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RVX-208, an Inhibitor of BET Transcriptional Regulators with Selectivity for the Second Bromodomain

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RVX-208, an Inhibitor of BET Transcriptional Regulators with Selectivity for the Second Bromodomain RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain Sarah Picauda, Christopher Wellsa, Ildiko Felletara, Deborah Brothertona, Sarah Martina, Pavel Savitskya, Beatriz Diez-Dacalb, Martin Philpotta, Chas Bountraa, Hannah Lingarda, Oleg Fedorova, Susanne Müllera, Paul E. Brennana, Stefan Knappa,c,1, and Panagis Filippakopoulosa,b,1 aStructural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom; bLudwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom; and cTarget Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7BN United Kingdom Edited by Angela M. Gronenborn, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved October 23, 2013 (received for review June 5, 2013) Bromodomains have emerged as attractive candidates for the (7), multiple myeloma (16), acute myeloid and mixed lineage development of inhibitors targeting gene transcription. Inhibitors leukemia (17), lung cancer (18), and glioblastoma (19). of the bromo and extraterminal (BET) family recently showed BET family members play an essential role in diverse cellular promising activity in diverse disease models. However, the pleio- processes, including general transcriptional elongation (20, 21), tropic nature of BET proteins regulating tissue-specific transcription replication (22), hematopoiesis (23), adipogenesis (24, 25), and has raised safety concerns and suggested that attempts should be spermatogenesis (26), suggesting that drug discovery efforts made for domain-specific targeting. Here, we report that RVX-208, should explore isoform, or even domain-specifictargeting,to a compound currently in phase II clinical trials, is a BET bromodo- avoid adverse effects of prolonged pan-BET inhibition during main inhibitor specific for second bromodomains (BD2s). Cocrystal treatment in different tissues. structures revealed binding modes of RVX-208 and its synthetic pre- The quinazolone RVX-208 (Fig. 1A) has been developed by cursor, and fluorescent recovery after photobleaching demon- Resverlogix Corporation for the treatment of cardiovascular strated that RVX-208 displaces BET proteins from chromatin. diseases associated with atherosclerosis (27, 28) and has more However, gene-expression data showed that BD2 inhibition only recently entered clinical studies on Alzheimer’s disease (29). BIOCHEMISTRY modestly affects BET-dependent gene transcription. Our data RVX-208 is a derivative of the plant polyphenol resveratrol fi demonstrate the feasibility of speci c targeting within the BET (3,4’,5-trihydroxy-transstilbene) that leads to an increase of family resulting in different transcriptional outcomes and high- plasma levels of the high-density lipid protein ApoA1. Increasing light the importance of BD1 in transcriptional regulation. ApoA1 levels has emerged as a promising approach for the treatment of atherosclerosis (30), and recent phase IIb clinical small molecule inhibitor | epigenetics | microarray | ApoA1 trial data using RVX-208 as an ApoA1 modulator have been encouraging (28). ApoA1 expression is regulated by BET pro- CHEMISTRY romodomains (BRDs) are protein-interaction modules that teins, and chemical inhibition of BET bromodomains has been Bare selectively recruited to e-N-acetylated lysine-containing associated with ApoA1 up-regulation on transcriptional and sequences. BRDs are present in 46 diverse, mostly nuclear pro- protein levels (9, 31, 32). As a consequence, a similar mode of teins functioning as effector domains of transcriptional regu- action has also been suggested for RVX-208, but no data char- lators, chromatin modulators, and chromatin-modifying enzymes acterizing the RVX-208/BET interaction have been published so (1). BRD-containing proteins have been implicated in the de- far. The promising clinical outcome of RVX-208 trials and the velopment of many diverse diseases, and the architecture of their presumed function of RVX-208 as a BRD inhibitor prompted us acetyl-lysine binding pocket makes them attractive targets for the fi development of potent and speci c inhibitors (2, 3). All BRD Significance modules share a conserved fold comprising a left-handed helical bundle creating a deep, largely hydrophobic and aromatic bind- fi Bromo and extraterminal (BET) proteins have diverse roles in ing pocket for the speci c recognition of peptide sequences fi e – regulating tissue-speci c transcriptional programs, raising safety containing one or more -N-acetylated lysine residues (1, 4 6). concerns for their inhibition and suggesting that targeting of In particular the bromo and extraterminal (BET) proteins, specific isoforms or even specific domains within this subfamily which comprise four members in human (BRD2, BRD3, BRD4, fi is important. We report the discovery and characterization of and the testis-speci c BRDT), recently received a lot of atten- RVX-208 as a domain-selective inhibitor of BETs and provide tion after highly potent and cell-active pan-BET inhibitors were a potential mechanism of action of a clinical compound that was – developed (7 10). BETs are transcriptional regulators that identified based on phenotypic screens. control expression of genes that play key regulatory roles in cellular proliferation, cell cycle progression, and apoptosis (11, Author contributions: C.B., H.L., O.F., S. Müller, P.E.B., S.K., and P.F. designed research; S.P., 12). Dysfunction of BET proteins has been associated with the C.W., I.F., D.B., S. Martin, P.S., B.D.-D., M.P., and P.F. performed research; S.P., C.W., B.D.-D., development of aggressive tumors, such as NUT midline carci- O.F., S.K., and P.F. analyzed data; and S.K. and P.F. wrote the paper. noma (NMC). In NMC, the N-terminal bromodomains of BRD3 The authors declare no conflict of interest. or BRD4 are fused in frame with the testis-specific protein NUT This article is a PNAS Direct Submission. (nuclear protein in testis), giving rise to an incurable fatal sub- Freely available online through the PNAS open access option. type of squamous carcinoma and in some cases tumors of other Data deposition: The crystal structures reported in this paper have been deposited in the tissue origin (13). Importantly, BETs play a critical role in tu- Protein Data Bank, www.pdb.org (4MR3–4MR6). Microarray data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. morigenesis also outside NMCs by driving the expression of GSE51143). genes that are essential for tumor growth and survival, such as 1 To whom correspondence may be addressed. E-mail: panagis.fi[email protected]. c-Myc (14) and Aurora B (15). The potent pan-BET inhibitors uk or [email protected]. + fi ( )-JQ1 and GSK1210151A (I-BET151) have exhibited signi cant This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. antitumor activity in murine models of NUT midline carcinoma 1073/pnas.1310658110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310658110 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 o ΔTm ( C) A RVX-208: R = -O(CH22 ) OH B BRD2/BD2 BRD3/BD2 R RVX-OH: R = -OH BRD3/BD1 > 4 BRD4/BD1 RVX-H: R = -H BRD4/BD2 BRD2/BD1 BRDT 3 - 4 /BD1 O N BRDT/BD2 2 - 3 1 - 2 FALZ GCN5L2 0 - 1 BAZ1A CECR2 PCAF NH EP300 < 0 CREBBP fi BRD8B Fig. 1. In vitro selectivity pro le of RVX-208. (A) WDR9/BD2 BRD8B PB1/BD1 PB1/BD3 /BD1 PB1/BD2 PHIP/BD2 /BD2 Structure of the inhibitor RVX-208 and its precursor OO PB1/BD4 BRWD3/BD2 ASH1L PB1/BD5 RVX-OH. (B) Selectivity of RVX-208 within the human C BAZ1B SMARCA2A BRD9 bromodomain family determined using a thermal 100 RVX-208 SMARCA2B BRD7 Δ obs μ BD1 PB1/BD6 SMARCA4 shift assay. Temperature shifts ( Tm in °C, at 10 M 75 BRPF3 BRPF1B BRWD3/BD1 compound concentration) are shown as spheres as BD2 BRD1 BRPF1A ATAD2 PHIP/BD1 50 RVX-OH KIAA1240 WDR9/BD1 indicated in the Inset. Screened proteins are shown MLL BD1 TRIM66 TRIM28 PRKCBP1 in bold. (C) Competitive displacement of a tetra- 25 BD2 BAZ2A TRIM33B BAZ2B ZMYND11 TAF1/BD1 acetylated histone H4 peptide (H4 – K5 /K8 / Inhibition (%) 1 20 ac ac 0 TRIM33A TIF1α SP110C TAF1L/BD1 K12ac/K16ac) from BD1 and BD2 of BRD3 using RVX- SP110A TAF1/BD2 -7 -6 -5 -4 SP100 TAF1L/BD2 208 or RVX-OH (as indicated in the Inset) in a bead- [compound] (log(M)) SP140 LOC93349 based proximity assay (ALPHA assay). (D) Isothermal D E titration calorimetry (ITC) binding study. Data col- 0.0 0.00 lected against the bromodomains of BRD3, showing raw injection heats for titrations of protein (BD1 or 0 0 BD2) into compound. The Inset shows the normal- -0.05 -1 -0.1 -2 ized binding enthalpies corrected for the heat of -2 BD1 -4 protein dilution as a function of binding site satu- cal/sec) -3 fi cal/sec) RVX-208 ration (symbols as indicated in the gure). Solid lines μ -6 -0.10 -4 μ RVX-OH represent a nonlinear least squares fit using a single- P ( -8 P ( BD2 Δ BD1 -5 -0.2 Δ -10 site binding model. (E) Isothermal titration calorimetry kcal/mole of injectant RVX-208 BD2 -6 (ITC) evaluation of RVX-208 and RVX-OH against the RVX-OH kcal/mole of injectant -0.15 -12 01234 bromodomains of BRD4. Data have been corrected BRD3 Molar Ratio BRD4 01234 Molar Ratio and displayed as described in D. All ITC titrations were 0 1020304050607080 -0.3 0 10203040506070 carried out in 50 mM Hepes, pH 7.5 (at 25 °C), 150 mM Time (min) Time (min) NaCl and 15 °C while stirring at 1,000 rpm. to study its role regulating BET-dependent transcription. In- minor changes (Fig. S1C) and to study its interaction with human terestingly, we found that RVX-208 is specific for BET bromodo- BRD proteins.
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