases: discovery and structural insights (September, pg 3175, 2010) FASEB JOURNAL, 25 (5), pp. 1775-1775. | Read more

Salah E, Ugochukwu E, Barr AJ, von Delft F, Knapp S, Elkins JM. 2011. Crystal structures of ABL-related gene (ABL2) in complex with imatinib, tozasertib (VX-680), and a type I inhibitor of the triazole carbothioamide class. J Med Chem, 54 (7), pp. 2359-2367. | Show Abstract | Read more

ABL2 (also known as ARG (ABL related gene)) is closely related to the well-studied Abelson kinase cABL. ABL2 is involved in human neoplastic diseases and is deregulated in solid tumors. Oncogenic gene translocations occur in acute leukemia. So far no structural information for ABL2 has been reported. To elucidate structural determinants for inhibitor interaction, we determined the cocrystal structure of ABL2 with the oncology drug imatinib. Interestingly, imatinib not only interacted with the ATP binding site of the inactive kinase but was also bound to the regulatory myristate binding site. This structure may therefore serve as a tool for the development of allosteric ABL inhibitors. In addition, we determined the structures of ABL2 in complex with VX-680 and with an ATP-mimetic type I inhibitor, which revealed an interesting position of the DFG motif intermediate between active and inactive conformations, that may also serve as a template for future inhibitor design.

Fedorov O, Huber K, Eisenreich A, Filippakopoulos P, King O, Bullock AN, Szklarczyk D, Jensen LJ, Fabbro D, Trappe J et al. 2011. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing. Chem Biol, 18 (1), pp. 67-76. | Show Abstract | Read more

There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).

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Philpott M, Yang J, Tumber T, Fedorov O, Uttarkar S, Filippakopoulos P, Picaud S, Keates T, Felletar I, Ciulli A et al. 2011. Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery MOLECULAR BIOSYSTEMS, 7 (10), pp. 2899-2908. | Show Abstract | Read more

Histone lysine acetylation is a key component of epigenetic regulation of gene transcription. Bromodomains, found in histone acetyl transferases and other chromatin-associated proteins, bind selectively to acetylated lysines, acting as "readers" of the histone code, and have recently been shown to contain a druggable binding pocket. Here we report the development of high-throughput assays that quantify the binding of bromodomains to acetylated histone peptides. We have used these assays to screen for histone binding partners of as yet uncharacterized bromodomains, adding to current knowledge of the histone code and expanding the repertoire of assays for chemical probe discovery. We have also demonstrated that these assays can be used to detect small molecule binding from the very weak to the nanomolar range. This assay methodology is thereby anticipated to provide the basis both for broader interactome profiling and for small molecule inhibitor discovery. © 2011 The Royal Society of Chemistry.

Deutsch GB, Zielonka EM, Coutandin D, Weber TA, Schäfer B, Hannewald J, Luh LM, Durst FG, Ibrahim M, Hoffmann J et al. 2011. DNA damage in oocytes induces a switch of the quality control factor TAp63α from dimer to tetramer. Cell, 144 (4), pp. 566-576. | Show Abstract | Read more

TAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ∼20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.

Feng L, Geisselbrecht Y, Blanck S, Wilbuer A, Atilla-Gokcumen GE, Filippakopoulos P, Kräling K, Celik MA, Harms K, Maksimoska J et al. 2011. Structurally sophisticated octahedral metal complexes as highly selective protein kinase inhibitors. J Am Chem Soc, 133 (15), pp. 5976-5986. | Show Abstract | Read more

The generation of synthetic compounds with exclusive target specificity is an extraordinary challenge of molecular recognition and demands novel design strategies, in particular for large and homologous protein families such as protein kinases with more than 500 members. Simple organic molecules often do not reach the necessary sophistication to fulfill this task. Here, we present six carefully tailored, stable metal-containing compounds in which unique and defined molecular geometries with natural-product-like structural complexity are constructed around octahedral ruthenium(II) or iridium(III) metal centers. Each of the six reported metal compounds displays high selectivity for an individual protein kinase, namely GSK3α, PAK1, PIM1, DAPK1, MLCK, and FLT4. Although being conventional ATP-competitive inhibitors, the combination of the unusual globular shape and rigidity characteristics, of these compounds facilitates the design of highly selective protein kinase inhibitors. Unique structural features of the octahedral coordination geometry allow novel interactions with the glycine-rich loop, which contribute significantly to binding potencies and selectivities. The sensitive correlation between metal coordination sphere and inhibition properties suggests that in this design, the metal is located at a "hot spot" within the ATP binding pocket, not too close to the hinge region where globular space is unavailable, and at the same time not too far out toward the solvent where the octahedral coordination sphere would not have a significant impact on potency and selectivity. This study thus demonstrates that inert (stable) octahedral metal complexes are sophisticated structural scaffolds for the design of highly selective chemical probes.

Zadjali F, Pike AC, Vesterlund M, Sun J, Wu C, Li SS, Rönnstrand L, Knapp S, Bullock AN, Flores-Morales A. 2011. Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase. J Biol Chem, 286 (1), pp. 480-490. | Show Abstract | Read more

The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564-574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (K(d) = 0.3 μm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.

Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I et al. 2010. Selective inhibition of BET bromodomains. Nature, 468 (7327), pp. 1067-1073. | Show Abstract | Read more

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Müller S, Knapp S. 2010. Targeting kinases for the treatment of inflammatory diseases. Expert Opin Drug Discov, 5 (9), pp. 867-881. | Show Abstract | Read more

IMPORTANCE OF THE FIELD: Inflammatory diseases are one of the major health issues and have become a major focus in the pharmaceutical and biotech industries. To date, drugs prescribed for treatment of these diseases target enzymes that are not specific to the immune system resulting in adverse effects. The main challenge of this research field is, therefore, identifying targets that act specifically on the diseased tissue. AREAS COVERED IN THIS REVIEW: This review summarizes drug discovery efforts on kinases that have been identified as key players mediating inflammation and autoimmune disorders. In particular, we discuss recent developments on well-established targets such as mammalian target of rapamycin, JAK3, spleen tyrosine kinase, p38α and lymphocyte specific kinase but provide also a perspective on emerging targets. WHAT THE READER WILL GAIN: The reader will obtain an overview of drug discovery efforts on kinases in inflammation, recent clinical and preclinical data and developed inhibitor scaffolds. In addition, the reader will be updated on issues in target validation of current drug targets and the potential of selected novel kinase targets in this important disease area. TAKE HOME MESSAGE: Cellular signaling networks that regulate inflammatory response are still poorly understood making rational selection of targets challenging. Recent data suggest that kinase targets that are specific to the immune system and mediate signals immediately downstream of surface receptors are most efficacious in the clinic.

Wang M, Mok MW, Harper H, Lee WH, Min J, Knapp S, Oppermann U, Marsden B, Schapira M. 2010. Structural genomics of histone tail recognition. Bioinformatics, 26 (20), pp. 2629-2630. | Show Abstract | Read more

SUMMARY: The structural genomics of histone tail recognition web server is an open access resource that presents within mini articles all publicly available experimental structures of histone tails in complex with human proteins. Each article is composed of interactive 3D slides that dissect the structural mechanism underlying the recognition of specific sequences and histone marks. A concise text html-linked to interactive graphics guides the reader through the main features of the interaction. This resource can be used to analyze and compare binding modes across multiple histone recognition modules, to evaluate the chemical tractability of binding sites involved in epigenetic signaling and design small molecule inhibitors. AVAILABILITY: http://www.thesgc.org/resources/histone_tails/ CONTACT: matthieu.schapira@utoronto.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Qi J, Filippakopoulos P, Picaud S, Smith W, Keates T, Morse E, Philpott M, Shaw K, Fedorov O, West N et al. 2010. Small-molecule bromodomain inhibitors for cancer therapy ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 240

Radwańska K, Tudor-Jones AA, Mizuno K, Pereira GS, Lucchesi W, Alfano I, Łach A, Kaczmarek L, Knapp S, Giese KP. 2010. Differential regulation of CaMKII inhibitor beta protein expression after exposure to a novel context and during contextual fear memory formation. Genes Brain Behav, 9 (6), pp. 648-657. | Show Abstract | Read more

Understanding of the molecular basis of long-term fear memory (fear LTM) formation provides targets in the treatment of emotional disorders. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is one of the key synaptic molecules involved in fear LTM formation. There are two endogenous inhibitor proteins of CaMKII, CaMKII N alpha and N beta, which can regulate CaMKII activity in vitro. However, the physiological role of these endogenous inhibitors is not known. Here, we have investigated whether CaMKII N beta protein expression is regulated after contextual fear conditioning or exposure to a novel context. Using a novel CaMKII N beta-specific antibody, CaMKII N beta expression was analysed in the naïve mouse brain as well as in the amygdala and hippocampus after conditioning and context exposure. We show that in naïve mouse forebrain CaMKII N beta protein is expressed at its highest levels in olfactory bulb, prefrontal and piriform cortices, amygdala and thalamus. The protein is expressed both in dendrites and cell bodies. CaMKII N beta expression is rapidly and transiently up-regulated in the hippocampus after context exposure. In the amygdala, its expression is regulated only by contextual fear conditioning and not by exposure to a novel context. In conclusion, we show that CaMKII N beta expression is differentially regulated by novelty and contextual fear conditioning, providing further insight into molecular basis of fear LTM.

Brault L, Gasser C, Bracher F, Huber K, Knapp S, Schwaller J. 2010. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica, 95 (6), pp. 1004-1015. | Show Abstract | Read more

The identification as cooperating targets of Proviral Integrations of Moloney virus in murine lymphomas suggested early on that PIM serine/threonine kinases play an important role in cancer biology. Whereas elevated levels of PIM1 and PIM2 were mostly found in hematologic malignancies and prostate cancer, increased PIM3 expression was observed in different solid tumors. PIM kinases are constitutively active and their activity supports in vitro and in vivo tumor cell growth and survival through modification of an increasing number of common as well as isoform-specific substrates including several cell cycle regulators and apoptosis mediators. PIM1 but not PIM2 seems also to mediate homing and migration of normal and malignant hematopoietic cells by regulating chemokine receptor surface expression. Knockdown experiments by RNA interference or dominant-negative acting mutants suggested that PIM kinases are important for maintenance of a transformed phenotype and therefore potential therapeutic targets. Determination of the protein structure facilitated identification of an increasing number of potent small molecule PIM kinase inhibitors with in vitro and in vivo anticancer activity. Ongoing efforts aim to identify isoform-specific PIM inhibitors that would not only help to dissect the kinase function but hopefully also provide targeted therapeutics. Here, we summarize the current knowledge about the role of PIM serine/threonine kinases for the pathogenesis and therapy of hematologic malignancies and solid cancers, and we highlight structural principles and recent progress on small molecule PIM kinase inhibitors that are on their way into first clinical trials.

Kwiatkowski N, Jelluma N, Filippakopoulos P, Soundararajan M, Manak MS, Kwon M, Choi HG, Sim T, Deveraux QL, Rottmann S et al. 2010. Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function. Nat Chem Biol, 6 (5), pp. 359-368. | Show Abstract | Read more

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

López-Ramos M, Prudent R, Moucadel V, Sautel CF, Barette C, Lafanechère L, Mouawad L, Grierson D, Schmidt F, Florent JC et al. 2010. New potent dual inhibitors of CK2 and Pim kinases: discovery and structural insights. FASEB J, 24 (9), pp. 3171-3185. | Show Abstract | Read more

Protein kinase casein kinase 2 (CK2) is a serine/threonine kinase with evidence of implication in growth dysregulation and apoptosis resistance, making it a relevant target for cancer therapy. Several CK2 inhibitors have been developed showing variable efficiency, emphasizing the need to expand the chemical diversity of those inhibitors. We report the identification and characterization of 2,8-difurandicarboxylic acid derivatives as a new class of nanomolar ATP-competitive inhibitors. Selectivity profiling pointed out proviral insertion Moloney virus kinases (Pim kinases) as the only other kinases that are significantly inhibited. By combining structure-activity relationship analysis with structural determination, we were able to determine the binding mode of these inhibitors for both kinases and to explain their strong inhibitory potency. Essential chemical features necessary for activity on both kinases were then identified. The described compounds are not cell permeable: however, they could provide a lead for developing novel inhibitors usable also in vivo. Given the similar but not redundant pathophysiological functions of CK2 and Pim family members, such inhibitors would provide new attractive leads for targeted cancer therapy. This work highlights that 2 functionally related kinases from different kinome branches display exquisite sensitivity to a common inhibitor.

Fedorov O, Müller S, Knapp S. 2010. The (un)targeted cancer kinome. Nat Chem Biol, 6 (3), pp. 166-169. | Read more

Barr AJ, Knapp S. 2010. Large-scale structural analysis of protein tyrosine phosphatases 2 pp. 871-876. | Show Abstract | Read more

Protein tyrosine phosphatases (PTPs) are essential regulators of signal transduction pathways, and control, together with protein tyrosine kinases (PTKs), the reversible phosphorylation of tyrosine residues. PTPs are, from a structural biology viewpoint, one of the most comprehensively covered protein families. This chapter focuses on the structural comparison of catalytic domains of the major PTP families, and summarizes implications of the available structural data for the understanding of PTP regulation and for the design of specific PTP inhibitors. Modular domains outside the catalytic domain also significantly regulate PTP function. Inactivation of PTPs by receptor dimerization is discussed as a key regulatory mechanism of PTP activity. This control mechanism was first suggested based on structural studies of the membrane proximal D1 domain of RPTP α. More structural data regarding phosphatases in complex with their substrates and regulators will be available in the near future, which will allow a more detailed insight into the regulation and substrate recognition of this diverse class of enzymes. © 2010 Elsevier Inc. All rights reserved.

Record CJ, Chaikuad A, Rellos P, Das S, Pike AC, Fedorov O, Marsden BD, Knapp S, Lee WH. 2010. Structural comparison of human mammalian ste20-like kinases. PLoS One, 5 (8), pp. e11905. | Show Abstract | Read more

BACKGROUND: The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains. PRINCIPAL FINDINGS: We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family. SIGNIFICANCE: The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Rellos P, Pike AC, Niesen FH, Salah E, Lee WH, von Delft F, Knapp S. 2010. Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation. PLoS Biol, 8 (7), pp. e1000426. | Show Abstract | Read more

UNLABELLED: Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.

Coutandin D, Löhr F, Niesen FH, Ikeya T, Weber TA, Schäfer B, Zielonka EM, Bullock AN, Yang A, Güntert P et al. 2009. Conformational stability and activity of p73 require a second helix in the tetramerization domain. Cell Death Differ, 16 (12), pp. 1582-1589. | Show Abstract | Read more

p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional alpha-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family.

Filippakopoulos P, Müller S, Knapp S. 2009. SH2 domains: modulators of nonreceptor tyrosine kinase activity. Curr Opin Struct Biol, 19 (6), pp. 643-649. | Show Abstract | Read more

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

Eswaran J, Patnaik D, Filippakopoulos P, Wang F, Stein RL, Murray JW, Higgins JM, Knapp S. 2009. Structure and functional characterization of the atypical human kinase haspin. Proc Natl Acad Sci U S A, 106 (48), pp. 20198-20203. | Show Abstract | Read more

The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP gamma-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.

Eswaran J, Knapp S. 2010. Insights into protein kinase regulation and inhibition by large scale structural comparison. Biochim Biophys Acta, 1804 (3), pp. 429-432. | Show Abstract | Read more

Protein structure determination of soluble globular protein domains has developed into an efficient routine technology which can now be applied to generate and analyze structures of entire human protein families. In the kinase area, several kinase families still lack comprehensive structural analysis. Nevertheless, Structural Genomics (SG) efforts contributed more than 40 kinase catalytic domain structures during the past 4 years providing a rich resource of information for large scale comparisons of kinase active sites. Moreover, many of the released structures are inhibitor complexes that offer chemical starting points for development of selective and potent inhibitors. Here we discuss the currently available structural data and strategies that can be utilized for the development of highly selective inhibitors.

Akué-Gédu R, Rossignol E, Azzaro S, Knapp S, Filippakopoulos P, Bullock AN, Bain J, Cohen P, Prudhomme M, Anizon F, Moreau P. 2009. Synthesis, kinase inhibitory potencies, and in vitro antiproliferative evaluation of new Pim kinase inhibitors. J Med Chem, 52 (20), pp. 6369-6381. | Show Abstract | Read more

Members of the Pim kinase family have been identified as promising targets for the development of antitumor agents. After a screening of pyrrolo[2,3-a]- and [3,2-a]carbazole derivatives toward 66 protein kinases, we identified pyrrolo[2,3-a]carbazole as a new scaffold to design potent Pim kinase inhibitors. In particular, compound 9 was identified as a low nM selective Pim inhibitor. Additionally, several pyrrolo[2,3-a]carbazole derivatives showed selectivity for Pim-1 and Pim-3 over Pim-2. In vitro antiproliferative activities of 9 and 28, the most potent Pim inhibitors identified, were evaluated toward three human solid cancer cell lines (PA1, PC3, and DU145) and one human fibroblast primary culture, revealing IC50 values in the micromolar range. Finally, the crystal structure of Pim-1 complexed with lead compound 9 was determined. The structure revealed a non-ATP mimetic binding mode with no hydrogen bonds formed with the kinase hinge region and explained the selectivity of pyrrolo[2,3-a]carbazole derivatives for Pim kinases.

Ugele M, Sasse F, Knapp S, Fedorov O, Zubriene A, Matulis D, Maier ME. 2009. Propionate analogues of zearalenone bind to Hsp90. Chembiochem, 10 (13), pp. 2203-2212. | Show Abstract | Read more

By replacement of an acetate with propionate through organic synthesis a range of zearalenone analogues were prepared. As key steps in the synthesis of the analogues we used the Noyori hydrogenation of methyl acetoacetate followed by Frater alkylation of the enantiomeric 3-hydroxybutyrates. This converted the second acetate to a propionate. Through the derived alkyne, chain extension led to 3-methylundec-10-en-2-ol derivatives. These were condensed with 2,4-dimethoxy-6-vinylbenzoic acid. Ring-closing metathesis of the obtained esters led to macrolactones, which were deproteced to give the zearalenone analogues. Several of the analogues showed cytotoxicity against the L929 mouse fibroblast cell line comparable to zearalenone (9 microM) itself. In the thermal-shift assay, two analogues 35 and ent-35 displayed stronger binding than the natural product geldanamycin to the chaperone Hsp90.

Grundler R, Brault L, Gasser C, Bullock AN, Dechow T, Woetzel S, Pogacic V, Villa A, Ehret S, Berridge G et al. 2009. Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration. J Exp Med, 206 (9), pp. 1957-1970. | Show Abstract | Read more

FLT3-ITD-mediated leukemogenesis is associated with increased expression of oncogenic PIM serine/threonine kinases. To dissect their role in FLT3-ITD-mediated transformation, we performed bone marrow reconstitution assays. Unexpectedly, FLT3-ITD cells deficient for PIM1 failed to reconstitute lethally irradiated recipients, whereas lack of PIM2 induction did not interfere with FLT3-ITD-induced disease. PIM1-deficient bone marrow showed defects in homing and migration and displayed decreased surface CXCR4 expression and impaired CXCL12-CXCR4 signaling. Through small interfering RNA-mediated knockdown, chemical inhibition, expression of a dominant-negative mutant, and/or reexpression in knockout cells, we found PIM1 activity to be essential for proper CXCR4 surface expression and migration of cells toward a CXCL12 gradient. Purified PIM1 led to the phosphorylation of serine 339 in the CXCR4 intracellular domain in vitro, a site known to be essential for normal receptor recycling. In primary leukemic blasts, high levels of surface CXCR4 were associated with increased PIM1 expression, and this could be significantly reduced by a small molecule PIM inhibitor in some patients. Our data suggest that PIM1 activity is important for homing and migration of hematopoietic cells through modification of CXCR4. Because CXCR4 also regulates homing and maintenance of cancer stem cells, PIM1 inhibitors may exert their antitumor effects in part by interfering with interactions with the microenvironment.

Müller S, Knapp S. 2009. Out of the box binding determines specificity of SH2 domain interaction. Structure, 17 (8), pp. 1040-1041. | Show Abstract | Read more

SH2 domains are phosphotyrosine specific interaction modules with largely overlapping sequence specificities. A recent structure by Bae et al. revealed that SH2 domain specificity can be mediated by secondary binding sites located outside the phosphotyrosine binding pocket.

Kimple AJ, Soundararajan M, Hutsell SQ, Roos AK, Urban DJ, Setola V, Temple BR, Roth BL, Knapp S, Willard FS, Siderovski DP. 2009. Structural determinants of G-protein alpha subunit selectivity by regulator of G-protein signaling 2 (RGS2). J Biol Chem, 284 (29), pp. 19402-19411. | Show Abstract | Read more

"Regulator of G-protein signaling" (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Galpha subunits (known as GTPase-accelerating protein or "GAP" activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Galpha(q) subunits. As many vasoconstrictive hormones signal via G(q) heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Galpha(q) over Galpha(i/o) substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Galpha(i)-directed binding and GAP activities without perturbing its association with Galpha(q). Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Galpha(i) at 2.8-A resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Galpha complexes highlighted the roles of these residues in wild type RGS2 that weaken Galpha(i) subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Galpha(q) GAP that modulates cardiovascular function.

Eswaran J, Soundararajan M, Knapp S. 2009. Targeting group II PAKs in cancer and metastasis. Cancer Metastasis Rev, 28 (1-2), pp. 209-217. | Show Abstract | Read more

The p21 activated kinases (PAKs) play an essential role in cell signaling and control a variety of cellular functions including cell motility, survival, angiogenesis and mitosis. PAKs are important regulators in growth factor signaling, cytoskeletal reorganization and growth factor-mediated cell migration. Overexpression of PAKs has been detected in many cancers and linked to increased migration potential, anchorage independent growth and metastasis. Six isoforms of PAKs are expressed in human and based on their regulatory properties they have been classified into group I (PAK1-3) and group II (PAK4-6). Besides the well studied group I family, members of the group II PAKs also emerged as interesting targets for the development of new inhibitors for cancer therapy. The availability of high resolution crystal structures for all group II PAKs and their fundamentally different regulatory properties when compared with group I enzymes has opened new opportunities for rational drug designing strategies. In this review, we summarize the results of recent advances of the function of group II PAKs in tumorigenesis and metastasis as well as opportunities for exploring the unique catalytic domain dynamics of this protein family for the design of group II PAK specific inhibitors.

Huck K, Feyen O, Niehues T, Rüschendorf F, Hübner N, Laws HJ, Telieps T, Knapp S, Wacker HH, Meindl A et al. 2009. Girls homozygous for an IL-2-inducible T cell kinase mutation that leads to protein deficiency develop fatal EBV-associated lymphoproliferation. J Clin Invest, 119 (5), pp. 1350-1358. | Show Abstract | Read more

The fatal immune dysregulation that sometimes follows EBV infection in boys has been linked to mutations in two X chromosome-encoded genes, SLAM-associated protein (SAP) and X-linked inhibitor of apoptosis (XIAP). In this study we describe 2 girls from a consanguineous Turkish family who died after developing severe immune dysregulation and therapy-resistant EBV-positive B cell proliferation following EBV infection. SNP array-based genome-wide linkage analysis revealed IL-2-inducible T cell kinase (ITK) as a candidate gene for this immunodeficiency syndrome. Both girls harbored a homozygous missense mutation that led to substitution of a highly conserved residue (R335W) in the SH2 domain of ITK. Characteristics of ITK deficiency in mouse models, such as absence of NKT cells and high levels of eomesodermin in CD8+ cells, were seen in either one or both of the girls. Two lines of evidence suggested that R335W caused instability of the ITK protein. First, in silico modeling of the mutant protein predicted destabilization of the SH2 domain. Additionally, Western blot analysis revealed that, unlike wild-type ITK, the R335W mutant was nearly undetectable when expressed in 293 T cells. Our results suggest that ITK deficiency causes what we believe to be a novel immunodeficiency syndrome that leads to a fatal inadequate immune response to EBV. Because ITK deficiency resembles EBV-associated lymphoproliferative disorders in boys, we suggest that this molecular cause should be considered during diagnosis and treatment.

Telieps T, Huck K, Feyen O, Niehues T, Rueschendorf F, Huebner N, Laws HJ, Knapp S, Wacker HH, Meindl A et al. 2009. Missense mutation in the inducible T-cell kinase (ITK) leading to immunodeficiency and fatal immune dysregulation after EBV infection KLINISCHE PADIATRIE, 221 (3), pp. 209-209.

Elkins JM, Amos A, Niesen FH, Pike AC, Fedorov O, Knapp S. 2009. Structure of dystrophia myotonica protein kinase. Protein Sci, 18 (4), pp. 782-791. | Show Abstract | Read more

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled-coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM-8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled-coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully-ordered and correctly positioned alphaC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C-terminal extension of the kinase domain is bound to the N-terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C-terminal extension compared to the closely related Rho-associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM-8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.

Bullock AN, Das S, Debreczeni JE, Rellos P, Fedorov O, Niesen FH, Guo K, Papagrigoriou E, Amos AL, Cho S et al. 2009. Kinase domain insertions define distinct roles of CLK kinases in SR protein phosphorylation. Structure, 17 (3), pp. 352-362. | Show Abstract | Read more

Splicing requires reversible phosphorylation of serine/arginine-rich (SR) proteins, which direct splice site selection in eukaryotic mRNA. These phosphorylation events are dependent on SR protein (SRPK) and cdc2-like kinase (CLK) families. SRPK1 phosphorylation of splicing factors is restricted by a specific docking interaction whereas CLK activity is less constrained. To understand functional differences between splicing factor targeting kinases, we determined crystal structures of CLK1 and CLK3. Intriguingly, in CLKs the SRPK1 docking site is blocked by insertion of a previously unseen helix alphaH. In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific beta7/8-hairpin insert. Thus, the unconstrained substrate interaction together with the determined active-site mediated substrate specificity allows CLKs to complete the functionally important hyperphosphorylation of splicing factors like ASF/SF2. In addition, despite high sequence conservation, we identified inhibitors with surprising isoform specificity for CLK1 over CLK3.

Huck K, Feyen O, Rueschendorf F, Knapp S, Borkhardt A. 2009. Identification of a novel primary immunodeficiency syndrome leading to an XLP-like phenotype in girls EUROPEAN JOURNAL OF PEDIATRICS, 168 (3), pp. 380-380.

Barr AJ, Ugochukwu E, Lee WH, King ON, Filippakopoulos P, Alfano I, Savitsky P, Burgess-Brown NA, Müller S, Knapp S. 2009. Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell, 136 (2), pp. 352-363. | Show Abstract | Read more

Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a "head-to-toe" dimerization model for RPTPgamma/zeta that is distinct from the "inhibitory wedge" model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.

Zimmermann TJ, Niesen FH, Pilka ES, Knapp S, Oppermann U, Maier ME. 2009. Discovery of a potent and selective inhibitor for human carbonyl reductase 1 from propionate scanning applied to the macrolide zearalenone. Bioorg Med Chem, 17 (2), pp. 530-536. | Show Abstract | Read more

In order to extend the chemical diversity available for organic polyketide synthesis, the concept of propionate scanning was developed. We observed that naturally occurring polyketides frequently comprise not only acetate, but also some propionate as building blocks. Therefore our approach consists of a systematic replacement of some of the acetate building blocks during synthesis by propionate moieties, resulting in additional methyl groups that may give rise to different properties of the polyketides. Here we present the results of a first 'proof of concept' study where a novel zearalenone analogue 5 was prepared that comprises an additional methyl group at C5'. Key steps in the synthesis of 5 include a Marshall-Tamaru reaction, a Suzuki cross-coupling reaction, and a Mitsunobu lactonization. Compared to the parent zearalenone (1), analogue 5 showed reduced binding to a panel of human protein kinases and no binding to human Hsp90. On the other hand, however, 5 turned out to be a potent (IC(50)=210 nM) inhibitor of human carbonyl reductase 1 (CBR1).

Scheeff ED, Eswaran J, Bunkoczi G, Knapp S, Manning G. 2009. Structure of the pseudokinase VRK3 reveals a degraded catalytic site, a highly conserved kinase fold, and a putative regulatory binding site. Structure, 17 (1), pp. 128-138. | Show Abstract | Read more

About 10% of all protein kinases are predicted to be enzymatically inactive pseudokinases, but the structural details of kinase inactivation have remained unclear. We present the first structure of a pseudokinase, VRK3, and that of its closest active relative, VRK2. Profound changes to the active site region underlie the loss of catalytic activity, and VRK3 cannot bind ATP because of residue substitutions in the binding pocket. However, VRK3 still shares striking structural similarity with VRK2, and appears to be locked in a pseudoactive conformation. VRK3 also conserves residue interactions that are surprising in the absence of enzymatic function; these appear to play important architectural roles required for the residual functions of VRK3. Remarkably, VRK3 has an "inverted" pattern of sequence conservation: although the active site is poorly conserved, portions of the molecular surface show very high conservation, suggesting that they form key interactions that explain the evolutionary retention of VRK3.

Bullock AN, Russo S, Amos A, Pagano N, Bregman H, Debreczeni JE, Lee WH, von Delft F, Meggers E, Knapp S. 2009. Crystal structure of the PIM2 kinase in complex with an organoruthenium inhibitor. PLoS One, 4 (10), pp. e7112. | Show Abstract | Read more

BACKGROUND: The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2. PRINCIPAL FINDINGS: Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases. SIGNIFICANCE: The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Gingras MC, Zhang YL, Kharitidi D, Barr AJ, Knapp S, Tremblay ML, Pause A. 2009. HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain. PLoS One, 4 (4), pp. e5105. | Show Abstract | Read more

BACKGROUND: The HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP). To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported. METHODOLOGY AND RESULTS: Here we report a rigorous enzymatic analysis demonstrating that the HD-PTP protein does not harbor tyrosine phosphatase or lipid phosphatase activity using the highly sensitive DiFMUP substrate and a panel of different phosphatidylinositol phosphates. We found that HD-PTP tyrosine phosphatase inactivity is caused by an evolutionary conserved amino acid divergence of a key residue located in the HD-PTP phosphatase domain since its back mutation is sufficient to restore the HD-PTP tyrosine phosphatase activity. Moreover, in agreement with a tumor suppressor activity, HD-PTP expression leads to colony growth reduction in human cancer cell lines, independently of its catalytic PTP activity status. CONCLUSION: In summary, we demonstrate that HD-PTP is a catalytically inactive protein tyrosine phosphatase. As such, we identify one residue involved in its inactivation and show that its colony growth reduction activity is independent of its PTP activity status in human cancer cell lines.

Atilla-Gokcumen GE, Pagano N, Streu C, Maksimoska J, Filippakopoulos P, Knapp S, Meggers E. 2008. Extremely tight binding of a ruthenium complex to glycogen synthase kinase 3. Chembiochem, 9 (18), pp. 2933-2936. | Read more

Filippakopoulos P, Kofler M, Hantschel O, Gish GD, Grebien F, Salah E, Neudecker P, Kay LE, Turk BE, Superti-Furga G et al. 2008. Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation. Cell, 134 (5), pp. 793-803. | Show Abstract | Read more

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Miller ML, Jensen LJ, Diella F, Jørgensen C, Tinti M, Li L, Hsiung M, Parker SA, Bordeaux J, Sicheritz-Ponten T et al. 2008. Linear motif atlas for phosphorylation-dependent signaling. Sci Signal, 1 (35), pp. ra2. | Show Abstract | Read more

Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains [Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14-3-3]. The atlas reveals new aspects of signaling systems, including the observation that tyrosine kinases mutated in cancer have lower specificity than their non-oncogenic relatives. The resource is maintained by an automated pipeline, which uses phylogenetic trees to structure the currently available in vivo and in vitro data to derive probabilistic sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info).

Eswaran J, Soundararajan M, Kumar R, Knapp S. 2008. UnPAKing the class differences among p21-activated kinases. Trends Biochem Sci, 33 (8), pp. 394-403. | Show Abstract | Read more

The p21-activated kinases (PAKs) are signal transducers, central to many vital cellular processes, including cell morphology, motility, survival, gene transcription and hormone signalling. The mammalian PAK family contains six serine/threonine kinases divided into two subgroups, group I (PAK 1-3) and group II (PAK4-6), based on their domain architecture and regulation. PAKs functioning as dynamic signalling nodes present themselves as attractive therapeutic targets in tumours, neurological diseases and infection. The recent findings across all PAKs, including newly reported structures, shed light on the cellular functions of PAKs, highlighting molecular mechanisms of activation, catalysis and substrate specificity. We believe that a comprehensive understanding of the entire PAK family is essential for developing strategies towards PAK-targeted therapeutics.

Baumli S, Lolli G, Lowe ED, Troiani S, Rusconi L, Bullock AN, Debreczeni JE, Knapp S, Johnson LN. 2008. The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation. EMBO J, 27 (13), pp. 1907-1918. | Show Abstract | Read more

The positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26 degrees of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis.

Pike AC, Rellos P, Niesen FH, Turnbull A, Oliver AW, Parker SA, Turk BE, Pearl LH, Knapp S. 2008. Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites. EMBO J, 27 (4), pp. 704-714. | Show Abstract | Read more

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies.

Marsden BD, Knapp S. 2008. Doing more than just the structure-structural genomics in kinase drug discovery. Curr Opin Chem Biol, 12 (1), pp. 40-45. | Show Abstract | Read more

Structural genomics (SG) has significantly increased the number of novel protein structures of targets with medical relevance. In the protein kinase area, SG has contributed >50% of all novel kinases structures during the past three years and determined more than 30 novel catalytic domain structures. Many of the released structures are inhibitor complexes and a number of them have identified new inhibitor binding modes and scaffolds. In addition, generated reagents, assays, and inhibitor screening data provide a diversity of chemogenomic data that can be utilized for early drug development. Here we discuss the currently available structural data for the kinase family considering novel structures as well as inhibitor complexes. Our analysis revealed that the structural coverage of many kinases families is still rather poor, and inhibitor complexes with diverse inhibitors are only available for a few kinases. However, we anticipate that with the current rate of structure determination and high throughput technologies developed by SG programs these gaps will be closed soon. In addition, the generated reagents will put SG initiatives in a unique position providing data beyond protein structure determination by identifying chemical probes, determining their binding modes and target specificity.

Structural Genomics Consortium, China Structural Genomics Consortium, Northeast Structural Genomics Consortium, Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S et al. 2008. Protein production and purification. Nat Methods, 5 (2), pp. 135-146. | Show Abstract | Read more

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Maksimoska J, Williams DS, Atilla-Gokcumen GE, Smalley KS, Carroll PJ, Webster RD, Filippakopoulos P, Knapp S, Herlyn M, Meggers E. 2008. Similar biological activities of two isostructural ruthenium and osmium complexes. Chemistry, 14 (16), pp. 4816-4822. | Show Abstract | Read more

In this study, we probe and verify the concept of designing unreactive bioactive metal complexes, in which the metal possesses a purely structural function, by investigating the consequences of replacing ruthenium in a bioactive half-sandwich kinase inhibitor scaffold by its heavier congener osmium. The two isostructural complexes are compared with respect to their anticancer properties in 1205 Lu melanoma cells, activation of the Wnt signaling pathway, IC(50) values against the protein kinases GSK-3beta and Pim-1, and binding modes to the protein kinase Pim-1 by protein crystallography. It was found that the two congeners display almost indistinguishable biological activities, which can be explained by their nearly identical three-dimensional structures and their identical mode of action as protein kinase inhibitors. This is a unique example in which the replacement of a metal in an anticancer scaffold by its heavier homologue does not alter its biological activity.

Eswaran J, Bernad A, Ligos JM, Guinea B, Debreczeni JE, Sobott F, Parker SA, Najmanovich R, Turk BE, Knapp S. 2008. Structure of the human protein kinase MPSK1 reveals an atypical activation loop architecture. Structure, 16 (1), pp. 115-124. | Show Abstract | Read more

The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

Wu X, Oppermann M, Berndt KD, Bergman T, Jörnvall H, Knapp S, Oppermann U. 2008. Thermal unfolding of the archaeal DNA and RNA binding protein Ssh10. Biochem Biophys Res Commun, 373 (4), pp. 482-487. | Show Abstract | Read more

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5-7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 degrees C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of DeltaH(cal) is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (DeltaG (T)) and results in a maximal DeltaG of only 9.5 kcal/mol at 320 K and a DeltaG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.

Fedorov O, Marsden B, Pogacic V, Rellos P, Müller S, Bullock AN, Schwaller J, Sundström M, Knapp S. 2007. A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases. Proc Natl Acad Sci U S A, 104 (51), pp. 20523-20528. | Show Abstract | Read more

Protein kinases play a pivotal role in cell signaling, and dysregulation of many kinases has been linked to disease development. A large number of kinase inhibitors are therefore currently under investigation in clinical trials, and so far seven inhibitors have been approved as anti-cancer drugs. In addition, kinase inhibitors are widely used as specific probes to study cell signaling, but systematic studies describing selectivity of these reagents across a panel of diverse kinases are largely lacking. Here we evaluated the specificity of 156 validated kinase inhibitors, including inhibitors used in clinical trials, against 60 human Ser/Thr kinases using a thermal stability shift assay. Our analysis revealed many unexpected cross-reactivities for inhibitors thought to be specific for certain targets. We also found that certain combinations of active-site residues in the ATP-binding site correlated with the detected ligand promiscuity and that some kinases are highly sensitive to inhibition using diverse chemotypes, suggesting them as preferred intervention points. Our results uncovered also inhibitor cross-reactivities that may lead to alternate clinical applications. For example, LY333'531, a PKCbeta inhibitor currently in phase III clinical trials, efficiently inhibited PIM1 kinase in our screen, a suggested target for treatment of leukemia. We determined the binding mode of this inhibitor by x-ray crystallography and in addition showed that LY333'531 induced cell death and significantly suppressed growth of leukemic cells from acute myeloid leukemia patients.

Bullock AN, Rodriguez MC, Debreczeni JE, Songyang Z, Knapp S. 2007. Structure of the SOCS4-ElonginB/C complex reveals a distinct SOCS box interface and the molecular basis for SOCS-dependent EGFR degradation. Structure, 15 (11), pp. 1493-1504. | Show Abstract | Read more

Tyrosine kinase signaling is tightly controlled by negative feedback inhibitors including suppressors of cytokine signaling (SOCS). SOCS assemble as SH2 domain substrate recognition modules in ElonginB/C-cullin ubiquitin ligases. In accordance, SOCS4 reduces STAT3 signaling from EGFR through increased receptor degradation. Variable C-termini in SOCS4-SOCS7 exclude these family members from a SOCS2-type domain arrangement in which a strictly conserved C terminus determines domain packing. The structure of the SOCS4-ElonginC-ElonginB complex reveals a distinct SOCS structural class. The N-terminal ESS helix functionally replaces the CIS/SOCS1-SOCS3 family C terminus in a distinct SH2-SOCS box interface that facilitates further interdomain packing between the extended N- and C-terminal regions characteristic for this subfamily. Using peptide arrays and calorimetry the STAT3 site in EGFR (pY(1092)) was identified as a high affinity SOCS4 substrate (K(D) = 0.5 microM) revealing a mechanism for EGFR degradation. SOCS4 also bound JAK2 and KIT with low micromolar affinity, whereas SOCS2 was specific for GH-receptor.

Bunkoczi G, Salah E, Filippakopoulos P, Fedorov O, Müller S, Sobott F, Parker SA, Zhang H, Min W, Turk BE, Knapp S. 2007. Structural and functional characterization of the human protein kinase ASK1. Structure, 15 (10), pp. 1215-1226. | Show Abstract | Read more

Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (K(d) approximately 0.2 microM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

Gileadi O, Knapp S, Lee WH, Marsden BD, Müller S, Niesen FH, Kavanagh KL, Ball LJ, von Delft F, Doyle DA et al. 2007. The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins. J Struct Funct Genomics, 8 (2-3), pp. 107-119. | Show Abstract | Read more

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.

Oliver AW, Knapp S, Pearl LH. 2007. Activation segment exchange: a common mechanism of kinase autophosphorylation? Trends Biochem Sci, 32 (8), pp. 351-356. | Show Abstract | Read more

The crystal structure of the kinase domain from human checkpoint kinase 2 (Chk2) has shown, for the first time, the reciprocal exchange of activation segments between two adjacent molecules and provides the molecular basis for understanding the observed mode of Chk2 kinase activation via trans-autophosphorylation. With further examples of activation segment exchanged kinase domains now publicly available (i.e. Ste20-like kinase, Ser/Thr kinase 10 and Death-associated protein kinase 3), we suggest that this phenomenon represents a common mechanism of activation amongst a particular subset of protein kinases, that is, those that are dimeric (either transiently or constitutively), that undergo activation by autophosphorylation and that have activation segment amino acid sequences that do not resemble those of their substrate consensus sequence.

Pogacic V, Bullock AN, Fedorov O, Filippakopoulos P, Gasser C, Biondi A, Meyer-Monard S, Knapp S, Schwaller J. 2007. Structural analysis identifies imidazo[1,2-b]pyridazines as PIM kinase inhibitors with in vitro antileukemic activity. Cancer Res, 67 (14), pp. 6916-6924. | Show Abstract | Read more

Much attention has recently been focused on PIM kinases as potential targets for the treatment of hematopoietic malignancies and some solid cancers. Using protein stability shift assays, we identified a family of imidazo[1,2-b]pyridazines to specifically interact with and inhibit PIM kinases with low nanomolar potency. The high-resolution crystal structure of a PIM1 inhibitor complex revealed that imidazo[1,2-b]pyridazines surprisingly interact with the NH(2)-terminal lobe helix alphaC rather than with the kinase hinge region. Thus, the identified inhibitors are ATP competitive but not ATP mimetic compounds, explaining their enhanced selectivity with respect to conventional type I kinase inhibitors. One of the identified imidazo[1,2-b]pyridazines (K00135) was further tested in several hematopoietic cellular systems. First, K00135 dose-dependently impaired survival of murine Ba/F3 cells that have been rendered cytokine independent by overexpression of human PIMs. Second, K00135 impaired survival and clonogenic growth of a panel of human acute leukemia cells. Third, exposure of K00135 significantly suppressed in vitro growth of leukemic blasts from five acute myelogenous leukemia patients but not of normal umbilical cord blood mononuclear cells. In vitro kinase assays and immunoblotting using lysates from human MV4;11 leukemic cells showed inhibition of phosphorylation of known PIM downstream targets, such as BAD and eukaryotic translation initiation factor 4E-binding protein 1, by K00135. Taken together, we report a family of small molecules that selectively interact and block PIM kinases and could serve as a lead to develop new targeted antileukemic therapeutics.

Baminger B, Ludwiczek ML, Kontaxis G, Knapp S, Konrat R. 2007. Protein-protein interaction site mapping using NMR-detected mutational scanning. J Biomol NMR, 38 (2), pp. 133-137. | Show Abstract | Read more

We demonstrate a novel NMR method for the mapping of protein-protein interaction sites. In our approach protein-protein binding sites are mapped by competition binding experiments using indirect NMR reporter technology and Ala positional scanning. The methodology provides high sensitivity, ease of implementation and high-throughput capabilities. The feasibility of the technique is demonstrated with an application to the beta-Catenin/Tcf4 complex.

Rennefahrt UE, Deacon SW, Parker SA, Devarajan K, Beeser A, Chernoff J, Knapp S, Turk BE, Peterson JR. 2007. Specificity profiling of Pak kinases allows identification of novel phosphorylation sites. J Biol Chem, 282 (21), pp. 15667-15678. | Show Abstract | Read more

The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks.

Rosettani P, Knapp S, Vismara MG, Rusconi L, Cameron AD. 2007. Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms. J Mol Biol, 368 (3), pp. 691-705. | Show Abstract | Read more

All eukaryotic cellular mRNAs contain a 5' m(7)GpppN cap. In addition to conferring stability to the mRNA, the cap is required for pre-mRNA splicing, nuclear export and translation by providing an anchor point for protein binding. In translation, the interaction between the cap and the eukaryotic initiation factor 4E (eIF4E) is important in the recruitment of the mRNAs to the ribosome. Human 4EHP (h4EHP) is a homologue of eIF4E. Like eIF4E it is able to bind the cap but it appears to play a different cellular role, possibly being involved in the fine-tuning of protein expression levels. Here we use X-ray crystallography and isothermal titration calorimetry (ITC) to investigate further the binding of cap analogues and peptides to h4EHP. m(7)GTP binds to 4EHP 200-fold more weakly than it does to eIF4E with the guanine base sandwiched by a tyrosine and a tryptophan instead of two tryptophan residues as seen in eIF4E. The tyrosine resides on a loop that is longer in h4EHP than in eIF4E. The consequent conformational difference between the proteins allows the tyrosine to mimic the six-membered ring of the tryptophan in eIF4E and adopt an orientation that is similar to that seen for equivalent residues in other non-homologous cap-binding proteins. In the absence of ligand the binding site is incompletely formed with one of the aromatic residues being disordered and the side-chain of the other adopting a novel conformation. A peptide derived from the eIF4E inhibitory protein, 4E-BP1 binds h4EHP 100-fold less strongly than eIF4E but in a similar manner. Overall the data, combined with sequence analyses of 4EHP from evolutionary diverse species, strongly support the hypothesis that 4EHP plays a physiological role utilizing both cap-binding and protein-binding functions but which is distinct from eIF4E.

Fedorov O, Sundström M, Marsden B, Knapp S. 2007. Insights for the development of specific kinase inhibitors by targeted structural genomics. Drug Discov Today, 12 (9-10), pp. 365-372. | Show Abstract | Read more

Many protein kinases are validated intervention points for drug development, however active site similarities often lead to a lack of selectivity and unwanted side effects in the clinic. To address this issue, it is desirable to increase the number of high resolution crystal structures and complexes with non-adenosine ligands available for the rational design of more selective inhibitors. Recent progress in protein crystallography and biotechnology has enabled structural genomics projects to target challenging proteins successfully, including protein kinases. As we discuss here, this effort has resulted in a considerable increase in the number of available high resolution structures and inhibitor complexes and has identified novel structural motifs that are available for drug development.

Rellos P, Ivins FJ, Baxter JE, Pike A, Nott TJ, Parkinson DM, Das S, Howell S, Fedorov O, Shen QY et al. 2007. Structure and regulation of the human Nek2 centrosomal kinase. J Biol Chem, 282 (9), pp. 6833-6842. | Show Abstract | Read more

The dimeric Ser/Thr kinase Nek2 regulates centrosome cohesion and separation through phosphorylation of structural components of the centrosome, and aberrant regulation of Nek2 activity can lead to aneuploid defects characteristic of cancer cells. Mutational analysis of autophosphorylation sites within the kinase domain identified by mass spectrometry shows a complex pattern of positive and negative regulatory effects on kinase activity that are correlated with effects on centrosomal splitting efficiency in vivo. The 2.2-A resolution x-ray structure of the Nek2 kinase domain in complex with a pyrrole-indolinone inhibitor reveals an inhibitory helical motif within the activation loop. This helix presents a steric barrier to formation of the active enzyme and generates a surface that may be exploitable in the design of specific inhibitors that selectively target the inactive state. Comparison of this "auto-inhibitory" conformation with similar arrangements in cyclin-dependent kinase 2 and epidermal growth factor receptor kinase suggests a role for dimerization-dependent allosteric regulation that combines with autophosphorylation and protein phosphatase 1c phosphatase activity to generate the precise spatial and temporal control required for Nek2 function in centrosomal maturation.

Wu X, Knapp S, Stamp A, Stammers DK, Jörnvall H, Dellaporta SL, Oppermann U. 2007. Biochemical characterization of TASSELSEED 2, an essential plant short-chain dehydrogenase/reductase with broad spectrum activities. FEBS J, 274 (5), pp. 1172-1182. | Show Abstract | Read more

The development of unisexual flowers in maize and other plants proceeds through selective elimination of floral organs in an initially bisexual floral meristem. The essential character of the tasselseed 2 gene (TS2) in this cell-death pathway has been established previously. Molecular cloning of TS2 reveals membership to the evolutionarily conserved superfamily of short-chain dehydrogenases/reductases, but its substrate specificity remained unknown. Recombinant TS2 protein was produced in Escherichia coli, and purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration experiments show that TS2 is a tetrameric enzyme. Thermal denaturation followed by circular dichroism spectroscopy reveals that TS2 binds NAD(H) and NAD(P)(H). Substrate screening demonstrates that TS2 converts steroids with specificities found at positions 3 and 17, and several dicarbonyl and quinone compounds, thus establishing TS2 as a plant 3beta/17beta-hydroxysteroid dehydrogenase and carbonyl/quinone reductase. Taken together, the genetic data and the substrate specificities determined suggest that TS2 converts specific plant compounds and acts as a prereceptor control mechanism, in a manner similar to that of mammalian hydroxysteroid dehydrogenases.

Eswaran J, Lee WH, Debreczeni JE, Filippakopoulos P, Turnbull A, Fedorov O, Deacon SW, Peterson JR, Knapp S. 2007. Crystal Structures of the p21-activated kinases PAK4, PAK5, and PAK6 reveal catalytic domain plasticity of active group II PAKs. Structure, 15 (2), pp. 201-213. | Show Abstract | Read more

p21-activated kinases have been classified into two groups based on their domain architecture. Group II PAKs (PAK4-6) regulate a wide variety of cellular functions, and PAK deregulation has been linked to tumor development. Structural comparison of five high-resolution structures comprising all active, monophosphorylated group II catalytic domains revealed a surprising degree of domain plasticity, including a number of catalytically productive and nonproductive conformers. Rearrangements of helix alphaC, a key regulatory element of kinase function, resulted in an additional helical turn at the alphaC N terminus and a distortion of its C terminus, a movement hitherto unseen in protein kinases. The observed structural changes led to the formation of interactions between conserved residues that structurally link the glycine-rich loop, alphaC, and the activation segment and firmly anchor alphaC in an active conformation. Inhibitor screening identified six potent PAK inhibitors from which a tri-substituted purine inhibitor was cocrystallized with PAK4 and PAK5.

Barr AJ, Knapp S. 2006. MAPK-specific tyrosine phosphatases: new targets for drug discovery? Trends Pharmacol Sci, 27 (10), pp. 525-530. | Show Abstract | Read more

Protein tyrosine phosphatases (PTPs) have key roles in a diverse range of cellular processes, and their dysregulation is associated with several human diseases. Many PTPs are recognized as potential drug targets; however, inhibitor development has focused only on a small number of enzymes, most notably PTP1B for type II diabetes and obesity, and MKP1 and CDC25 for cancer. The future challenge of selective-inhibitor development for PTPs will be significantly facilitated by the recent rapid progress in the structural biology of the 'PTPome'. In this article, we focus on the family of mitogen-activated protein kinase (MAPK)-specific tyrosine phosphatases--PTPN5 [also called striatal-enriched phosphatase (STEP)], PTPN7 (also called hematopoietic PTP) and PTPRR (also called PC12 PTP or STEP-like PTP)--and discuss approaches for achieving selectivity for the MAPK-PTPs at the molecular level using recently determined high-resolution X-ray crystal structures. We believe that the development of specific inhibitors would provide a valuable set of experimental pharmacological tools for investigating the physiological role of these phosphatases and exploring their emerging role in human disease.

Marsden BD, Sundstrom M, Knapp S. 2006. High-throughput structural characterisation of therapeutic protein targets. Expert Opin Drug Discov, 1 (2), pp. 123-136. | Show Abstract | Read more

The overarching scientific objective for high-throughput structural biology projects targeting human proteins or relevant proteins of human pathogens is to generate a detailed structural insight of biologically relevant structural features of soluble or membrane protein families of therapeutic relevance. These data will better enable structure-guided drug discovery in pharmaceutical and biotechechnology companies. In particular, detailed structural descriptions for entire protein families will be highly valuable for the design of drugs and other bioactive compounds that are selective for individual members or groups of proteins within protein families. In addition to determined structural information, targeted high-throughput structural biology will also provide reagents, methodologies, technologies and small molecules that bind selectively to targets that are suitable as intervention points in the treatment of disease.

Barr AJ, Debreczeni JE, Eswaran J, Knapp S. 2006. Crystal structure of human protein tyrosine phosphatase 14 (PTPN14) at 1.65-A resolution. Proteins, 63 (4), pp. 1132-1136. | Read more

Eswaran J, Debreczeni JE, Longman E, Barr AJ, Knapp S. 2006. The crystal structure of human receptor protein tyrosine phosphatase kappa phosphatase domain 1. Protein Sci, 15 (6), pp. 1500-1505. | Show Abstract | Read more

The receptor-type protein tyrosine phosphatases (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain consists of tandem PTP domains, of which the D1 domain is enzymatically active. RPTPkappa is a member of the R2A/IIb subfamily of RPTPs along with RPTPmu, RPTPrho, and RPTPlambda. Here, we have determined the crystal structure of catalytically active, monomeric D1 domain of RPTPkappa at 1.9 A. Structural comparison with other PTP family members indicates an overall classical PTP architecture of twisted mixed beta-sheets flanked by alpha-helices, in which the catalytically important WPD loop is in an unhindered open conformation. Though the residues forming the dimeric interface in the RPTPmu structure are all conserved, they are not involved in the protein-protein interaction in RPTPkappa. The N-terminal beta-strand, formed by betax association with betay, is conserved only in RPTPs but not in cytosolic PTPs, and this feature is conserved in the RPTPkappa structure forming a beta-strand. Analytical ultracentrifugation studies show that the presence of reducing agents and higher ionic strength are necessary to maintain RPTPkappa as a monomer. In this family the crystal structure of catalytically active RPTPmu D1 was solved as a dimer, but the dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. In agreement, we show that RPTPkappa is monomeric in solution and crystal structure.

Bullock AN, Debreczeni JE, Edwards AM, Sundström M, Knapp S. 2006. Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proc Natl Acad Sci U S A, 103 (20), pp. 7637-7642. | Show Abstract | Read more

Growth hormone (GH) signaling is tightly controlled by ubiquitination of GH receptors, phosphorylation levels, and accessibility of binding sites for downstream signaling partners. Members of the suppressors of cytokine signaling (SOCS) family function as key regulators at all levels of this pathway, and mouse knockout studies implicate SOCS2 as the primary suppressor. To elucidate the structural basis for SOCS2 function, we determined the 1.9-A crystal structure of the ternary complex of SOCS2 with elongin C and elongin B. The structure defines a prototypical SOCS box ubiquitin ligase with a Src homology 2 (SH2) domain as a substrate recognition motif. Overall, the SOCS box and SH2 domain show a conserved spatial domain arrangement with the BC box and substrate recognition domain of the von Hippel-Lindau (VHL) tumor suppressor protein, suggesting a common mechanism of ubiquitination in these cullin-dependent E3 ligases. The SOCS box binds elongin BC in a similar fashion to the VHL BC box and shows extended structural conservation with the F box of the Skp2 ubiquitin ligase. A previously unrecognized feature of the SOCS box is revealed with the burial of the C terminus, which packs together with the N-terminal extended SH2 subdomain to create a stable interface between the SOCS box and SH2 domain. This domain organization is conserved in SOCS1-3 and CIS1, which share a strictly conserved length of their C termini, but not in SOCS4, 5, and 7, which have extended C termini defining two distinct classes of inter- and intramolecular SOCS box interactions.

Kavanagh KL, Guo K, Dunford JE, Wu X, Knapp S, Ebetino FH, Rogers MJ, Russell RG, Oppermann U. 2006. The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs. Proc Natl Acad Sci U S A, 103 (20), pp. 7829-7834. | Show Abstract | Read more

Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. Because of their bone-selective pharmacokinetics, nitrogen-containing bisphosphonates (N-BPs), currently used as clinical inhibitors of bone-resorption diseases, target osteoclast farnesyl pyrophosphate synthase (FPPS) and inhibit protein prenylation. FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. To understand the molecular events involved in inhibition of FPPS by N-BPs, we used protein crystallography, enzyme kinetics, and isothermal titration calorimetry. We report here high-resolution x-ray structures of the human enzyme in complexes with risedronate and zoledronate, two of the leading N-BPs in clinical use. These agents bind to the dimethylallyl/geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-BP cyclic nitrogen with Thr-201 and Lys-200 suggest that these inhibitors achieve potency by positioning their nitrogen in the proposed carbocation-binding site. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight binding character, indicating that isomerization of an initial enzyme-inhibitor complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N-BPs to the apoenzyme is entropy-driven, presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of these important drugs.

Eswaran J, von Kries JP, Marsden B, Longman E, Debreczeni JE, Ugochukwu E, Turnbull A, Lee WH, Knapp S, Barr AJ. 2006. Crystal structures and inhibitor identification for PTPN5, PTPRR and PTPN7: a family of human MAPK-specific protein tyrosine phosphatases. Biochem J, 395 (3), pp. 483-491. | Show Abstract | Read more

Protein tyrosine phosphatases PTPN5, PTPRR and PTPN7 comprise a family of phosphatases that specifically inactivate MAPKs (mitogen-activated protein kinases). We have determined high-resolution structures of all of the human family members, screened them against a library of 24000 compounds and identified two classes of inhibitors, cyclopenta[c]quinolinecarboxylic acids and 2,5-dimethylpyrrolyl benzoic acids. Comparative structural analysis revealed significant differences within this conserved family that could be explored for the design of selective inhibitors. PTPN5 crystallized, in two distinct crystal forms, with a sulphate ion in close proximity to the active site and the WPD (Trp-Pro-Asp) loop in a unique conformation, not seen in other PTPs, ending in a 3(10)-helix. In the PTPN7 structure, the WPD loop was in the closed conformation and part of the KIM (kinase-interaction motif) was visible, which forms an N-terminal aliphatic helix with the phosphorylation site Thr66 in an accessible position. The WPD loop of PTPRR was open; however, in contrast with the structure of its mouse homologue, PTPSL, a salt bridge between the conserved lysine and aspartate residues, which has been postulated to confer a more rigid loop structure, thereby modulating activity in PTPSL, does not form in PTPRR. One of the identified inhibitor scaffolds, cyclopenta[c]quinoline, was docked successfully into PTPRR, suggesting several possibilities for hit expansion. The determined structures together with the established SAR (structure-activity relationship) propose new avenues for the development of selective inhibitors that may have therapeutic potential for treating neurodegenerative diseases in the case of PTPRR or acute myeloblastic leukaemia targeting PTPN7.

Debreczeni JE, Bullock AN, Atilla GE, Williams DS, Bregman H, Knapp S, Meggers E. 2006. Ruthenium half-sandwich complexes bound to protein kinase Pim-1. Angew Chem Int Ed Engl, 45 (10), pp. 1580-1585. | Show Abstract | Read more

(Figure Presented) Keeping in shape with half a sandwich: The structure of a picomolar organoruthenium inhibitor bound to the ATP-binding site of the protein kinase Pim-1 (see picture) demonstrates that the ruthenium center has solely a structural role in organizing the organic ligands in the three-dimensional receptor space, thus yielding a structure that is complementary in shape and functional group presentation to the active site of Pim-1. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Marsden BD, Sundstrom M, Knapp S. 2006. High-throughput structural characterisation of therapeutic protein targets Expert Opinion on Drug Discovery, 1 (2), pp. 123-136. | Show Abstract | Read more

The overarching scientific objective for high-throughput structural biology projects targeting human proteins or relevant proteins of human pathogens is to generate a detailed structural insight of biologically relevant structural features of soluble or membrane protein families of therapeutic relevance. These data will better enable structure-guided drug discovery in pharmaceutical and biotechechnology companies. In particular, detailed structural descriptions for entire protein families will be highly valuable for the design of drugs and other bioactive compounds that are selective for individual members or groups of proteins within protein families. In addition to determined structural information, targeted high-throughput structural biology will also provide reagents, methodologies, technologies and small molecules that bind selectively to targets that are suitable as intervention points in the treatment of disease. © 2006 Informa UK Ltd.

Trosset JY, Dalvit C, Knapp S, Fasolini M, Veronesi M, Mantegani S, Gianellini LM, Catana C, Sundström M, Stouten PF, Moll JK. 2006. Inhibition of protein-protein interactions: the discovery of druglike beta-catenin inhibitors by combining virtual and biophysical screening. Proteins, 64 (1), pp. 60-67. | Show Abstract | Read more

The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.

Bullock AN, Debreczeni J, Amos AL, Knapp S, Turk BE. 2005. Structure and substrate specificity of the Pim-1 kinase. J Biol Chem, 280 (50), pp. 41675-41682. | Show Abstract | Read more

The Pim kinases are a family of three vertebrate protein serine/threonine kinases (Pim-1, -2, and -3) belonging to the CAMK (calmodulin-dependent protein kinase-related) group. Pim kinases are emerging as important mediators of cytokine signaling pathways in hematopoietic cells, and they contribute to the progression of certain leukemias and solid tumors. A number of cytoplasmic and nuclear proteins are phosphorylated by Pim kinases and may act as their effectors in normal physiology and in disease. Recent crystallographic studies of Pim-1 have identified unique structural features but have not provided insight into how the kinase recognizes its target substrates. Here, we have conducted peptide library screens to exhaustively determine the sequence specificity of active site-mediated phosphorylation by Pim-1 and Pim-3. We have identified the major site of Pim-1 autophosphorylation and find surprisingly that it maps to a novel site that diverges from its consensus phosphorylation motif. We have solved the crystal structure of Pim-1 bound to a high affinity peptide substrate in complexes with either the ATP analog AMP-PNP or the bisindolylmaleimide kinase inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide HCl. These structures reveal an unanticipated mode of recognition for basic residues upstream of the phosphorylation site, distinct from that of other kinases with similar substrate specificity. The structures provide a rationale for the unusually high affinity of Pim kinases for peptide substrates and suggest a general mode for substrate binding to members of the CAMK group.

Bullock AN, Debreczeni JE, Fedorov OY, Nelson A, Marsden BD, Knapp S. 2005. Structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM-1) kinase. J Med Chem, 48 (24), pp. 7604-7614. | Show Abstract | Read more

The kinase PIM-1 plays a pivotal role in cytokine signaling and is implicated in the development of a number of tumors. The three-dimensional structure of PIM-1 is characterized by an unique hinge region which lacks a second hydrogen bond donor and makes it particularly important to determine how inhibitors bind to this kinase. We determined the structures of PIM-1 in complex with bisindolylmaleimide (BIM-1) and established the structure-activity relationship (SAR) for this inhibitor class. In addition, we screened a kinase targeted library and identified a number of high affinity inhibitors of PIM-1 such as imidazo[1,2-b]pyridazines, pyrazolo[1,5-a]pyrimidines, and members of the flavonoid family. In this paper we present an initial SAR of the identified scaffolds determined on the basis of a thermostability shift assay, calorimetric binding data, and biochemical assays which may find applications for the treatment of PIM-1 dependent cancer types.

Cristiani C, Rusconi L, Perego R, Schiering N, Kalisz HM, Knapp S, Isacchi A. 2005. Regulation of the wild-type and Y1235D mutant Met kinase activation. Biochemistry, 44 (43), pp. 14110-14119. | Show Abstract | Read more

Met receptor tyrosine kinase plays a crucial role in the regulation of a large number of cellular processes and, when deregulated by overexpression or mutations, leads to tumor growth and invasion. The Y1235D mutation identified in metastases was shown to induce constitutive activation and a motile-invasive phenotype on transduced carcinoma cells. Wild-type Met activation requires phosphorylation of both Y1234 and Y1235 in the activation loop. We mapped the major phosphorylation sites in the kinase domain of a recombinant Met protein and identified the known residues Y1234 and Y1235 as well as a new phosphorylation site at Y1194 in the hinge region. Combining activating and silencing mutations at these sites, we characterized in depth the mechanism of activation of wild-type and mutant Met proteins. We found that the phosphotyrosine mimetic mutation Y1235D is sufficient to confer constitutive kinase activity, which is not influenced by phosphorylation at Y1234. However, the specific activity of this mutant was lower than that observed for fully activated wild-type Met and induced less phosphorylation of Y1349 in the signaling site, indicating that this mutation cannot entirely compensate for a phosphorylated tyrosine at this position. The Y1194F silencing mutation yielded an enzyme that could be activated to a similar extent as the wild type but with significantly slower activation kinetics, underlying the importance of this residue, which is conserved among different tyrosine kinase receptors. Finally, we observed different interactions of wild-type and mutant Met with the inhibitor K252a that may have therapeutic implications for the selective inhibition of this kinase.

Tarricone C, Perrina F, Monzani S, Massimiliano L, Kim MH, Derewenda ZS, Knapp S, Tsai LH, Musacchio A. 2004. Coupling PAF signaling to dynein regulation: structure of LIS1 in complex with PAF-acetylhydrolase. Neuron, 44 (5), pp. 809-821. | Show Abstract | Read more

Mutations in the LIS1 gene cause lissencephaly, a human neuronal migration disorder. LIS1 binds dynein and the dynein-associated proteins Nde1 (formerly known as NudE), Ndel1 (formerly known as NUDEL), and CLIP-170, as well as the catalytic alpha dimers of brain cytosolic platelet activating factor acetylhydrolase (PAF-AH). The mechanism coupling the two diverse regulatory pathways remains unknown. We report the structure of LIS1 in complex with the alpha2/alpha2 PAF-AH homodimer. One LIS1 homodimer binds symmetrically to one alpha2/alpha2 homodimer via the highly conserved top faces of the LIS1 beta propellers. The same surface of LIS1 contains sites of mutations causing lissencephaly and overlaps with a putative dynein binding surface. Ndel1 competes with the alpha2/alpha2 homodimer for LIS1, but the interaction is complex and requires both the N- and C-terminal domains of LIS1. Our data suggest that the LIS1 molecule undergoes major conformational rearrangement when switching from a complex with the acetylhydrolase to the one with Ndel1.

Knapp S, Müller S, Digilio G, Bonaldi T, Bianchi ME, Musco G. 2004. The long acidic tail of high mobility group box 1 (HMGB1) protein forms an extended and flexible structure that interacts with specific residues within and between the HMG boxes. Biochemistry, 43 (38), pp. 11992-11997. | Show Abstract | Read more

HMGB1 (high mobility group B1) is a conserved chromosomal protein composed of two similar DNA binding domains (HMG box A and box B) linked by a short basic stretch to an acidic C-terminal tail of 30 residues. The acidic tail modulates the DNA binding properties of HMGB1, and its length differentiates the various HMGB family members. We synthesized a peptide that corresponds to the acidic tail in HMGB1 (T-peptide) and studied its binding to the single boxes and to the fragment corresponding to tailless HMGB1 (designated as AB(bt) fragment). CD spectroscopy showed that T-peptide stabilizes significantly the AB(bt) fragment and that the complex has an identical thermal stability as full-length HMGB1. Calorimetric and NMR data showed that T-peptide binds with a dissociation constant of 9 microM to box A and much more weakly to box B. (1)H-(15)N HSQC spectra of full-length HMGB1 and of the AB(bt) fragment are very similar; the small chemical shift differences that exist correspond to those residues of the AB(bt) fragment that were affected by the addition of the T-peptide. We conclude that the T-peptide mimics closely the acidic tail and that the basic stretch and the acidic tail form an extended and flexible segment. The tail interacts with specific residues in the boxes and shields them from other interactions.

Schiering N, Knapp S, Marconi M, Flocco MM, Cui J, Perego R, Rusconi L, Cristiani C. 2003. Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a. Proc Natl Acad Sci U S A, 100 (22), pp. 12654-12659. | Show Abstract | Read more

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Bertrand JA, Thieffine S, Vulpetti A, Cristiani C, Valsasina B, Knapp S, Kalisz HM, Flocco M. 2003. Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors. J Mol Biol, 333 (2), pp. 393-407. | Show Abstract | Read more

GSK-3beta is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3beta may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3beta, we determined crystal structures of unphosphorylated GSK-3beta in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3beta in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3beta inhibitors.

Faria TQ, Knapp S, Ladenstein R, Maçanita AL, Santos H. 2003. Protein stabilisation by compatible solutes: effect of mannosylglycerate on unfolding thermodynamics and activity of ribonuclease A. Chembiochem, 4 (8), pp. 734-741. | Show Abstract | Read more

Differential scanning calorimetry, optical spectroscopy, and activity measurements were used to investigate the effect of mannosylglycerate, a negatively charged osmolyte widely distributed among thermophilic and hyperthermophilic archaea and bacteria, on the thermal unfolding of ribonuclease A (RNase A). For comparison, assays in the presence of trehalose, a canonical solute in mesophiles, and potassium chloride were also carried out. A thermodynamic analysis was performed by using differential scanning calorimetry data. The changes in the heat capacity for unfolding were similar for the different solutes examined. Mannosylglycerate was an efficient thermostabiliser of RNase A and induced an increase of 6 degrees C mole(-1) in the melting temperature. Moreover, the performance of mannosylglycerate as a stabiliser depended on the net charge of the molecule, with the maximal effect being observed at pH values above 4.5. Analysis of the enthalpic and entropic contributions to unfolding, derived from calorimetric data, revealed that the stabilisation rendered by mannosylglycerate is primarily achieved through a decrease in the unfolding entropy. Also, the number of protons taken up by RNase A upon denaturation in the presence of mannosylglycerate was considerably higher than with other solutes, a result consistent with a more rigid structure of the native protein. Mannosylglycerate (potassium salt) inhibited the activity of RNase A, albeit to a smaller extent than KCl, and acted as an efficient suppressor of aggregation of the denatured protein, thereby having a remarkable beneficial effect on the inactivation of RNase A upon thermal denaturation. The results are discussed in view of the physiological role of this charged compatible solute.

Lolli G, Thaler F, Valsasina B, Roletto F, Knapp S, Uggeri M, Bachi A, Matafora V, Storici P, Stewart A et al. 2003. Inhibitor affinity chromatography: profiling the specific reactivity of the proteome with immobilized molecules. Proteomics, 3 (7), pp. 1287-1298. | Show Abstract | Read more

An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat-shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug.

Fasolini M, Wu X, Flocco M, Trosset JY, Oppermann U, Knapp S. 2003. Hot spots in Tcf4 for the interaction with beta-catenin. J Biol Chem, 278 (23), pp. 21092-21098. | Show Abstract | Read more

The interaction of beta-catenin with T-cell factor (Tcf) 4 plays a central role in the Wnt signaling pathway and has been discussed as a possible site of intervention for the development of anti-cancer drugs. In this study, we performed Ala-scanning mutagenesis of all Tcf4 residues in the Tcf-beta-catenin interface and studied the binding energetics of these mutants using isothermal titration calorimetry. Binding of Tcf4 was found to be highly cooperative. Single site mutations of most Tcf4 residues resulted in a significant reduction in binding enthalpies but in similar binding constants as compared with wild type Tcf4. Interestingly, this was also true for residues that are disordered in the reported crystal structures. The mutation D16A caused the largest reduction in binding constant (50-fold) accompanied by a large unfavorable enthalpy change (DeltaDeltaHobs) of +8 kcal/mol at 25 degrees C. In contrast, the mutation of the Tcf residues Glu24 and Glu28, which have been proposed as an interaction hot spot due to their location in a field of strong positive electrostatic potential on the beta-catenin surface (charge button), resulted only in a significant reduction of binding enthalpies, which were largely compensated for by unfavorable entropic contributions to the binding. Other mutations that significantly reduced Tcf binding constants were D11A and alanine mutations of the hydrophobic residues Leu41, Val44, and Leu48. The measured thermodynamic data are discussed with the available structural information of Tcf-beta-catenin crystal structures and allow us to propose possible sites for development of Tcf antagonists.

Filling C, Berndt KD, Benach J, Knapp S, Prozorovski T, Nordling E, Ladenstein R, Jörnvall H, Oppermann U. 2002. Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem, 277 (28), pp. 25677-25684. | Show Abstract | Read more

Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.

Dalvit C, Flocco M, Knapp S, Mostardini M, Perego R, Stockman BJ, Veronesi M, Varasi M. 2002. High-throughput NMR-based screening with competition binding experiments. J Am Chem Soc, 124 (26), pp. 7702-7709. | Show Abstract | Read more

The Achilles heel of ligand-based NMR screening methods is their failure to detect high-affinity ligands and molecules that bind covalently to the receptor. We have developed a novel approach for performing high-throughput screening with NMR spectroscopy that overcomes this limitation. The method also permits detection of potential high-affinity molecules that are only marginally soluble, thus significantly enlarging the diversity of compounds amenable to NMR screening. The techniques developed utilize transverse and/or selective longitudinal relaxation parameters in combination with competition binding experiments. Mathematical expressions are derived for proper setup of the NMR experiments and for extracting an approximate value of the binding constant for the identified ligand from a single-point measurement. With this approach it is possible to screen thousands of compounds in a short period of time against protein or DNA and RNA fragments. The methodology can also be applied for screening plant and fungi extracts.

Dalvit C, Fasolini M, Flocco M, Knapp S, Pevarello P, Veronesi M. 2002. NMR-Based screening with competition water-ligand observed via gradient spectroscopy experiments: detection of high-affinity ligands. J Med Chem, 45 (12), pp. 2610-2614. | Show Abstract | Read more

Water-ligand observed via gradient spectroscopy (WaterLOGSY) represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. The method is useful for the detection of compounds binding to a receptor with binding affinity in the micromolar range. The Achille's heel of the method, as with all the techniques that detect the ligand resonances, is its inability to identify strong ligands with slow dissociation rates. We show here that the use of a reference compound with a known K(D) in the micromolar range together with properly designed competition binding experiments (c-WaterLOGSY) permits the detection of strong binders. A derived mathematical expression is used for the selection of the appropriate setup NMR experimental conditions and for an approximate determination of the binding constant. The experiment requires low ligand concentration, therefore allowing its application in the identification of potential strong inhibitors that are only marginally soluble. The technique is particularly suitable for rapid screening of chemical mixtures and plant or fungi extracts.

Sironi L, Mapelli M, Knapp S, De Antoni A, Jeang KT, Musacchio A. 2002. Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint. EMBO J, 21 (10), pp. 2496-2506. | Show Abstract | Read more

The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2-Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1-Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled-coil to form two connected sub-complexes with Mad2. The Mad2 C-terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular 'safety belts'. We show that Mad1 is a competitive inhibitor of the Mad2-Cdc20 complex, and propose that the Mad1-Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1-Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1-Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1-Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C-terminal tail is required for ligand release from Mad2, and that the 'safety belt' mechanism may prolong the lifetime of Mad2-ligand complexes.

Müller S, Bianchi ME, Knapp S. 2001. Thermodynamics of HMGB1 interaction with duplex DNA. Biochemistry, 40 (34), pp. 10254-10261. | Show Abstract | Read more

The high mobility group protein HMGB1 is a small, highly abundant protein that binds to DNA in a non-sequence-specific manner. HMGB1 consists of 2 DNA binding domains, the HMG boxes A and B, followed by a short basic region and a continuous stretch of 30 glutamate or aspartate residues. Isothermal titration calorimetry was used to characterize the binding of HMGB1 to the double-stranded model DNAs poly(dAdT).(dTdA) and poly(dGdC).(dCdG). To elucidate the contribution of the different structural motifs to DNA binding, calorimetric measurements were performed comparing the single boxes A and B, the two boxes plus or minus the basic sequence stretch (AB(bt) and AB), and the full-length HMGB1 protein. Thermodynamically, binding of HMGB1 and all truncated constructs to duplex DNA was characterized by a positive enthalpy change at 15 degrees C. From the slopes of the temperature dependence of the binding enthalpies, heat capacity changes of -0.129 +/- 0.02 and -0.105 +/- 0.05 kcal mol(-1) K(-1) were determined for box A and full-length HMGB1, respectively. Significant differences in the binding characteristics were observed using full-length HMGB1, suggesting an important role for the acid tail in modulating DNA binding. Moreover, full-length HMGB1 binds differently these two DNA templates: binding to poly(dAdT).(dTdA) was cooperative, had a larger apparent binding site size, and proceeded with a much larger unfavorable binding enthalpy than binding to poly(dGdC).(dCdG).

Knapp S, Zamai M, Volpi D, Nardese V, Avanzi N, Breton J, Plyte S, Flocco M, Marconi M, Isacchi A, Caiolfa VR. 2001. Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin. J Mol Biol, 306 (5), pp. 1179-1189. | Show Abstract | Read more

The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.

Consalvi V, Chiaraluce R, Giangiacomo L, Scandurra R, Christova P, Karshikoff A, Knapp S, Ladenstein R. 2000. Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima. Protein Eng, 13 (7), pp. 501-507. | Show Abstract | Read more

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme. The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.

Knapp S, Ladenstein R, Galinski EA. 1999. Extrinsic protein stabilization by the naturally occurring osmolytes beta-hydroxyectoine and betaine. Extremophiles, 3 (3), pp. 191-198. | Show Abstract | Read more

Thermodynamic aspects of protein stabilization by two widespread naturally occurring osmolytes, beta-hydroxyectoine and betaine, were studied using differential scanning calorimetry (DSC) and bovine ribonuclease A (RNase A) as a model protein. The osmolyte beta-hydroxyectoine purified from Marinococcus was found to be a very efficient stabilizer. At a concentration of 3M it increased the melting temperature of RNase A (Tm) by more than 12K and gave rise to a stability increase of 10.6kJ/mol at room temperature. The heat capacity difference between the folded and unfolded state (deltaC(p)) was found to be significantly increased. Betaine stabilized RNase A only at concentrations less than 3M. Also, here deltaCp was found to be increased. Calculation of the number of water molecules that additionally bind to unfolded RNase A resulted in surprisingly low numbers for both osmolytes. The significant stabilization of RNase A by beta-hydroxyectoine makes this osmolyte an interesting stabilizer in biotechnological processes in which enzymes are applied in the presence of denaturants or at high temperature.

Vécsey-Semjén B, Knapp S, Möllby R, van der Goot FG. 1999. The staphylococcal alpha-toxin pore has a flexible conformation. Biochemistry, 38 (14), pp. 4296-4302. | Show Abstract | Read more

The alpha-toxin from Staphylococcus aureus undergoes several conformational changes from the time it is released from the bacterium to the moment it forms a channel in the plasma membrane of its target cell. It is initially a soluble monomer, which undergoes membrane binding and oligomerization into a heptameric ring and finally inserts into the lipid bilayer to form a pore. Here we have analyzed the stability of different forms of the alpha-toxin (monomer as well as heptamers in solution, bound to the membrane and membrane-inserted) by differential scanning calorimetry and limited proteolysis. Data presented here show that, in contrast to both the membrane-bound prepore complex and the monomer in solution, the membrane-inserted alpha-toxin channel does not undergo cooperative unfolding and is highly susceptible to proteases. These observations suggest that the channel has a looser conformation. Interestingly, resistance to proteases could be recovered upon solubilization of the channel, indicating that the loss of rigid tertiary packing only occurred upon membrane insertion. Far-UV CD data, however, suggest that the transmembrane beta-barrel must be stably folded and that therefore only the Cap and Rim domains of the channel are loosely packed. All together, our data show that the alpha-toxin channel is not a rigid complex within the membrane but adopts a rather flexible conformation.

Oppermann UC, Knapp S, Bonetto V, Ladenstein R, Jörnvall H. 1998. Isolation and structure of repressor-like proteins from the archaeon Sulfolobus solfataricus. Co-purification of RNase A with Sso7c. FEBS Lett, 432 (3), pp. 141-144. | Show Abstract | Read more

The thermostable histone-like protein Sso7c (Sso for Sulfolobus solfataricus) from the archaeon Sulfolobus solfataricus was purified from the supernatant of acid-soluble cell lysates. Reverse phase HPLC of an apparently homogeneous Sso7c protein fraction from Mono S chromatography resulted in resolution of three further peaks. Sequence analysis revealed one of these components to be bovine RNase A, originating from the culture medium and explaining the RNA hydrolyzing activities of Sso7 preparations previously described. Sequence analysis of pure Sso7c showed an epsilon-Lys methylation pattern identical to that of Sso7d and a single Gln --> Glu mutational difference at position 13. The remaining two proteins obtained after HPLC separation were identified as homologues of bacterial repressor-like proteins. Thus, the existence of repressor-like proteins was demonstrated at the protein level in archaea, raising the question of structural and functional consequences of these proteins on the otherwise eukaryotic-like basal transcriptional machinery in archaea.

Knapp S, Mattson PT, Christova P, Berndt KD, Karshikoff A, Vihinen M, Smith CI, Ladenstein R. 1998. Thermal unfolding of small proteins with SH3 domain folding pattern. Proteins, 31 (3), pp. 309-319. | Show Abstract | Read more

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities.

Knapp S, Karshikoff A, Berndt KD, Christova P, Atanasov B, Ladenstein R. 1996. Thermal unfolding of the DNA-binding protein Sso7d from the hyperthermophile Sulfolobus solfataricus. J Mol Biol, 264 (5), pp. 1132-1144. | Show Abstract | Read more

Thermal unfolding of the small hyperthermophilic DNA-binding protein Sso7d was studied by circular dichroism spectroscopy and differential scanning calorimetry. The unfolding transition can be described by a reversible two state process. Maximum stability was observed in the region between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperature between 370.8 to 371.9 K and an unfolding enthalpy between 62.9 and 65.4 kcal/mol. The heat capacity differences between the native and the heat denatured states obtained by differential scanning calorimetry (620 cal/(molK)) and circular dichroism spectroscopy (580 cal/(mol K)) resulted in comparable values. The thermodynamic reason for the high melting temperature of Sso7d is the shallow stability curve with a broad free energy maximum, corresponding to the relatively small heat capacity change which was obtained. The calculated stability curve shows that Sso7d has, despite of its high melting temperature, an only moderate intrinsic stability, which reaches its maximum (approximately 7 kcal/mol) at 282 K. Sso7d is particularly poorly stabilized (approximately 1 kcal/mol) at the maximum physiological growth temperature of Sulfolobus solfataricus. Sso7d has furthermore untypically low specific enthalpy (0.99 kcal/(mol residue)) and entropy (2.99 cal/(mol K)) values at convergence temperatures. No significant differences in thermal stability of the partially methylated Sso7d from Sulfolobus solfataricus and the cloned non-methylated form of the protein expressed in Escherichia coli were observed.

Knapp S, Schmidt-Krey I, Hebert H, Bergman T, Jörnvall H, Ladenstein R. 1994. The molecular chaperonin TF55 from the Thermophilic archaeon Sulfolobus solfataricus. A biochemical and structural characterization. J Mol Biol, 242 (4), pp. 397-407. | Show Abstract | Read more

The purification and characterization of a new type of thermostable chaperonin from the archaebacterium Sulfolobus solfataricus is described. The chaperonin forms a hetero-oligomeric complex of two different, but closely related, subunits, which we have assigned TF55-alpha and TF55-beta. Their N-terminal sequences and amino acid residue compositions are reported. Two-dimensional projections of the chaperonin have been reconstructed from electron microscopy images, showing a 9-fold symmetrical complex, about 17.5 nm in height and 16 nm in diameter, with a central cavity of 4.5 nm. The complex is resistant to denaturing agents at room temperature and only pH values lower than 2 lead to dissociation. The separated subunits do not reassemble spontaneously but require Mg2+ and ATP for complex formation. Both subunits are necessary for formation of the TF55 oligomer. Significant structural changes have been observed after phosphorylation, thus providing evidence for a structural mobility during the chaperonin-assisted folding process of a protein. The phosphorylation reaction is modulated by potassium and magnesium ions. Magnesium seems to have an inhibitory effect, whereas potassium enhances this reaction.

KLUG J, KNAPP S, CASTRO I, BEATO M. 1994. 2 DISTINCT FACTORS BIND TO THE RABBIT UTEROGLOBIN TATA-BOX REGION AND ARE REQUIRED FOR EFFICIENT TRANSCRIPTION MOLECULAR AND CELLULAR BIOLOGY, 14 (9), pp. 6208-6218.

Klug J, Knapp S, Castro I, Beato M. 1994. Two distinct factors bind to the rabbit uteroglobin TATA-box region and are required for efficient transcription. Mol Cell Biol, 14 (9), pp. 6208-6218. | Show Abstract | Read more

The rabbit uteroglobin gene is expressed in a variety of epithelial cell types like the lung Clara cells and the glandular and luminal epithelial cells of the endometrium. Expression in Clara cells is on a high constitutive level, whereas expression in the rabbit endometrium is under tight hormonal control. One important element of the rabbit uteroglobin gene mediating its efficient transcription in two epithelial cell lines from human endometrium (Ishikawa) and lung (NCI-H441) is its noncanonical TATA box (TACA). Here, we show that two factors (TATA core factor [TCF] and TATA palindrome factor [TPF]) different from the TATA-box binding protein bind to the DNA major groove at two adjacent sites within the uteroglobin TATA-box region and that one of them (TCF) is specifically expressed in cell lines derived from uteroglobin-expressing tissues. The binding sites for TCF and TPF, respectively, are both required for efficient transcription in Ishikawa and NCI-H441 cells. Mutation of the TACA box, which we show is a poor TATA box in functional terms, to a canonical TATA motif does not affect TCF and TPF binding. Therefore, we suggest that the function of the unusual cytosine could be to reduce rabbit uteroglobin expression in cells lacking TCF and that the interaction of TATA-box binding protein with the weak TACA site is facilitated in TCF- and TPF-positive cells.

Picaud S, Leonards K, Lambert JP, Dovey O, Wells C, Fedorov O, Monteiro O, Fujisawa T, Wang CY, Lingard H et al. 2016. Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia. Sci Adv, 2 (10), pp. e1600760. | Show Abstract | Read more

Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.

Knapp S, Arruda P, Blagg J, Burley S, Drewry DH, Edwards A, Fabbro D, Gillespie P, Gray NS, Kuster B et al. 2013. A public-private partnership to unlock the untargeted kinome. Nat Chem Biol, 9 (1), pp. 3-6. | Read more

Fish PV, Filippakopoulos P, Bish G, Brennan PE, Bunnage ME, Cook AS, Federov O, Gerstenberger BS, Jones H, Knapp S et al. 2012. Identification of a chemical probe for bromo and extra C-terminal bromodomain inhibition through optimization of a fragment-derived hit. J Med Chem, 55 (22), pp. 9831-9837. | Show Abstract | Read more

The posttranslational modification of chromatin through acetylation at selected histone lysine residues is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The significance of this subset of the epigenetic code is interrogated and interpreted by an acetyllysine-specific protein-protein interaction with bromodomain reader modules. Selective inhibition of the bromo and extra C-terminal domain (BET) family of bromodomains with a small molecule is feasible, and this may represent an opportunity for disease intervention through the recently disclosed antiproliferative and anti-inflammatory properties of such inhibitors. Herein, we describe the discovery and structure-activity relationship (SAR) of a novel, small-molecule chemical probe for BET family inhibition that was identified through the application of structure-based fragment assessment and optimization techniques. This has yielded a potent, selective compound with cell-based activity (PFI-1) that may further add to the understanding of BET family function within the bromodomains.

Filippakopoulos P, Picaud S, Mangos M, Keates T, Lambert JP, Barsyte-Lovejoy D, Felletar I, Volkmer R, Müller S, Pawson T et al. 2012. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell, 149 (1), pp. 214-231. | Show Abstract | Read more

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.

Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I et al. 2010. Selective inhibition of BET bromodomains. Nature, 468 (7327), pp. 1067-1073. | Show Abstract | Read more

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Kwiatkowski N, Jelluma N, Filippakopoulos P, Soundararajan M, Manak MS, Kwon M, Choi HG, Sim T, Deveraux QL, Rottmann S et al. 2010. Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function. Nat Chem Biol, 6 (5), pp. 359-368. | Show Abstract | Read more

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

Fedorov O, Müller S, Knapp S. 2010. The (un)targeted cancer kinome. Nat Chem Biol, 6 (3), pp. 166-169. | Read more

Rellos P, Pike AC, Niesen FH, Salah E, Lee WH, von Delft F, Knapp S. 2010. Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation. PLoS Biol, 8 (7), pp. e1000426. | Show Abstract | Read more

UNLABELLED: Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.

Eswaran J, Patnaik D, Filippakopoulos P, Wang F, Stein RL, Murray JW, Higgins JM, Knapp S. 2009. Structure and functional characterization of the atypical human kinase haspin. Proc Natl Acad Sci U S A, 106 (48), pp. 20198-20203. | Show Abstract | Read more

The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP gamma-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.

Barr AJ, Ugochukwu E, Lee WH, King ON, Filippakopoulos P, Alfano I, Savitsky P, Burgess-Brown NA, Müller S, Knapp S. 2009. Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell, 136 (2), pp. 352-363. | Show Abstract | Read more

Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a "head-to-toe" dimerization model for RPTPgamma/zeta that is distinct from the "inhibitory wedge" model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.

Filippakopoulos P, Kofler M, Hantschel O, Gish GD, Grebien F, Salah E, Neudecker P, Kay LE, Turk BE, Superti-Furga G et al. 2008. Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation. Cell, 134 (5), pp. 793-803. | Show Abstract | Read more

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Eswaran J, Soundararajan M, Kumar R, Knapp S. 2008. UnPAKing the class differences among p21-activated kinases. Trends Biochem Sci, 33 (8), pp. 394-403. | Show Abstract | Read more

The p21-activated kinases (PAKs) are signal transducers, central to many vital cellular processes, including cell morphology, motility, survival, gene transcription and hormone signalling. The mammalian PAK family contains six serine/threonine kinases divided into two subgroups, group I (PAK 1-3) and group II (PAK4-6), based on their domain architecture and regulation. PAKs functioning as dynamic signalling nodes present themselves as attractive therapeutic targets in tumours, neurological diseases and infection. The recent findings across all PAKs, including newly reported structures, shed light on the cellular functions of PAKs, highlighting molecular mechanisms of activation, catalysis and substrate specificity. We believe that a comprehensive understanding of the entire PAK family is essential for developing strategies towards PAK-targeted therapeutics.

Fedorov O, Marsden B, Pogacic V, Rellos P, Müller S, Bullock AN, Schwaller J, Sundström M, Knapp S. 2007. A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases. Proc Natl Acad Sci U S A, 104 (51), pp. 20523-20528. | Show Abstract | Read more

Protein kinases play a pivotal role in cell signaling, and dysregulation of many kinases has been linked to disease development. A large number of kinase inhibitors are therefore currently under investigation in clinical trials, and so far seven inhibitors have been approved as anti-cancer drugs. In addition, kinase inhibitors are widely used as specific probes to study cell signaling, but systematic studies describing selectivity of these reagents across a panel of diverse kinases are largely lacking. Here we evaluated the specificity of 156 validated kinase inhibitors, including inhibitors used in clinical trials, against 60 human Ser/Thr kinases using a thermal stability shift assay. Our analysis revealed many unexpected cross-reactivities for inhibitors thought to be specific for certain targets. We also found that certain combinations of active-site residues in the ATP-binding site correlated with the detected ligand promiscuity and that some kinases are highly sensitive to inhibition using diverse chemotypes, suggesting them as preferred intervention points. Our results uncovered also inhibitor cross-reactivities that may lead to alternate clinical applications. For example, LY333'531, a PKCbeta inhibitor currently in phase III clinical trials, efficiently inhibited PIM1 kinase in our screen, a suggested target for treatment of leukemia. We determined the binding mode of this inhibitor by x-ray crystallography and in addition showed that LY333'531 induced cell death and significantly suppressed growth of leukemic cells from acute myeloid leukemia patients.

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