An Advanced Tool To Interrogate BRD9

Viewpoint pubs.acs.org/jmc An Advanced Tool To Interrogate BRD9 Rezaul M. Karim‡,† and Ernst Schönbrunn*,†,‡ † Drug Discovery Department, Moffitt Cancer Center, Tampa, Florida 33612, United States Department of Molecular Medicine, University of South Florida, Tampa, Florida 33612, United States Downloaded via 54.161.69.107 on June 17, 2020 at 13:03:56 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ‡ ABSTRACT: Selective inhibitors of bromodomain-containing protein 9 (BRD9) may have therapeutic potential in the treatment of human malignancies and inflammatory diseases. A selective small molecule inhibitor that is well tolerated and has proper pharmacokinetic properties is required to explore the function of BRD9 in diseases. BI-9564 (2) is a cell permeable and noncytotoxic BRD9 inhibitor provided to the scientific community to explore BRD9 biology and determine its potential as a drug target. B bromodomains outside the BET family, the relevance of these proteins in certain disease states, and their potential as drug targets. In this issue of Journal of Medicinal Chemistry, Martin et al. report the development of a selective, in vivo active BRD9 inhibitor with promising potential as a chemical probe.4 BRD9 is a subunit of the mammalian SWI/SNF chromatin remodeling complex, but its biological function is unknown. High prevalence of mutations of SWI/SNF subunits in human tumors5 and overexpression of BRD9 in several cancers suggest BRD9 as a potential target for cancer drugs.6 Martin et al. describe the structure-guided development of 2 (BI-9564), from hit compounds identified by fragment based and virtual screening to the design of potent BRD9 inhibitors. Various methods and techniques were applied to demonstrate binding activity and to determine the structural basis of inhibition, target engagement in the cell, and target selectivity through profiling against panels of bromodomains, kinases, and GPCRs. romodomains are highly conserved epigenetic “reader” modules that specifically recognize N-acetylated lysine (KAc) residues on histones and other proteins. Bromodomaincontaining proteins modulate numerous functions including gene transcription and chromatin remodeling and play fundamental roles in cell proliferation and division. A number of BRD-containing proteins, particularly those of the bromodomain and extraterminal (BET) family, have been linked to tumorigenesis and inflammatory diseases. The landmark discoveries of potent small molecule inhibitors of BET bromodomains1,2 provided for the first time chemical tools to explore the function of proteins such as BRD4 in disease states. Since then, several BET inhibitors have entered clinical trials for oncology and cardiovascular indications. More recently, novel inhibitors targeting non-BET bromodomains for which the physiological functions are not well understood have been reported.3 Such inhibitors are valuable probes to unravel the function of Figure 1. Structures of inhibitors 1 and 2 in comparison with previously reported BRD9 inhibitors. The atoms labeled magenta directly interact with Asn100. The blue highlighted methoxy moiety in 1 contributes to increased selectivity for BRD9 over BRD7. Received: April 11, 2016 Published: April 27, 2016 © 2016 American Chemical Society 4459 DOI: 10.1021/acs.jmedchem.6b00550 J. Med. Chem. 2016, 59, 4459−4461 Journal of Medicinal Chemistry Viewpoint Figure 2. Cocrystal structures reveal a subsite critical to the selectivity of inhibitors for BRD9. (A, B) Detailed interaction patterns of the isomers 2 and 1 in the KAc site of BRD9, respectively. Black dotted lines indicate H-bonding, green dotted lines VDW and π interactions, and cyan spheres are water molecules. The arrow denotes the conformational change of Phe47 in a subsite that appears to modulate BRD9 selectivity. (C−F) Surface presentations of structurally aligned BRD9 complexes with 2 (PDB code 5F1H), 1 (PDB code 5EU1), I-BRD9 (PDB code 4UIW), and LP99 (PDB code 5IGN), respectively. Inhibitor 2 displayed binding and inhibitory potential against BRD9 with low nanomolar Kd and IC50 values, similar to the previously reported BRD9 inhibitors LP99,7 compound 28,8 and I-BRD99 (Figure 1). Key interactions of the BRD9-2 complex are hydrogen bonding with Asn100, a conserved bromodomain residue critical for the interaction with KAc residues, π-stacking with Tyr106, T-stacking with Phe44, and CH−π interactions with Ile53 (Figure 2A). Importantly, 2 is >10-fold more selective for BRD9 over the highly homologues bromodomain BRD7, which has been implied as a tumor suppressor and is downregulated in cancer cells.10 The high similarity of the KAc binding site across bromodomain containing proteins presents a formidable challenge in the development of selective inhibitors. Most potent BRD4 inhibitors target all members of the BET family with similar potency, irrespective of the high chemical diversity among the pharmacophores and warheads identified to date. It is equally challenging to develop intrafamily selective inhibitors of bromodomains from other branches on the phylogenetic tree, such as BRD9 and BRD7, which exhibit high sequence and structure similarity of the KAc sites. Of the BRD9 inhibitors reported to date,4,7−9,11,12 2 is the most comprehensively studied. Structure−activity relationship studies elegantly identified a hot spot in the KAc site of BRD9 around residue Phe47 that responds to variations of the dimethoxyphenyl moiety with large effects on selectivity (Figure 2A−D). Inhibitor 1 (BI-7273), an isomer of 2, 4460 DOI: 10.1021/acs.jmedchem.6b00550 J. Med. Chem. 2016, 59, 4459−4461 Journal of Medicinal Chemistry Viewpoint (4) Martin, L. J.; Koegl, M.; Bader, G.; Cockcroft, X. L.; Fedorov, O.; Fiegen, D.; Gerstberger, T.; Hofmann, M. H.; Hohmann, A. F.; Kessler, D.; Knapp, S.; Knesl, P.; Kornigg, S.; Muller, S.; Nar, H.; Rogers, C.; Rumpel, K.; Schaaf, O.; Steurer, S.; Tallant, C.; Vakoc, C. R.; Zeeb, M.; Zoephel, A.; Pearson, M.; Boehmelt, G.; McConnell, D. Structure-based design of an in vivo active selective BRD9 inhibitor. J. Med. Chem. 2016, DOI: 10.1021/acs.jmedchem.5b01865. (5) Kadoch, C.; Hargreaves, D. C.; Hodges, C.; Elias, L.; Ho, L.; Ranish, J.; Crabtree, G. R. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy. Nat. Genet. 2013, 45, 592−601. (6) Hohmann, A. F.; Vakoc, C. R. A rationale to target the SWI/SNF complex for cancer therapy. Trends Genet. 2014, 30, 356−363. (7) Clark, P. G.; Vieira, L. C.; Tallant, C.; Fedorov, O.; Singleton, D. C.; Rogers, C. M.; Monteiro, O. P.; Bennett, J. M.; Baronio, R.; Muller, S.; Daniels, D. L.; Mendez, J.; Knapp, S.; Brennan, P. E.; Dixon, D. J. LP99: Discovery and synthesis of the first selective BRD7/9 bromodomain inhibitor. Angew. Chem., Int. Ed. 2015, 54, 6217−6221. (8) Hay, D. A.; Rogers, C. M.; Fedorov, O.; Tallant, C.; Martin, S.; Monteiro, O. P.; Muller, S.; Knapp, S.; Schofield, C. J.; Brennan, P. E. Design and synthesis of potent and selective inhibitors of BRD7 and BRD9 bromodomains. MedChemComm 2015, 6, 1381−1386. (9) Theodoulou, N. H.; Bamborough, P.; Bannister, A. J.; Becher, I.; Bit, R. A.; Che, K. H.; Chung, C. W.; Dittmann, A.; Drewes, G.; Drewry, D. H.; Gordon, L.; Grandi, P.; Leveridge, M.; Lindon, M.; Michon, A. M.; Molnar, J.; Robson, S. C.; Tomkinson, N. C.; Kouzarides, T.; Prinjha, R. K.; Humphreys, P. G. Discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition. J. Med. Chem. 2016, 59, 1425−1439. (10) Drost, J.; Mantovani, F.; Tocco, F.; Elkon, R.; Comel, A.; Holstege, H.; Kerkhoven, R.; Jonkers, J.; Voorhoeve, P. M.; Agami, R.; Del Sal, G. BRD7 is a candidate tumour suppressor gene required for p53 function. Nat. Cell Biol. 2010, 12, 380−389. (11) Fedorov, O.; Lingard, H.; Wells, C.; Monteiro, O. P.; Picaud, S.; Keates, T.; Yapp, C.; Philpott, M.; Martin, S. J.; Felletar, I.; Marsden, B. D.; Filippakopoulos, P.; Muller, S.; Knapp, S.; Brennan, P. E. [1,2,4]triazolo[4,3-a]phthalazines: inhibitors of diverse bromodomains. J. Med. Chem. 2014, 57, 462−476. (12) Picaud, S.; Strocchia, M.; Terracciano, S.; Lauro, G.; Mendez, J.; Daniels, D. L.; Riccio, R.; Bifulco, G.; Bruno, I.; Filippakopoulos, P. 9H-purine scaffold reveals induced-fit pocket plasticity of the BRD9 bromodomain. J. Med. Chem. 2015, 58, 2718−2736. (13) Sun, B.; Shah, B.; Fiskus, W.; Qi, J.; Rajapakshe, K.; Coarfa, C.; Li, L.; Devaraj, S. G.; Sharma, S.; Zhang, L.; Wang, M. L.; Saenz, D. T.; Krieger, S.; Bradner, J. E.; Bhalla, K. N. Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib. Blood 2015, 126, 1565−1574. (14) Baker, E. K.; Taylor, S.; Gupte, A.; Sharp, P. P.; Walia, M.; Walsh, N. C.; Zannettino, A. C.; Chalk, A. M.; Burns, C. J.; Walkley, C. R. BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells. Sci. Rep. 2015, 5, 10120. (15) Stuhlmiller, T. J.; Miller, S. M.; Zawistowski, J. S.; Nakamura, K.; Beltran, A. S.; Duncan, J. S.; Angus, S. P.; Collins, K. A.; Granger, D. A.; Reuther, R. A.; Graves, L. M.; Gomez, S. M.; Kuan, P. F.; Parker, J. S.; Chen, X.; Sciaky, N.; Carey, L. A.; Earp, H. S.; Jin, J.; Johnson, G. L. Inhibition of lapatinib-induced kinome reprogramming in ERBB2positive breast cancer by targeting BET family bromodomains. Cell Rep. 2015, 11, 390−404. displayed subnanomolar binding potential for BRD9 but inhibited BRD7 to the same extent and therefore lacked selectivity. While the o-methoxy of 2 attracts the side chain of Phe47, the m-methoxy of 1 pushes Phe47 away from the KAc site. Notably, Phe47 adopts the same conformation in complex with all BRD9 inhibitors except 2 (Figure 2E,F). Inhibitors 1 and 2 showed no activity against BET bromodomains, due to the structural differences of the KAc binding sites imposing steric hindrance. CECR2 was the only in vitro off-target identified outside the BRD9/BRD7 subfamily, but cellular inhibition of CECR2 was not observed. Studying the effect of 1 and 2 in parallel may provide information on synergistic or antagonistic effects of BRD9−BRD7 inhibition in malignant and normal cell lines, patient samples, and/or in vivo models. What sets 2 apart from other reported BRD9 inhibitors is the evaluation of in vivo properties including pharmacokinetic profiling, as well as demonstration of efficacy in cell lines and in a disseminated mouse model of AML. Significantly, 2 will be made available to the broad scientific community, providing an invaluable resource to align and streamline biological studies by different laboratories on the effect of BRD9 inhibition in diverse disease models. Growing evidence suggests that targeting bromodomains with selective small molecule inhibitors is a viable strategy to control epigenetic events. While most kinase inhibitors drastically disrupt vital signaling pathways or cell cycle events, inhibitors of bromodomains outside the BET family may exert more subtle responses over a longer period of time. Recent reports on strong synergistic action of kinase and BET bromodomain inhibitors for a variety of cancers13−15 suggest that inhibitors of bromodomains outside the BET family may synergize with other drugs as well. Compounds such as 2 are perfectly suited to determine the function of BRD9 and its potential as a drug target. ■ AUTHOR INFORMATION Corresponding Author *E-mail: ernst.schonbrunn@moffitt.org. Phone: 813-745-4703. ABBREVIATIONS USED AML, acute myeloid leukemia; BET, bromodomain and extra-terminal; BRD, bromodomain; BRD4, bromodomaincontaining protein 4; BRD7, bromodomain-containing protein 7; BRD9, bromodomain-containing protein 9; CECR2, cat eye syndrome chromosome region, candidate 2; GPCR, G-proteincoupled receptor; KAc, N-acetylated lysine; SWI/SNF, switch/ sucrose nonfermentable ■ ■ REFERENCES (1) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.; Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.; West, N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La Thangue, N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.; Bradner, J. E. Selective inhibition of BET bromodomains. Nature 2010, 468, 1067−1073. (2) Nicodeme, E.; Jeffrey, K. L.; Schaefer, U.; Beinke, S.; Dewell, S.; Chung, C. W.; Chandwani, R.; Marazzi, I.; Wilson, P.; Coste, H.; White, J.; Kirilovsky, J.; Rice, C. M.; Lora, J. 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