Prof Stefan Knapp

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

Name Department Institution Country
Prof Benjamin Turk Department of Pharmacology Yale University School of Medicine United States
Prof Laurent Meijer ManRos Therapeutics France
Prof Andrew L Hopkins Division of Biological Chemistry and Drug Discovery University of Dundee United Kingdom
Prof Franz Bracher Department of Pharmacy Ludwig-Maximilians-Universität München Germany
Prof James E. Bradner Dana-Farber Cancer Institute Harvard Medical School United States
Prof Juerg Schwaller Deartment of Haematology Basel University Hospital Switzerland
Prof Nathanael S. Gray Dana Farber Cancer Institute Harvard Medical School United States
Prof Jonathan Morris School of Chemistry University of New South Wales Australia
Prof Andrew Holland Department of Molecular Biology & Genetics Johns Hopkins University School of Medicine United States
Prof Friedrich W. Herberg Biochemistry University of Kassel Germany
Prof Asko Uri Chemistry University of Tartu Estonia
Prof Giulio Superti-Furga Research Center for Molecular Medicine Austria
Prof David Bates University of Bristol United Kingdom
Prof Paul Fish UCL School of Pharmacy United States
Prof Cheryl Arrowsmith University of Toronto Canada
Dr Swen Hoelder Institute of Cancer Research United Kingdom
Prof Tatjana Stankovic University of Birmingham United Kingdom
Dr Alex Bullock Structural Genomics Consortium University of Oxford United Kingdom
Dr Panagis Filippakopoulos Structural Genomics Consortium University of Oxford United Kingdom
Dr Frank von Delft Structural Genomics Consortium University of Oxford United Kingdom
Prof Xin Lu Oxford Ludwig Institute University of Oxford United Kingdom
Dr Stephan Feller (RDM) University of Oxford United Kingdom
Dr Paul Brennan Structural Genomics Consortium University of Oxford United Kingdom
Dr Susanne Muller-Knapp Structural Genomics Consortium University of Oxford United Kingdom
Prof Benedikt M Kessler Target Discovery Institute University of Oxford United Kingdom
Daniel Ebner Target Discovery Institute University of Oxford United Kingdom
Dr Stuart Conway Department of Chemistry University of Oxford United Kingdom

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

Fish PV, Filippakopoulos P, Bish G, Brennan PE, Bunnage ME, Cook AS, Federov O, Gerstenberger BS 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. Read 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. Hide abstract

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

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

Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T et al. 2010. Selective inhibition of BET bromodomains. Nature, 468 (7327), pp. 1067-1073. Read 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. Hide abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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. Read 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. Hide abstract

Modular Role of the ET Domain of BET proteins in Transcription

Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT in human) belong to the class of bromodomains (BRDs), the only known protein module that selectively recognizes acetylated lysine residues found on histones and other diverse proteins (Filippakopoulos and Knapp, 2012; Filippakopoulos et al., 2012; Muller et al., 2011). BETs contain two conserved N-terminal), an extra terminal domain (ET) and a more divergent C-terminal recruitment domain (CT motif or CTM). They stimulate ...

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Role of the NUT and its Fusion to BET Proteins in Cancer

Proteins of the bromo and extra terminal (BET) family are key mediators for the initiation of transcription elongation (Bres et al., 2008). Chromosomal translocations that involve BET proteins have been identified in an aggressive form of human squamous carcinoma (French et al., 2003). In these translocations the two N-terminal BET bromodomains are fused in-frame with the NUT (nuclear protein in testis) protein giving rise to the so-called NUT midline carcinoma (NMC). Functional studies in ...

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Structural Role of BET proteins in Transcription Initiation

Proteins of the bromo and extra terminal (BET) family are key mediators for the assembly of the positive transcription elongation factor b complex (P-TEFb), an event required for the initiation of transcription elongation (Bres et al., 2008). The P-TEFb core complex is composed of cyclin-dependent kinase-9 (CDK9) and its activator cyclin T, and it functions to activate RNA polymerase II (RNAPol-II). BET proteins (BRD2, BRD3, BRD4 and BRDT in human) contain two conserved N-terminal bromodomains ...

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