Chemical Biology Publications 2014

Wang J, Knapp S, Pyne NJ, Pyne S, Elkins JM. 2014. Crystal Structure of Sphingosine Kinase 1 with PF-543. ACS Med Chem Lett, 5 (12), pp. 1329-1333. | Citations: 32 (Scopus) | Show Abstract | Read more

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

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

Chaikuad A, Petros AM, Fedorov O, Xu J, Knapp S. 2014. Structure-based approaches towards identification of fragments for the low-druggability ATAD2 bromodomain MEDCHEMCOMM, 5 (12), pp. 1843-1848. | Citations: 33 (Scopus) | Show Abstract | Read more

<p>Fragments for the development of ATAD2 bromodomain inhibitors have been identified and characterized by NMR and co-crystallization.</p>

Abdul Azeez KR, Knapp S, Fernandes JMP, Klussmann E, Elkins JM. 2014. The crystal structure of the RhoA-AKAP-Lbc DH-PH domain complex. Biochem J, 464 (2), pp. 231-239. | Citations: 12 (Web of Science Lite) | Show Abstract | Read more

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

Chaikuad A, Tacconi EMC, Zimmer J, Liang Y, Gray NS, Tarsounas M, Knapp S. 2014. A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics. Nat Chem Biol, 10 (10), pp. 853-860. | Citations: 77 (Web of Science Lite) | Show Abstract | Read more

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

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

Knapp S, Sundström M. 2014. Recently targeted kinases and their inhibitors-the path to clinical trials. Curr Opin Pharmacol, 17 (1), pp. 58-63. | Citations: 30 (Scopus) | Show Abstract | Read more

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

Knapp S, Sundstrom M. 2014. Recently targeted kinases and their inhibitors - the path to clinical trials CURRENT OPINION IN PHARMACOLOGY, 17 pp. 58-63. | Citations: 29 (Web of Science Lite) | Read more

Nagarajan S, Hossan T, Alawi M, Najafova Z, Indenbirken D, Bedi U, Taipaleenmäki H, Ben-Batalla I, Scheller M, Loges S et al. 2014. Bromodomain Protein BRD4 Is Required for Estrogen Receptor-Dependent Enhancer Activation and Gene Transcription Cell Reports, 8 (2), pp. 460-469. | Citations: 68 (Scopus) | Show Abstract | Read more

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

Philpott M, Rogers CM, Yapp C, Wells C, Lambert J-P, Strain-Damerell C, Burgess-Brown NA, Gingras A-C, Knapp S, Müller S. 2014. Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching. Epigenetics Chromatin, 7 (1), pp. 14. | Citations: 40 (Scopus) | Show Abstract | Read more

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

Nagarajan S, Hossan T, Alawi M, Najafova Z, Indenbirken D, Bedi U, Taipaleenmäki H, Ben-Batalla I, Scheller M, Loges S et al. 2014. Bromodomain protein BRD4 is required for estrogen receptor-dependent enhancer activation and gene transcription. Cell Rep, 8 (2), pp. 460-469. | Citations: 63 (Web of Science Lite) | Show Abstract | Read more

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

Zhao Z, Wu H, Wang L, Liu Y, Knapp S, Liu Q, Gray NS. 2014. Exploration of type II binding mode: A privileged approach for kinase inhibitor focused drug discovery? ACS Chem Biol, 9 (6), pp. 1230-1241. | Citations: 159 (Scopus) | Show Abstract | Read more

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

Hay DA, Fedorov O, Martin S, Singleton DC, Tallant C, Wells C, Picaud S, Philpott M, Monteiro OP, Rogers CM et al. 2014. Discovery and optimization of small-molecule ligands for the CBP/p300 bromodomains. J Am Chem Soc, 136 (26), pp. 9308-9319. | Citations: 130 (Scopus) | 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.

Rudolf AF, Skovgaard T, Knapp S, Jensen LJ, Berthelsen J. 2014. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination. PLoS One, 9 (6), pp. e98800. | Citations: 23 (Scopus) | Show Abstract | Read more

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

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. | Citations: 63 (Scopus) | Show Abstract | Read more

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

Filippakopoulos P, Knapp S. 2014. Targeting bromodomains: epigenetic readers of lysine acetylation. Nat Rev Drug Discov, 13 (5), pp. 337-356. | Citations: 525 (Scopus) | Show Abstract | Read more

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

Maiolica A, de Medina-Redondo M, Schoof EM, Chaikuad A, Villa F, Gatti M, Jeganathan S, Lou HJ, Novy K, Hauri S et al. 2014. Modulation of the chromatin phosphoproteome by the Haspin protein kinase. Mol Cell Proteomics, 13 (7), pp. 1724-1740. | Citations: 17 (Web of Science Lite) | Show Abstract | Read more

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

Brady DC, Crowe MS, Turski ML, Hobbs GA, Yao X, Chaikuad A, Knapp S, Xiao K, Campbell SL, Thiele DJ, Counter CM. 2014. Copper is required for oncogenic BRAF signalling and tumorigenesis. Nature, 509 (7501), pp. 492-496. | Citations: 153 (Scopus) | Show Abstract | Read more

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

Huber KVM, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A, Jemth A-S, Göktürk C, Sanjiv K, Strömberg K et al. 2014. Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature, 508 (7495), pp. 222-227. | Citations: 189 (Scopus) | Show Abstract | Read more

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

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. | Citations: 26 (Scopus) | Show Abstract | Read more

<p>Novel technologies were developed and used for the synthesis and evaluation of new triazolopyridazine BRD9 inhibitors.</p>

Cowan-Jacob SW, Jahnke W, Knapp S. 2014. Novel approaches for targeting kinases: allosteric inhibition, allosteric activation and pseudokinases. Future Med Chem, 6 (5), pp. 541-561. | Citations: 45 (Scopus) | Show Abstract | Read more

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

Ekambaram R, Enkvist E, Manoharan GB, Ugandi M, Kasari M, Viht K, Knapp S, Issinger O-G, Uri A. 2014. Benzoselenadiazole-based responsive long-lifetime photoluminescent probes for protein kinases. Chem Commun (Camb), 50 (31), pp. 4096-4098. | Citations: 10 (Scopus) | Show Abstract | Read more

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

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. | Citations: 160 (Scopus) | 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.

Mueller S, Knapp S. 2014. Discovery of BET bromodomain inhibitors and their role in target validation MEDCHEMCOMM, 5 (3), pp. 288-296. | Citations: 27 (Web of Science Lite) | Show Abstract | Read more

<p>Publicly available bromodomain inhibitors led to discoveries of key functions of BET-proteins in disease and development of new therapeutic strategies.</p>

Davis MI, Lea W, Simeonov A, Auld D. 2014. Literature Search and Review ASSAY and Drug Development Technologies, 12 (2), pp. 101-109. | Read more

Liu S, Knapp S, Ahmed AA. 2014. The structural basis of PI3K cancer mutations: from mechanism to therapy. Cancer Res, 74 (3), pp. 641-646. | Citations: 29 (Scopus) | Show Abstract | Read more

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

Selner NG, Luechapanichkul R, Chen X, Neel BG, Zhang Z-Y, Knapp S, Bell CE, Pei D. 2014. Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases. Biochemistry, 53 (2), pp. 397-412. | Citations: 12 (Scopus) | Show Abstract | Read more

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

Chaikuad A, Keates T, Vincke C, Kaufholz M, Zenn M, Zimmermann B, Gutiérrez C, Zhang R-G, Hatzos-Skintges C, Joachimiak A et al. 2014. Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies. Biochem J, 459 (1), pp. 59-69. | Citations: 22 (Scopus) | Show Abstract | Read more

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


Costa L, Casimiro S, Gupta S, Knapp S, Pillai MR, Toi M, Badwe R, Carmo-Fonseca M, Kumar R. 2014. The global cancer genomics consortium’s third annual symposium: From oncogenomics to cancer care Genes and Cancer, 5 (3-4), pp. 64-70. | Citations: 1 (Scopus) | Show Abstract

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

Chen C, Ha BH, Thévenin AF, Lou HJ, Zhang R, Yip KY, Peterson JR, Gerstein M, Kim PM, Filippakopoulos P et al. 2014. Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity. Mol Cell, 53 (1), pp. 140-147. | Citations: 37 (Web of Science Lite) | Show Abstract | Read more

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

Fedorov O, Lingard H, Wells C, Monteiro OP, Picaud S, Keates T, Yapp C, Philpott M, Martin SJ, Felletar I et al. 2014. [1,2,4]triazolo[4,3-a]phthalazines: inhibitors of diverse bromodomains. J Med Chem, 57 (2), pp. 462-476. | Citations: 52 (Scopus) | 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.

van Ameijde J, Overvoorde J, Knapp S, den Hertog J, Ruijtenbeek R, Liskamp RMJ. 2014. A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates. Anal Biochem, 448 (1), pp. 9-13. | Citations: 6 (Scopus) | Show Abstract | Read more

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

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