register interest

Dr Oleg Fedorov

Research Area: Protein Science and Structural Biology
Technology Exchange: Crystallography, Drug discovery, Mass spectrometry, Microscopy (Confocal) and Protein interaction
Scientific Themes: Protein Science & Structural Biology and Cancer Biology

Molecular Biophysics and Biochemistry group in SGC/TDI Oxford specialises on chemical probe discovery projects, especially in the field of Epigenetics. Main target areas are currently acetyl-lysine reader domains and histone demethylases. The group supports the wide variety of screening assays developed for most of the targets. The in-depth characterization of chemical probe candidates relies on a series of biophysical techniques established for accurate measurements of thermodynamics of and kinetic parameters of binding. The group also supports and extend the focused libraries of small molecular weight ligands for proteins of interest.

Name Department Institution Country
Professor Paul Brennan Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Dr Kilian Huber Structural Genomics Consortium Oxford University, NDM Research Building United Kingdom
Dr Brian D Marsden Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Dr Jon Elkins Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Moustakim M, Riedel K, Schuller M, Gehring AP, Monteiro OP, Martin SP, Fedorov O, Heer J, Dixon DJ, Elkins JM et al. 2018. Discovery of a novel allosteric inhibitor scaffold for polyadenosine-diphosphate-ribose polymerase 14 (PARP14) macrodomain 2. Bioorg Med Chem, 26 (11), pp. 2965-2972. | Show Abstract | Read more

The polyadenosine-diphosphate-ribose polymerase 14 (PARP14) has been implicated in DNA damage response pathways for homologous recombination. PARP14 contains three (ADP ribose binding) macrodomains (MD) whose exact contribution to overall PARP14 function in pathology remains unclear. A medium throughput screen led to the identification of N-(2(-9H-carbazol-1-yl)phenyl)acetamide (GeA-69, 1) as a novel allosteric PARP14 MD2 (second MD of PARP14) inhibitor. We herein report medicinal chemistry around this novel chemotype to afford a sub-micromolar PARP14 MD2 inhibitor. This chemical series provides a novel starting point for further development of PARP14 chemical probes.

Rahm F, Viklund J, Trésaugues L, Ellermann M, Giese A, Ericsson U, Forsblom R, Ginman T, Günther J, Hallberg K et al. 2018. Creation of a Novel Class of Potent and Selective MutT Homologue 1 (MTH1) Inhibitors Using Fragment-Based Screening and Structure-Based Drug Design. J Med Chem, 61 (6), pp. 2533-2551. | Show Abstract | Read more

Recent literature has both suggested and questioned MTH1 as a novel cancer target. BAY-707 was just published as a target validation small molecule probe for assessing the effects of pharmacological inhibition of MTH1 on tumor cell survival, both in vitro and in vivo. (1) In this report, we describe the medicinal chemistry program creating BAY-707, where fragment-based methods were used to develop a series of highly potent and selective MTH1 inhibitors. Using structure-based drug design and rational medicinal chemistry approaches, the potency was increased over 10,000 times from the fragment starting point while maintaining high ligand efficiency and drug-like properties.

Bulbrook D, Brazier H, Mahajan P, Kliszczak M, Fedorov O, Marchese FP, Aubareda A, Chalk R, Picaud S, Strain-Damerell C et al. 2018. Tryptophan-Mediated Interactions between Tristetraprolin and the CNOT9 Subunit Are Required for CCR4-NOT Deadenylase Complex Recruitment. J Mol Biol, 430 (5), pp. 722-736. | Show Abstract | Read more

The zinc-finger protein tristetraprolin (TTP) binds to AU-rich elements present in the 3' untranslated regions of transcripts that mainly encode proteins of the inflammatory response. TTP-bound mRNAs are targeted for destruction via recruitment of the eight-subunit deadenylase complex "carbon catabolite repressor protein 4 (CCR4)-negative on TATA-less (NOT)," which catalyzes the removal of mRNA poly-(A) tails, the first obligatory step in mRNA decay. Here we show that a novel interaction between TTP and the CCR4-NOT subunit, CNOT9, is required for recruitment of the deadenylase complex. In addition to CNOT1, CNOT9 is now included in the identified CCR4-NOT subunits shown to interact with TTP. We find that both the N- and C-terminal domains of TTP are involved in an interaction with CNOT9. Through a combination of SPOT peptide array, site-directed mutagenesis, and bio-layer interferometry, we identified several conserved tryptophan residues in TTP that serve as major sites of interaction with two tryptophan-binding pockets of CNOT9, previously found to interact with another modulator GW182. We further demonstrate that these interactions are also required for recruitment of the CCR4-NOT complex and TTP-directed decay of an mRNA containing an AU-rich element in its 3'-untranslated region. Together the results reveal new molecular details for the TTP-CNOT interaction that shape an emerging mechanism whereby TTP targets inflammatory mRNAs for deadenylation and decay.

Sansook S, Ocasio CA, Day IJ, Tizzard GJ, Coles SJ, Fedorov O, Bennett JM, Elkins JM, Spencer J. 2018. Correction: Synthesis of kinase inhibitors containing a pentafluorosulfanyl moiety. Org Biomol Chem, 16 (2), pp. 336. | Show Abstract | Read more

Correction for 'Synthesis of kinase inhibitors containing a pentafluorosulfanyl moiety' by Supojjanee Sansook et al., Org. Biomol. Chem., 2017, 15, 8655-8660.

Fernández-Montalván AE, Berger M, Kuropka B, Koo SJ, Badock V, Weiske J, Puetter V, Holton SJ, Stöckigt D, Ter Laak A et al. 2017. Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action. ACS Chem Biol, 12 (11), pp. 2730-2736. | Show Abstract | Read more

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.

Sansook S, Ocasio CA, Day IJ, Tizzard GJ, Coles SJ, Fedorov O, Bennett JM, Elkins JM, Spencer J. 2017. Synthesis of kinase inhibitors containing a pentafluorosulfanyl moiety ORGANIC & BIOMOLECULAR CHEMISTRY, 15 (40), pp. 8655-8660. | Read more

Sansook S, Ocasio CA, Day IJ, Tizzard GJ, Coles SJ, Fedorov O, Bennett JM, Elkins JM, Spencer J. 2017. Synthesis of kinase inhibitors containing a pentafluorosulfanyl moiety. Org Biomol Chem, 15 (40), pp. 8655-8660. | Show Abstract | Read more

A series of 3-methylidene-1H-indol-2(3H)-ones substituted with a 5- or 6-pentafluorosulfanyl group has been synthesized by a Knoevenagel condensation reaction of SF5-substituted oxindoles with a range of aldehydes. The resulting products were characterized by X-ray crystallography studies and were tested for biological activity versus a panel of cell lines and protein kinases. Some exhibited single digit nM activity.

Gerken PA, Wolstenhulme JR, Tumber A, Hatch SB, Zhang Y, Müller S, Chandler SA, Mair B, Li F, Nijman SMB et al. 2017. Discovery of a Highly Selective Cell-Active Inhibitor of the Histone Lysine Demethylases KDM2/7. Angew Chem Int Ed Engl, 56 (49), pp. 15555-15559. | Show Abstract | Read more

Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.

Meier JC, Tallant C, Fedorov O, Witwicka H, Hwang S-Y, van Stiphout RG, Lambert J-P, Rogers C, Yapp C, Gerstenberger BS et al. 2017. Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation. ACS Chem Biol, 12 (10), pp. 2619-2630. | Show Abstract | Read more

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.

Carr SM, Munro S, Sagum CA, Fedorov O, Bedford MT, La Thangue NB. 2017. Tudor-domain protein PHF20L1 reads lysine methylated retinoblastoma tumour suppressor protein. Cell Death Differ, 24 (12), pp. 2139-2149. | Show Abstract | Read more

The retinoblastoma tumour suppressor protein (pRb) classically functions to regulate early cell cycle progression where it acts to enforce a number of checkpoints in response to cellular stress and DNA damage. Methylation at lysine (K) 810, which occurs within a critical CDK phosphorylation site and antagonises a CDK-dependent phosphorylation event at the neighbouring S807 residue, acts to hold pRb in the hypo-phosphorylated growth-suppressing state. This is mediated in part by the recruitment of the reader protein 53BP1 to di-methylated K810, which allows pRb activity to be effectively integrated with the DNA damage response. Here, we report the surprising observation that an additional methylation-dependent interaction occurs at K810, but rather than the di-methyl mark, it is selective for the mono-methyl K810 mark. Binding of the mono-methyl PHF20L1 reader to methylated pRb occurs on E2F target genes, where it acts to mediate an additional level of control by recruiting the MOF acetyltransferase complex to E2F target genes. Significantly, we find that the interplay between PHF20L1 and mono-methyl pRb is important for maintaining the integrity of a pRb-dependent G1-S-phase checkpoint. Our results highlight the distinct roles that methyl-lysine readers have in regulating the biological activity of pRb.

Igoe N, Bayle ED, Tallant C, Fedorov O, Meier JC, Savitsky P, Rogers C, Morias Y, Scholze S, Boyd H et al. 2017. Design of a Chemical Probe for the Bromodomain and Plant Homeodomain Finger-Containing (BRPF) Family of Proteins. J Med Chem, 60 (16), pp. 6998-7011. | Show Abstract | Read more

The bromodomain and plant homeodomain finger-containing (BRPF) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Here, we describe NI-57 (16) as new pan-BRPF chemical probe of the bromodomain (BRD) of the BRPFs. Inhibitor 16 preferentially bound the BRD of BRPF1 and BRPF2 over BRPF3, whereas binding to BRD9 was weaker. Compound 16 has excellent selectivity over nonclass IV BRD proteins. Target engagement of BRPF1B and BRPF2 with 16 was demonstrated in nanoBRET and FRAP assays. The binding of 16 to BRPF1B was rationalized through an X-ray cocrystal structure determination, which showed a flipped binding orientation when compared to previous structures. We report studies that show 16 has functional activity in cellular assays by modulation of the phenotype at low micromolar concentrations in both cancer and inflammatory models. Pharmacokinetic data for 16 was generated in mouse with single dose administration showing favorable oral bioavailability.

Bouché L, Christ CD, Siegel S, Fernández-Montalván AE, Holton SJ, Fedorov O, Ter Laak A, Sugawara T, Stöckigt D, Tallant C et al. 2017. Benzoisoquinolinediones as Potent and Selective Inhibitors of BRPF2 and TAF1/TAF1L Bromodomains. J Med Chem, 60 (9), pp. 4002-4022. | Show Abstract | Read more

Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure-activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2- or TAF1-histone H3.3 or H4 interaction assay.

Santamaria S, Fedorov O, McCafferty J, Murphy G, Dudhia J, Nagase H, Yamamoto K. 2017. Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1. MAbs, 9 (4), pp. 595-602. | Show Abstract | Read more

The potent aggrecanase ADAMTS-5 is constitutively secreted by chondrocytes, but it is rapidly endocytosed in normal cartilage via the cell surface endocytic receptor LRP1. Therefore it is difficult to detect the total ADAMTS-5 activity produced. In this study, we isolated a monoclonal anti-ADAMTS-5 antibody 1B7 that blocks LRP1-mediated internalization without affecting the aggrecanolytic activity. Addition of 1B7 to cultured human chondrocytes revealed the full aggrecanolytic activity of ADAMTS-5 generated by the cells. 1B7 is a useful tool to estimate the ADAMTS-5 activity and to identify its potential roles in the tissues.

Tumber A, Nuzzi A, Hookway ES, Hatch SB, Velupillai S, Johansson C, Kawamura A, Savitsky P, Yapp C, Szykowska A et al. 2017. Potent and Selective KDM5 Inhibitor Stops Cellular Demethylation of H3K4me3 at Transcription Start Sites and Proliferation of MM1S Myeloma Cells. Cell Chem Biol, 24 (3), pp. 371-380. | Show Abstract | Read more

Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe2+-dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 μM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation.

Hatch SB, Yapp C, Montenegro RC, Savitsky P, Gamble V, Tumber A, Ruda GF, Bavetsias V, Fedorov O, Atrash B et al. 2017. Assessing histone demethylase inhibitors in cells: lessons learned. Epigenetics Chromatin, 10 (1), pp. 9. | Show Abstract | Read more

BACKGROUND: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active 'probe' molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells. RESULTS: A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition. CONCLUSIONS: High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.

Bataille CJR, Brennan MB, Byrne S, Davies SG, Durbin M, Fedorov O, Huber KVM, Jones AM, Knapp S, Liu G et al. 2017. Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family. Bioorg Med Chem, 25 (9), pp. 2657-2665. | Show Abstract | Read more

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75μM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.

Igoe N, Bayle ED, Fedorov O, Tallant C, Savitsky P, Rogers C, Owen DR, Deb G, Somervaille TCP, Andrews DM et al. 2017. Design of a Biased Potent Small Molecule Inhibitor of the Bromodomain and PHD Finger-Containing (BRPF) Proteins Suitable for Cellular and in Vivo Studies. J Med Chem, 60 (2), pp. 668-680. | Show Abstract | Read more

The BRPF (bromodomain and PHD finger-containing) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Evaluation of the BRPF family as a potential drug target is at an early stage although there is an emerging understanding of a role in acute myeloid leukemia (AML). We report the optimization of fragment hit 5b to 13-d as a biased, potent inhibitor of the BRD of the BRPFs with excellent selectivity over nonclass IV BRD proteins. Evaluation of 13-d in a panel of cancer cell lines showed a selective inhibition of proliferation of a subset of AML lines. Pharmacokinetic studies established that 13-d had properties compatible with oral dosing in mouse models of disease (Fpo 49%). We propose that NI-42 (13-d) is a new chemical probe for the BRPFs suitable for cellular and in vivo studies to explore the fundamental biology of these proteins.

Moustakim M, Clark PGK, Trulli L, Fuentes de Arriba AL, Ehebauer MT, Chaikuad A, Murphy EJ, Mendez-Johnson J, Daniels D, Hou C-FD et al. 2017. Discovery of a PCAF Bromodomain Chemical Probe. Angew Chem Int Ed Engl, 56 (3), pp. 827-831. | Show Abstract | Read more

The p300/CBP-associated factor (PCAF) and related GCN5 bromodomain-containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine-based L-45 (dubbed L-Moses) as the first potent, selective, and cell-active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)-(-)-norephedrine furnished L-45 in enantiopure form. L-45 was shown to disrupt PCAF-Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co-crystal structure of L-45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L-45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell-permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Olzscha H, Fedorov O, Kessler BM, Knapp S, La Thangue NB. 2017. CBP/p300 Bromodomains Regulate Amyloid-like Protein Aggregation upon Aberrant Lysine Acetylation. Cell Chem Biol, 24 (1), pp. 9-23. | Show Abstract | Read more

Lysine acetylation is becoming increasingly recognized as a general biological principle in cellular homeostasis, and is subject to abnormal control in different human pathologies. Here, we describe a global effect on amyloid-like protein aggregation in human cells that results from aberrant lysine acetylation. Bromodomain reader proteins are involved in the aggregation process and, using chemical biology and gene silencing, we establish that p300/CBP bromodomains are necessary for aggregation to occur. Moreover, protein aggregation disturbs proteostasis by impairing the ubiquitin proteasome system (UPS) and protein translation, resulting in decreased cell viability. p300/CBP bromodomain inhibitors impede aggregation, which coincides with enhanced UPS function and increased cell viability. Aggregation of a pathologically relevant form of huntingtin protein is similarly affected by p300/CBP inhibition. Our results have implications for understanding the molecular basis of protein aggregation, and highlight the possibility of treating amyloid-like pathologies and related protein folding diseases with bromodomain inhibitor-based strategies.

Picaud S, Leonards K, Lambert J-P, Dovey O, Wells C, Fedorov O, Monteiro O, Fujisawa T, Wang C-Y, 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.

Myrianthopoulos V, Gaboriaud-Kolar N, Tallant C, Hall M-L, 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).

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.

Moustakim M, Clark PGK, Hay DA, Dixon DJ, Brennan PE. 2016. Chemical probes and inhibitors of bromodomains outside the BET family. Medchemcomm, 7 (12), pp. 2246-2264. | Show Abstract | Read more

In the last five years, the development of inhibitors of bromodomains has emerged as an area of intensive worldwide research. Emerging evidence has implicated a number of non-BET bromodomains in the onset and progression of diseases such as cancer, HIV infection and inflammation. The development and use of small molecule chemical probes has been fundamental to pre-clinical evaluation of bromodomains as targets. Recent efforts are described highlighting the development of potent, selective and cell active non-BET bromodomain inhibitors and their therapeutic potential. Over half of typical bromodomains now have reported ligands, but those with atypical binding site residues remain resistant to chemical probe discovery efforts.

Montenegro RC, Clark PGK, 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.

Sdelci S, Lardeau C-H, 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.

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.

Martin LJ, Koegl M, Bader G, Cockcroft X-L, 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.

Cox OB, Krojer T, Collins P, Monteiro O, Talon R, Bradley A, Fedorov O, Amin J, Marsden BD, Spencer J et al. 2016. A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain. Chem Sci, 7 (3), pp. 2322-2330. | Show Abstract | Read more

Research into the chemical biology of bromodomains has been driven by the development of acetyl-lysine mimetics. The ligands are typically anchored by binding to a highly conserved asparagine residue. Atypical bromodomains, for which the asparagine is mutated, have thus far proven elusive targets, including PHIP(2) whose parent protein, PHIP, has been linked to disease progression in diabetes and cancers. The PHIP(2) binding site contains a threonine in place of asparagine, and solution screening have yielded no convincing hits. We have overcome this hurdle by combining the sensitivity of X-ray crystallography, used as the primary fragment screen, with a strategy for rapid follow-up synthesis using a chemically-poised fragment library, which allows hits to be readily modified by parallel chemistry both peripherally and in the core. Our approach yielded the first reported hit compounds of PHIP(2) with measurable IC50 values by an AlphaScreen competition assay. The follow-up libraries of four poised fragment hits improved potency into the sub-mM range while showing good ligand efficiency and detailed structural data.

Wagner T, Greschik H, Burgahn T, Schmidtkunz K, Schott A-K, McMillan J, Baranauskienė L, Xiong Y, Fedorov O, Jin J et al. 2016. Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform. Nucleic Acids Res, 44 (9), pp. e88. | Show Abstract | Read more

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein-protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.

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.

Bavetsias V, Lanigan RM, Ruda GF, Atrash B, McLaughlin MG, Tumber A, Mok NY, Le Bihan Y-V, Dempster S, Boxall KJ et al. 2016. 8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors. J Med Chem, 59 (4), pp. 1388-1409. | Show Abstract | Read more

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

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.

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.

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.

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.

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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.

Clark PGK, Vieira LCC, 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 PGK, Vieira LCC, 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.

Chen P, Chaikuad A, Bamborough P, Bantscheff M, Bountra C, Chung C-W, 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.

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.

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.

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),

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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

Troeberg L, Lazenbatt C, Anower-E-Khuda MF, Freeman C, Federov O, Habuchi H, Habuchi O, Kimata K, Nagase H. 2014. Sulfated glycosaminoglycans control the extracellular trafficking and the activity of the metalloprotease inhibitor TIMP-3. Chem Biol, 21 (10), pp. 1300-1309. | Show Abstract | Read more

Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.

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

Carr SM, Munro S, Zalmas L-P, Fedorov O, Johansson C, Krojer T, Sagum CA, Bedford MT, Oppermann U, La Thangue NB. 2014. Lysine methylation-dependent binding of 53BP1 to the pRb tumor suppressor. Proc Natl Acad Sci U S A, 111 (31), pp. 11341-11346. | Show Abstract | Read more

The retinoblastoma tumor suppressor protein pRb is a key regulator of cell cycle progression and mediator of the DNA damage response. Lysine methylation at K810, which occurs within a critical Cdk phosphorylation motif, holds pRb in the hypophosphorylated growth-suppressing state. We show here that methyl K810 is read by the tandem tudor domain containing tumor protein p53 binding protein 1 (53BP1). Structural elucidation of 53BP1 in complex with a methylated K810 pRb peptide emphasized the role of the 53BP1 tandem tudor domain in recognition of the methylated lysine and surrounding residues. Significantly, binding of 53BP1 to methyl K810 occurs on E2 promoter binding factor target genes and allows pRb activity to be effectively integrated with the DNA damage response. Our results widen the repertoire of cellular targets for 53BP1 and suggest a previously unidentified role for 53BP1 in regulating pRb tumor suppressor activity.

Ciceri P, Müller S, O'Mahony A, Fedorov O, Filippakopoulos P, Hunt JP, Lasater EA, Pallares G, Picaud S, Wells C et al. 2014. Corrigendum: Dual kinase-bromodomain inhibitors for rationally designed polypharmacology. Nat Chem Biol, 10 (8), pp. 692. | Read more

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.

Fedorov O. 2014. What is the future of bromodomains in targeted drug development? Future Med Chem, 6 (10), pp. 1101-1103. | Read more

Rooney TPC, 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.

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.

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 JRC, 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.

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.

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.

Zheng S, Moehlenbrink J, Lu Y-C, Zalmas L-P, Sagum CA, Carr S, McGouran JF, Alexander L, Fedorov O, Munro S et al. 2013. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1. Mol Cell, 52 (1), pp. 37-51. | Show Abstract | Read more

The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.

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.

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.

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.

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.

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Zhang W, Tyl M, Ward R, Sobott F, Maman J, Murthy AS, Watson AA, Fedorov O, Bowman A, Owen-Hughes T et al. 2013. Structural plasticity of histones H3-H4 facilitates their allosteric exchange between RbAp48 and ASF1 Nature Structural and Molecular Biology, 20 (1), pp. 29-35. | Show Abstract | Read more

The mechanisms by which histones are disassembled and reassembled into nucleosomes and chromatin structure during DNA replication, repair and transcription are poorly understood. A better understanding of the processes involved is, however, crucial if we are to understand whether and how histone variants and post-translationally modified histones are inherited in an epigenetic manner. To this end we have studied the interaction of the histone H3-H4 complex with the human retinoblastoma-associated protein RbAp48 and their exchange with a second histone chaperone, anti-silencing function protein 1 (ASF1). Exchange of histones H3-H4 between these two histone chaperones has a central role in the assembly of new nucleosomes, and we show here that the H3-H4 complex has an unexpected structural plasticity, which is important for this exchange. © 2013 Nature America, Inc. All rights reserved.

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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 Bioorganic and Medicinal Chemistry, 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. © 2013 Elsevier Ltd. All rights reserved.

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.

Kempen HJ, Bellus D, Fedorov O, Nicklisch S, Filippakopoulos P, Picaud S, Knapp S. 2013. Stimulation of Hepatic Apolipoprotein A-I Production by Novel Thieno-Triazolodiazepines: Roles of the Classical Benzodiazepine Receptor, PAF Receptor, and Bromodomain Binding. Lipid Insights, 6 pp. 47-54. | Show Abstract | Read more

Expression and secretion of apolipoprotein A-I (apoA-I) by cultured liver cells can be markedly stimulated by triazolodiazepines (TZDs). It has been shown previously that the thieno-TZD Ro 11-1464 increases plasma levels of apoA-I and in vivomacrophage reverse cholesterol transport in mice. However, these effects were only seen at high doses, at which the compound could act on central benzodiazepine (BZD) receptors or platelet activating factor (PAF) receptors, interfering with its potential utility. In this work, we describe 2 new thieno-TZDs MDCO-3770 and MDCO-3783, both derived from Ro 11-1464. These compounds display the same high efficacy on apoA-I production, metabolic stability, and lack of cytotoxicity in cultured hepatocytes as Ro 11-1464, but they do not bind to the central BZD receptor and PAF receptor. The quinazoline RVX-208 was less efficacious in stimulating apoA-I production and displayed signs of cytotoxicity. Certain TZDs stimulating apoA-I production are now known to be inhibitors of bromodomain (BRD) extra-terminal (BET) proteins BRDT, BRD2, BRD3, and BRD4, and this inhibition was inferred as a main molecular mechanism for their effect on apoA-I expression. We show here that the thieno-TZD (+)-JQ1, a potent BET inhibitor, strongly stimulated apoA-I production in Hep-G2 cells, but that its enantiomer (-)-JQ1, which has no BET inhibitor activity, also showed considerable effect on apoA-I production. MDCO-3770 and MDCO-3783 also inhibited BRD3 and BRD4 in vitro, with potency somewhat below that of (+)-JQ1. We conclude that the effect of thieno-TZDs on apoA-I expression is not due to inhibition of the BZD or PAF receptors and is not completely explained by transcriptional repression by BET proteins.

Zhang W, Tyl M, Ward R, Sobott F, Maman J, Murthy AS, Watson AA, Fedorov O, Bowman A, Owen-Hughes T et al. 2013. Structural plasticity of histones H3-H4 facilitates their allosteric exchange between RbAp48 and ASF1. Nat Struct Mol Biol, 20 (1), pp. 29-35. | Show Abstract | Read more

The mechanisms by which histones are disassembled and reassembled into nucleosomes and chromatin structure during DNA replication, repair and transcription are poorly understood. A better understanding of the processes involved is, however, crucial if we are to understand whether and how histone variants and post-translationally modified histones are inherited in an epigenetic manner. To this end we have studied the interaction of the histone H3-H4 complex with the human retinoblastoma-associated protein RbAp48 and their exchange with a second histone chaperone, anti-silencing function protein 1 (ASF1). Exchange of histones H3-H4 between these two histone chaperones has a central role in the assembly of new nucleosomes, and we show here that the H3-H4 complex has an unexpected structural plasticity, which is important for this exchange.

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.

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.

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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.

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.

Artz JD, Wernimont AK, Allali-Hassani A, Zhao Y, Amani M, Lin Y-H, Senisterra G, Wasney GA, Fedorov O, King O et al. 2011. The Cryptosporidium parvum kinome. BMC Genomics, 12 (1), pp. 478. | Show Abstract | Read more

BACKGROUND: Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase. RESULTS: The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀ values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation. CONCLUSIONS: Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.

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.

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.

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.

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.

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

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

Record CJ, Chaikuad A, Rellos P, Das S, Pike ACW, 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.

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.

Elkins JM, Amos A, Niesen FH, Pike ACW, 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.

Soundararajan M, Turnbull A, Fedorov O, Johansson C, Doyle DA. 2008. RhoB can adopt a Mg2+ free conformation prior to GEF binding. Proteins, 72 (1), pp. 498-505. | Read more

Soundararajan M, Willard FS, Kimple AJ, Turnbull AP, Ball LJ, Schoch GA, Gileadi C, Fedorov OY, Dowler EF, Higman VA et al. 2008. Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits. Proc Natl Acad Sci U S A, 105 (17), pp. 6457-6462. | Show Abstract | Read more

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.

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.

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.

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.

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 D-M, 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.

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.

Fedorov OY, Higman VA, Schmieder P, Leidert M, Diehl A, Elkins J, Soundararajan M, Oschkinat H, Ball LJ. 2007. Resonance assignment of the RGS domain of human RGS10. J Biomol NMR, 38 (2), pp. 191. | Read more

Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ, Wasney GA, Yeung R, Arrowsmith C, Ball LJ, Berglund H et al. 2006. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci U S A, 103 (43), pp. 15835-15840. | Show Abstract | Read more

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.

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.

Fedoroff OY, Townson SA, Golovanov AP, Baron M, Avis JM. 2004. The structure and dynamics of tandem WW domains in a negative regulator of notch signaling, Suppressor of deltex. J Biol Chem, 279 (33), pp. 34991-35000. | Show Abstract | Read more

WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.

Duan W, Rangan A, Vankayalapati H, Kim MY, Zeng Q, Sun D, Han H, Fedoroff OY, Nishioka D, Rha SY et al. 2001. Design and synthesis of fluoroquinophenoxazines that interact with human telomeric G-quadruplexes and their biological effects. Mol Cancer Ther, 1 (2), pp. 103-120. | Show Abstract

In this study we have identified a new structural motif for a ligand with G-quadruplex interaction that results in biological effects associated with G-quadruplex-interactive compounds. Fluoroquinolones have been reported to possess weak telomerase inhibitory activity in addition to their better known bacterial gyrase poisoning. Starting with a fluoroquinobenzoxazine, which has modest potency in a human topoisomerase II assay, we have designed a more potent inhibitor of telomerase that has lost its topoisomerase II poisoning activity. This fluoroquinophenoxazine (FQP) interacts with G-quadruplex structures to inhibit the progression of Taq polymerase in a G-quadruplex polymerase stop assay. In addition, we demonstrate by 1H NMR studies that this compound interacts with telomeric G-quadruplex structures by external stacking to the G-tetrad with both the unimolecular fold-over and the parallel G-quadruplex structures. A photocleavage assay confirms the FQP interaction site, which is located off center of the external tetrad but within the loop region. Molecular modeling using simulated annealing was performed on the FQP-parallel G-quadruplex complex to determine the optimum FQP orientation and key molecular interactions with the telomeric G-quadruplex structure. On the basis of the results of these studies, two additional FQP analogues were synthesized, which were designed to test the importance of these key interactions. These analogues were evaluated in the Taq polymerase stop assay for G-quadruplex interaction. The data from this study and the biological evaluation of these three FQPs, using cytotoxicity and a sea urchin embryo system, were in accord with the predicted more potent telomeric G-quadruplex interactions of the initial lead compound and one of the analogues. On the basis of these structural and biological studies, the design of more potent and selective telomeric G-quadruplex-interactive compounds can be envisaged.

Rangan A, Fedoroff OY, Hurley LH. 2001. Induction of duplex to G-quadruplex transition in the c-myc promoter region by a small molecule. J Biol Chem, 276 (7), pp. 4640-4646. | Show Abstract | Read more

A major control element of the human c-myc oncogene is the nuclease-hypersensitive purine/pyrimidine-rich sequence. This double-stranded DNA fragment, corresponding to the 27-base pair segment in the nuclease-hypersensitive element of the c-myc promoter region, forms a stable Watson-Crick double helix under physiological conditions. However, this duplex DNA can be effectively converted to G-quadruplex DNA by a small molecular weight ligand. Both intermolecular and intramolecular G-quadruplex forms can be induced by this ligand. Similar transitional changes are also observed with the duplex telomeric sequence from the Oxytricha species. These results provide additional support to the idea that G-quadruplex structures may play structural roles in vivo and also provide insight into novel methodologies for rational drug design. These structurally altered DNA elements might serve as regulatory signals in gene expression or in telomere dynamics and hence are promising targets for drug action.

Fedoroff OY, Rangan A, Chemeris VV, Hurley LH. 2000. Cationic porphyrins promote the formation of i-motif DNA and bind peripherally by a nonintercalative mechanism. Biochemistry, 39 (49), pp. 15083-15090. | Show Abstract | Read more

Telomeric C-rich strands can form a noncanonical intercalated DNA structure known as an i-motif. We have studied the interactions of the cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) with the i-motif forms of several oligonucleotides containing telomeric sequences. TMPyP4 was found to promote the formation of the i-motif DNA structure. On the basis of (1)H NMR studies, we have created a model of the i-motif-TMPyP4 complex that is consistent with all the available experimental data. Two-dimensional NOESY data prompted us to conclude that TMPyP4 binds specifically to the edge of the intercalated DNA core by a nonintercalative mechanism. Since we have shown that TMPyP4 binds to and stabilizes the G-quadruplex form of the complementary G-rich telomeric strand, this study raises the intriguing possibility that TMPyP4 can trigger the formation of unusual DNA structures in both strands of the telomeres, which may in turn explain the recently documented biological effects of TMPyP4 in cancer cells.

Fedoroff OY, Salazar M, Han H, Chemeris VV, Kerwin SM, Hurley LH. 1998. NMR-Based model of a telomerase-inhibiting compound bound to G-quadruplex DNA. Biochemistry, 37 (36), pp. 12367-12374. | Show Abstract | Read more

The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

Cerritelli SM, Fedoroff OY, Reid BR, Crouch RJ. 1998. A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs. Nucleic Acids Res, 26 (7), pp. 1834-1840. | Show Abstract | Read more

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.

Fedoroff OY, Ge Y, Reid BR. 1997. Solution structure of r(gaggacug):d(CAGTCCTC) hybrid: implications for the initiation of HIV-1 (+)-strand synthesis. J Mol Biol, 269 (2), pp. 225-239. | Show Abstract | Read more

The three-dimensional solution structure of the hybrid duplex r(gaggacug):d(CAGTCCTC) has been determined by two-dimensional NMR, distance geometry (DG), restrained molecular dynamics (rMD) and NOE back-calculation methods. This hybrid, consisting of a purine-rich RNA strand and a pyrimidine-rich DNA strand, is related to the polypurine (+)-strand primer formed after (-)-strand DNA synthesis and RNase H degradation of the viral RNA strand and contains the site of a specific cleavage by reverse transcription (RT) RNase H at the end of the HIV-1 polypurine tract. This polypurine primer is an important intermediate in the formation of virally encoded double-stranded DNA prior to HIV-1 retrovirus integration. The correct processing of this primer is vital in the life cycle of the human immunodeficiency virus type (HIV-1) retrovirus. The structure of the r(gaggacug):d(CAGTCCTC) hybrid, as determined in solution by NMR, is intermediate between canonical A-type and B-type double helices, and has mixed structural characteristics. It is quantitatively different from the previously determined solution structures of other RNA-DNA hybrids, particularly in the width and shape of the major groove, which is wider than the major groove of other hybrids and is close to the dimension of the major groove of B-type DNA duplexes. The structure of this hybrid duplex contains a prominent bend in the double helix with a magnitude and direction similar to the bend in Okazaki fragments. The structural features of the present duplex may explain the unique interactions of this sequence with HIV-1 RT during both (-)-strand and (+)-strand DNA synthesis.

Fedoroff OYu, Salazar M, Reid BR. 1996. Structural variation among retroviral primer-DNA junctions: solution structure of the HIV-1 (-)-strand Okazaki fragment r(gcca)d(CTGC).d(GCAGTGGC). Biochemistry, 35 (34), pp. 11070-11080. | Show Abstract | Read more

The three-dimensional solution structure of the hybrid-chimeric duplex r(gcca)d(CTGC).d(GCAGTGGC) has been determined by two-dimensional NMR, restrained molecular dynamics (rMD), and NOE back-calculation methods. This chimera, consisting of a chimeric RNA-DNA strand and its complementary DNA strand, is formed after priming (-)-strand DNA synthesis by tRNA(Lys3) and subsequent (+)-strand DNA synthesis by reverse transcriptase and is an obligatory intermediate in the formation of double-stranded DNA prior to HIV-1 retrovirus integration. The duplex consists of two different types of double helix: a hybrid form (H-form) and a B-form structure connected by a junction. It is chemically similar to several other Okazaki fragments whose structures have been previously determined in our laboratory. However, some structural parameters are not the same and were found to be sequence dependent. In particular, the sugar conformations at the DNA base pair proximal to the hybrid segment vary from O4'-endo to C2'-endo depending on the base composition. The position of the transition from the relatively wide groove of H-form to the narrow groove of B-form is also sequence dependent, occurring either exactly at the RNA-DNA junction or within the purely DNA segment of the chimera-as is the case in the structure of the present HIV-1 (-)-strand primer. This structural change produces a kink at the DNA-DNA step adjacent to the RNA-DNA junction in the HIV-1 (-)-strand primer. The sequence dependence of structures of RNA-DNA chimeric duplexes may be responsible for the variable cleavage pattern of different Okazaki fragments by reverse transcriptase RNase H.

Salazar M, Fedoroff OY, Reid BR. 1996. Structure of chimeric duplex junctions: solution conformation of the retroviral Okazaki-like fragment r(ccca)d(AATGA).d(TCATTTGGG) from Moloney murine leukemia virus. Biochemistry, 35 (25), pp. 8126-8135. | Show Abstract | Read more

We have determined the solution structure of the synthetic chimeric duplex r(ccca)d(AATGA).d(TCATTTGGG) by two-dimensional NMR, distance geometry, restrained molecular dynamics, and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The chimeric strand of this duplex consists of the last four residues of the tRNA(Pro) primer for (-) strand DNA synthesis of Moloney murine leukemia virus and the first five residues of the (-) strand DNA produced by extending this primer; the complementary DNA strand corresponds to the (+) strand product from this template. The hybrid section of this chimeric duplex assumes a structure similar to that found for pure hybrid duplexes of mixed sequence, while the DNA section assumes a conformation closer to B-form DNA. There is significant distortion of the duplex at the hybrid-DNA junction which is manifested in marked changes in the helical parameters buckle, roll, and tip, changes in glycosidic torsion angles, and changes in the backbone torsion angles delta, epsilon, and zeta. The sugar conformations also undergo large changes, from heteromerous puckers in the hybrid section to a more B-form in the DNA section. Furthermore, the intrastrand phosphate separation in the chimeric strand is more typical of A-form duplexes in the RNA section but more like B-form duplexes in the DNA section. In the DNA section the minor groove width changes gradually from B-form at the periphery and approaches hybrid-like dimensions closer to the junction. The structural discontinuities act synergistically to produce a bend of 18 +/- 3 degrees at the junction. The global structure of this sequence is similar to that previously found in the chemically analogous Okazaki fragment r(gcg)d(TATACCC).d(GGGTATACGC) in solution. Such structure homology suggests a possible link between structure and function with respect to the recognition and cleavage of the junction RNA residues in both retroviral chimeras and Okazaki fragments during reverse transcription and normal DNA replication.

Salazar M, Fedoroff OYu, Zhu L, Reid BR. 1994. The solution structure of the r(gcg)d(TATACCC):d(GGGTATACGC) Okazaki fragment contains two distinct duplex morphologies connected by a junction. J Mol Biol, 241 (3), pp. 440-455. | Show Abstract | Read more

Okazaki fragments are important intermediates in DNA replication. Chimeric duplexes that are structurally equivalent to Okazaki fragments also occur during reverse transcription of RNA retroviruses. Such duplexes consist of an RNA-DNA chimeric strand base-paired to a pure DNA strand; hence they have a hybrid duplex "left half" covalently linked to a "right half" that is pure DNA. We have determined the solution structure of the synthetic Okazaki fragment r(gcg)d(TATACCC):d(GGGTATACGC) by means of two-dimensional NMR, restrained molecular dynamics and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The large negative x-displacement and large positive inclination in the hybrid section of the duplex are structural characteristics similar to those found in pure hybrid duplexes. However, the DNA sugar puckers and the width and depth of the minor groove in the pure DNA section are more like B-form DNA, especially beyond the junction. Thus, this Okazaki fragment duplex assumes a conformation in solution that is a chimeric mixture of hybrid-form (H-form) and B-form structures and the overall molecule cannot be classified as either an A-form or a B-form duplex. The co-existence of these two different conformations in a single duplex gives rise to a structural discontinuity with a bend of approximately 18.1 (+/- 0.4) degrees at the junction between the hybrid and DNA segments that may be important for reverse transcriptase binding and RNase H cleavage of such molecules. Despite the fact that the solution structure is quite different from the all A-form structure reported recently for the exact same molecule in the crystalline state, a surprising number of local helical parameters were found to be quite similar to those reported for the crystal structure.

Chou SH, Cheng JW, Fedoroff O, Reid BR. 1994. DNA sequence GCGAATGAGC containing the human centromere core sequence GAAT forms a self-complementary duplex with sheared G.A pairs in solution. J Mol Biol, 241 (3), pp. 467-479. | Show Abstract | Read more

The DNA sequence dGCGAATGAGC has a well-resolved, two-dimensional nuclear Overhauser (NOESY) spectrum that is suitable for high quality solution structure determination by NMR methods; in solution this sequence forms a stable self-complementary duplex containing sheared G.A base-pairs. A total of 220 distance constraints derived from time-dependent NOE measurements were collected and refined by repeated back-calculation of the NOESY spectra. Distance information from imino proton studies and from exclusive two-dimensional correlated spectroscopy (E. COSY) and/or linewidth analysis was included in the structure calculation using the program DSPACE 4.2, followed by restrained energy minimization with the program DISCOVER using the AMBER force field. The energies of the distance geometry (DG) structures decreased rapidly in the first few cycles and approached -510 +/- 3 kcal after 1000 cycles of conjugate gradient minimization (about 540 kcal lower than in the initial DG structures). All 15 final DG structures converged to a single family of closely related structures with pair-wise r.m.s.d. values of 0.96 +/- 0.34 A, which was further reduced by energy minimization to 0.70 +/- 0.35 A. Rather unusual structural features of the duplex are revealed in the final structures. The results indicate that, in addition to normal sequences with standard base-pairing, unusual nucleic acid structures can also be determined in solution with quite high precision by NMR/distance geometry methods.

Olzscha H, Fedorov O, Kessler BM, Knapp S, La Thangue NB. 2017. CBP/p300 Bromodomains Regulate Amyloid-like Protein Aggregation upon Aberrant Lysine Acetylation. Cell Chem Biol, 24 (1), pp. 9-23. | Show Abstract | Read more

Lysine acetylation is becoming increasingly recognized as a general biological principle in cellular homeostasis, and is subject to abnormal control in different human pathologies. Here, we describe a global effect on amyloid-like protein aggregation in human cells that results from aberrant lysine acetylation. Bromodomain reader proteins are involved in the aggregation process and, using chemical biology and gene silencing, we establish that p300/CBP bromodomains are necessary for aggregation to occur. Moreover, protein aggregation disturbs proteostasis by impairing the ubiquitin proteasome system (UPS) and protein translation, resulting in decreased cell viability. p300/CBP bromodomain inhibitors impede aggregation, which coincides with enhanced UPS function and increased cell viability. Aggregation of a pathologically relevant form of huntingtin protein is similarly affected by p300/CBP inhibition. Our results have implications for understanding the molecular basis of protein aggregation, and highlight the possibility of treating amyloid-like pathologies and related protein folding diseases with bromodomain inhibitor-based strategies.

Sdelci S, Lardeau C-H, 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.

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.

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.

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.

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.

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.

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

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.

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.

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).

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.

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