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). 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- 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.

19754–19759 | PNAS | December 3, 2013 | vol. 110 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1310658110 Downloaded by guest on October 1, 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- -3 RVX-208 ration (symbols as indicated in the ﬁgure). Solid lines

μ cal/sec) -6 -0.10 -4 μ cal/sec) RVX-OH ﬁ -8 represent a nonlinear least squares t using a single- BD2 Δ P ( BD1 -5 -0.2 Δ P ( -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 BIOCHEMISTRY 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 specificforBETbromodo- BRD proteins. mains and showed preferred binding to their second bromodomains (BD2s). Highest selectivity was observed for BD2 of BRD2 (23- RVX-208 Is a Potent Inhibitor of Second BET Bromodomains. To es- CHEMISTRY fold) and BRD3 (21-fold). RVX-208 displaces BRD3 from tablish a selectivity profile for RVX-208, we used temperature-shift Δ chromatin at higher concentrations, and inhibition of BET bro- assays ( Tm) carried out on 44 of the 61 human bromodomains and modomains in the liver hepatocellular carcinoma cell line HepG2 found that its effect on BRD temperature stabilization is limited to resulted in weak regulation of only a subset of BET target genes. the BET subfamily. Interestingly, we observed significant stabiliza- The study suggests that first and second BDs of BET family tion of only second bromodomains (BD2s) of BET proteins (Fig. 1B members can be selectively targeted despite high levels of sequence and Table S1), suggesting that RVX-208 selectively targets BD2s. In homology resulting in distinct transcriptional outcomes. contrast, the synthetic precursor RVX-OH, which lacks the hydroxylether substitution (Fig. 1A), also specifically interacted with Results BET family members, although the differences in ΔTm between Human BET proteins have a modular architecture comprising BD1s and BD2s were diminished. In addition, RVX-OH showed two N-terminal BRD modules, an extraterminal (ET) domain, a number of weak ΔTm shifts on BRD9, CECR2, and some other and a C-terminal motif (BRD4 and BRDT only) (Fig. S1A). BRDs (Table S1). AlphaScreen assays carried out using BRD3, for Their two highly conserved BRD modules share a high degree of which a large difference in ΔTm values was observed, confirmed the sequence homology. Sequence homology is most pronounced interactions and demonstrated competitive displacement of histone comparing all four BD1s or BD2s, respectively, resulting in H4 acetyl-lysine containing peptides by RVX-208. The IC50 values clustering of these interaction modules in sequence- and struc- derived from the AlphaScreen data were 87 ± 10 μMand0.510± ture-based phylogenetic trees (1). Interestingly, sequence com- 0.041 μM for BD1 and BD2, respectively, thus about 170-fold se- parisons revealed three residue positions in close proximity to lectivity (Fig. 1C). In agreement with ΔTm data, RVX-OH exhibi- the acetyl-lysine peptide binding site that differ between BD1 and ted a smaller window of selectivity between the two BRD3 BD2 domains: with the exception of BRDT, the residue position bromodomains (IC50,11.4± 5.9 μMand0.379± 0.101 μMforBD1 corresponding to BRD4/BD1 Q85 is a lysine residue in BD2s; the and BD2, respectively) (Fig. 1C). Isothermal titration calorimetry position corresponding to the BRD4/BD1 residue D144 is a histi- (ITC) led to determination of accurate binding constants in solution dine residue in all BET BD2s; the position corresponding to the for both RVX-OH and RVX-208 for all eight BET bromodomains BRD4/BD1 residue I146 is a valine residue in BD2s. The location (Fig. 1 D and E and Tables S2 and S3). The interaction of RVX-208 of these residues suggested that these sequence variations may be with BET bromodomains is driven by large negative binding en- explored for the development of inhibitors that specifically rec- thalpy changes resulting in a KD of 4.06 ± 0.16 μM for BD1 and ognize one of the two BET BRDs (Fig. S1B). more than 20-fold stronger binding to BD2 (0.194 ± 0.013 μM) in The established role of RVX-208 up-regulating the BET- the case of BRD3. ITC data measured on other BET bromodo- regulated HDL protein ApoA1 without associated antiproliferative mains confirmed BD2 selectivity of RVX-208 but resulted in less- effects prompted us to synthesize this compound following the pronounced differences in binding affinities. In contrast, RVX-OH synthetic route disclosed by Resverlogix Corporation [previously showed KD values between 0.15 and 1.4 μM, with no significant established by Hansen—examples no. 4 and no 7 (33)] with differences in ligand affinity for the two BRDs of BRD3. The more

Picaud et al. PNAS | December 3, 2013 | vol. 110 | no. 49 | 19755 Downloaded by guest on October 1, 2021 pronounced differences observed in AlphaScreen assays might be OH complexe with the BD1 of BRD4 (Fig. S2C). In this cocrystal due to differences in peptide affinity for BD1 and BD2 domains. structure, the ligand’s free hydroxyl group from the phenyl ring system acts as an acetyl-lysine mimetic moiety, forming a hydrogen RVX-208 Presents a Template for BD2 Rearrangement upon Binding. bond with N140. Surprisingly, RVX-OH inverts its binding mode The observed selectivity profile for RVX-208 and the lack of in the cocrystal complex with the BD2 of BRD2 assuming a similar selectivity of the closely related compound RVX-OH prompted interaction as observed in RVX-208 where the quinaxolinone us to determine the high-resolution cocrystal structures of RVX- function acts as the acetyl-lysine mimetic moiety (Fig. S2D and Fig. 208 and RVX-OH with representative BET bromodomains (Fig. 2 S3A). Both RVX-OH and RVX-208 are very well resolved in the and Fig. S2). As predicted from our AlphaScreen data, RVX-208 high-resolution crystal structures (Fig. S3 B and C). The shift in bound to the acetyl-lysine binding pocket in a peptide-competitive binding mode of this inhibitor is also evident by the thermody- manner. In the cocrystal structure of the first bromodomain of namic data of this interaction. The RVX-OH interaction with BRD4, the carbonyl oxygen and one of the nitrogen atoms of the BD1s is characterized by a large negative binding enthalpy change quinazolinone ring system act as an acetyl-lysine mimetic moiety, for all BD1 interactions (∼−7to−10 kcal/mol) that is opposed by forming a hydrogen bond with the conserved asparagine residue a negative entropy term (TΔS ∼−2 kcal/mol). In contrast, in- (N140), as well as a water-mediated hydrogen bond with Y97 (Fig. teraction with BD2s gives rise to a modest negative enthalpy ∼− 2A and Fig. S2A). The hydroxy-ethylether moiety points out of the change ( 3.5 kcal/mol) associated with a favorable positive en- Δ ∼ + + acetyl-lysine binding pocket and makes only a few contacts with tropy change (T S 4to 5kcal/mol)(Table S3). However, the the bromodomain surface. The WPF shelf, which contributes different binding modes of these two closely related inhibitors fi fi complicate the interpretation of the structural reasons for the signi cantly to the af nity of phenyl-isoxazole and methyl-triazolo ’ inhibitors (9, 34), is not occupied by the inhibitor but is occupied observed selectivity of RVX-208. To further test the template s by an ethylene glycol solvent molecule. RVX-208 makes no direct binding mode, we synthesized RVX-H, an analog lacking the Kac- mimetic free hydroxyl group and tested its ability to stabilize BET interactions with residues unique to BD1 except to a water- BRDs in thermal melt experiments. As expected, this scaffold mediated hydrogen bond with Q85. The binding mode of RVX- significantly lost its affinity for BET BD1s (Table S1). We conclude 208 is largely conserved in BD2 domains, mimicking that of a that differences in binding affinity are due to small structural histone substrate with the ligand occupying the entire channel used rearrangements and differences in binding mode that may include by a single acetyl-lysine. However, the BD2 unique residue H433 fl contributions of BD2 unique residues such as H433 in the case of in BRD2 ips into the acetyl-lysine binding site packing against the the BD2 of BRD2, as well as possible differences in dynamic phenyl ring of the inhibitor, providing a possible explanation for properties of BET bromodomains. the tighter affinity for BD2 domains (Fig. 2B and Fig. S2B). In- terestingly, the RVX-208 binding mode is not conserved in RVX- Selective Inhibition of BD2 Displaces BETs from Chromatin. Given the weak interaction of RVX-208 with BET BD1s, we were in- terested in establishing whether this inhibitor can dissociate full- length BET proteins from acetylated chromatin. To this end, ZA-loop N140 D144 A R3 D145 we established a FRAP (fluorescence recovery after photo- Y97 R3 Y139 o bleaching) assay using full-length human GFP-BRD3 transiently BC-loop 45 K5ac L94 ZA-loop K5ac I146 transfected into U2OS osteosarcoma cells. Exposure of these K8ac cells to the potent pan-BET inhibitor PFI-1 (10, 35) led to sig- Y97 M149 K8ac nificant reduction of recovery times of the photobleached nu- L92 clear region, suggesting efficient dissociation of BRD3 from αA H4K5ac/K8ac chromatin (Fig. 3), even at concentrations as low as 100 nM, αZ RVX-208 W81 αC BRD4/BD1 Q85 given that the in vitro dissociation constant for this inhibitor is 80 V87 P82 nM for the BD1 of BRD3 and 76 nM for BD2 (35). At 250 nM, Y428 B L383 recovery times reached a plateau that did not decrease further at ZA-loop H433 H4K5ac/K12ac Y386 R3 H433 RVX-208 higher concentrations of the inhibitor. RVX-208 exhibited K5ac BRD2/BD2 slightly weaker activity, displacing BRD3 at concentration of 500 K8 H433 L381 nM and higher, thus demonstrating that both bromodomains are needed for efficient interactions with nucleosomes. V376 V435 D434 BET Transcriptional Regulation Is Mainly Mediated by First Bromo- αA αC domains in Liver. We next investigated the transcriptional regula- P371 αZ K12ac K374 W370 M438 tion effect on gene expression in human liver carcinoma HepG2 cells by BET bromodomains, by either inhibiting both domains at fi Fig. 2. Cocrystal structures of RVX-208 and RVX-OH with the rst and sec- the same time using the pan-BET inhibitor (+)-JQ1 (or JQ1 for ond bromdomains of human BET proteins. (A) Overview of RVX-208 binding simplicity) or RVX-208 seeking to inhibit mainly the second onto BD1 of BRD4 (Left). A detail of the boxed area is shown on the Right (rotated 45° counterclockwise), highlighting the acetyl-lysine mimetic bind- bromodomain (BD2). A microarray study of cells treated with either inhibitor for 4 h revealed large differences in gene ex- ing of the inhibitor, compared with a histone H4 di-acetyl peptide (H4K5ac/ K8ac, PDB ID no. 3UVW, shown in stick representation, colored in blue), pression, with the pan-BET inhibitor JQ1 strongly affecting engaging the protein by directly interacting with the conserved asparagine transcription of genes with almost a 10-fold difference compared (N140 in BRD4/BD1) in addition to a water-mediated hydrogen bond to Y97. with the BD2-specific inhibitor RVX-208. Although inhibition of Residues that differ between BD1 and BD2 are highlighted in red. (B) both BD1 and BD2 affected the gene expression of 754 genes Comparison of the binding mode of RVX-208 and a histone H4 peptide onto within a 1.5-fold window, only 46 genes were affected by the BD2 of BRD2 (Left). The inhibitor engages the protein in the same orienta- inhibition of only BD2 using RVX-208 (Fig. 4A and Fig. S4A). tion to that observed in the structure of a di-acetylated H4 peptide (H4K5ac/ Indeed, the top genes that seem to be affected by each inhibitor K12ac, PDB ID code 2E3K, colored as in A). A blow-up of the boxed area highlights the stabilization of the inhibitor complex, achieved by an inwards have small overlap, and the fold change in their expression is motion of H433 from the BC-loop region toward the front of the acetyl-lysine systematically higher when both domains are inhibited, using binding pocket, a unique feature of second site-BET bromodomains (Right). JQ1 (Fig. 4 B–D). This trend was also observed when we looked Residues that differ from the N-terminal BET BRDs are highlighted in red. at the effect on the expression of the top 1,000 statistically

19756 | www.pnas.org/cgi/doi/10.1073/pnas.1310658110 Picaud et al. Downloaded by guest on October 1, 2021 AB1.0 established role of JQ1 down-regulating c-MYC we found that pan-BET inhibition by JQ1, but not by RVX-208, led to tran- Vehicle 0.8 scriptional down-regulation of the c-MYC oncogene in liver cells RVX-208 0.6 (Fig. S4D). Interestingly ApoA1, a reported downstream target of Vehicle 100 nM 100 nM BET proteins (9), was not affected by RVX-208 at the concen- 0.4

RVX-208 250 nM trations tested (27). 500 nM

5000 nM 0.2 Normalized Intensity 5000 nM We found that ApoA1 RNA levels were not affected in a time- 0.0 or dose-dependent manner whereas ApoA1 protein levels were

100 nM 0 5 10 15 20 25 30 35 + A–D Time (sec) only slightly affected by ( )-JQ1 treatment (Fig. S5 ). Using PFI-1 C a luciferase reporter, we were also unable to observe a significant 1.0 5000 nM effect on ApoA1 transcription using a range of inhibitor con- Pre 1.4 sec 5.3 sec 9.2 sec 38.9 sec E F D 0.8 centrations (Fig. S5 and ). Taken together, our data suggest 6 RVX-208 * PFI-1 PFI-1 that the transcriptional effect of BET proteins on their target 5 * 0.6 Vehicle genes is mainly dependent on both BDs, which seem to drive 4 *** *** 1/2 *** 100 nM 3 *** *** 0.4 interactions with histones in the context of chromatin, suggesting *** 250 nM 2 that they are not functionally redundant. Indeed, deletion of only 0.2 500 nM 1 Normalized Intensity 5000 nM fi Recovery t (sec) BD1 in BRDT is suf cient to impair spermatogenesis in mice (26), 0 0.0 0 100 250 500 5000 0 5 10 15 20 25 30 35 supporting the dominant role of BD1 in transcription control. Compound Concentration (nM) Time (sec) Discussion Fig. 3. Fluorescence recovery after photobleaching (FRAP) evaluation of human BRD3 dissociation from chromatin. (A) Nuclei of DMSO treated (Top), BET proteins act as multidomain docking platforms that have RVX-208 treated (Middle), or PFI-1 treated (Bottom) cells. Target regions of a general role in transcription elongation by recruiting the pos- photobleaching are indicated by a white circle. (Scale bars: 10 μm region.) (B itive transcription elongation factor b (P-TEFb) to acetylated and C) RVX-208 and PFI-1 accelerate fluorescence recovery in FRAP experi- chromatin (36, 37), as well as tissue-specific functions. The cell ments performed in U2OS cells transfected with full-length GFP-BRD3 in type-specific roles have been highlighted by recent reports on a concentration-dependent manner. (B) RVX-208 can displace the protein BRDT-dependent transcription programs that regulate sper- from chromatin at concentrations above 500 nM whereas PFI-1 displaces the matogenesis (37–39) and BRD3-dependent recruitment of protein when used at 100 nM. (D) Quantitative comparison of time to half- fl GATA1 in hematopoietic cells regulating maturation of ery- BIOCHEMISTRY maximal uorescence recovery for FRAP studies using RVX-208 (blue bars) throid, megakaryocyte, and mast cell lineages (23, 40), as well as and PFI-1 (red bars) as a function of ligand concentration. Data represent the mean ± SEM (n = 15) and are annotated with P values as obtained from BRD2-dependent roles regulating differentiation of adipose tis- a two-tailed t test (*P < 0.05; ***P < 0.001). sue (25) and neurons (41). Application of BET inhibitors outside the area of oncology, given the pleiotropic nature of BET transcriptional regulation, strongly suggests a requirement for more significant genes (with an adjusted P value less than 0.05) for each selective targeting with respect to isoform and/or domains, to avoid

inhibitor; genes that are strongly up-/down-regulated when both adverse effects. Currently developed inhibitors are highly BET- CHEMISTRY domains are inhibited by JQ1 are only weakly regulated when the specific but show no or little isoform or domain specificity (7–10). second domain is inhibited by RVX-208 (Fig. S4B) whereas genes In this work, we have established a first step toward isoform that are strongly up-/down-regulated when the second domain is selective inhibition by characterizing interactions of RVX-208 inhibited by RVX-208 exhibit an even higher degree of regulation with BET bromodomains, a template currently in phase I/II when both domains are inhibited by JQ1 (Fig. S4C). To further clinical trials for the treatment of cardiovascular diseases. Im- probe and verify the differences in gene expression initiated by portantly, RVX-208 preferentially binds to the second bromo- BD1 or BD2, we selected a subset of four genes for qPCR studies domain found on BET proteins, exhibiting selectivity over BD1 K μ that were strongly down-regulated (AREG, EREG, INHBE, and of up to 23-fold. With a D of 195 nM against BD2 and 4 M NR1H4) and four genes that were up-regulated (BRD2, against BD1 of BRD3, we were able to show in vitro competitive MYBL1, MYLIP, and ZNF117). The effects of pan-BET versus displacement of a histone H4 tetra-acetylated peptide. In- BD2-selective inhibition were monitored in a time-dependent terestingly, cocrystal structures showed that RVX-208 induced (Fig. 4E) and dose-dependent (Fig. 4F) manner. In the first a conformational switch upon binding to BD2, resulting in aro- instance, we observed a striking difference in gene expression matic stacking accompanied by large entropic changes, as determined by isothermal titration calorimetry, which may explain when both BDs were inhibited by JQ1, with little or no effect when in part its preference for BD2 over BD1 and suggests that dy- only BD2 was inhibited with RVX-208. This effect was verified by namic properties of bromodomains need to be considered in the the dose experiment as well, despite the fact that we chose a 10- design of domain-specific inhibitors. BET BD1s and BD2s show fold window between the two inhibitors in an effort to account for fi a high degree of sequence similarity but differ in their recogni- their differences in af nity for the two domains. Despite the lower tion of acetylated target sequences (1). It is therefore likely that concentrations used for JQ1 (0.3–1.0 μM), as opposed to higher – μ each BET bromodomain has a distinct function although the doses of RVX-208 (0.78 25.0 M), inhibition of the second do- combined effect seems to be conferring a regulatory output in fi main was not suf cient to drive a strong transcriptional response. gene transcription. We observed in this study that targeting the To establish that these effects are not inhibitor-dependent, we BD2 had only a modest effect on transcription, an observation conducted a similar experiment and monitored gene expression by supported by previous studies of BRD3 that showed that its re- quantitative real-time PCR on a set of genes using either the pan- cruitment to acetylated sites on GATA1 is mediated by BD1 BET inhibitors (+)-JQ1 and PFI-1 in addition to the BD2 in- (40). Notably, deletion of the first bromodomain in BRDT in hibitor RVX-208. We included also the precursor RVX-OH and mice is sufficient to confer sterility by blocking BRDT-dependent used (−)-JQ1 as an inactive control compound. As expected, we sperm maturation (39). Unfortunately, no genetic knockout stud- observed strong up- or down-regulation when both BD1 and BD2 ies have been published that target only the BD2 in any of the where inhibited by (+)-JQ1, PFI-1, or RVX-OH and a smaller BET family members, but biochemical studies and data pre- effect when only BD2 was inhibited by RVX-208 (Fig. S4D) sented here suggest a modulating role of BET function by whereas the inactive variant, (−)-JQ1, had no significant effect on BD2. In a recent study, Wu et al. showed that phosphorylation the expression of the selected genes. In agreement with the well- of flanking regions of BD2 in BRD4 triggers an intramolecular

Picaud et al. PNAS | December 3, 2013 | vol. 110 | no. 49 | 19757 Downloaded by guest on October 1, 2021 Differentially Expressed Up RVX-208 2 A B 1 Genes (> 1.5-fold) (+)-JQ1 1 0 Total 28 -1 -2 Log Fold (+)-JQ1 Change 370 (+)-JQ1 TNS4 AREG TXNIP HNF1A GCNT3 RVX-208 DHRS2 CNNM4 HEXIM1 ZSWIM6 SLC16A6 4 42 712 2 (+)-JQ1 1 0 Fig. 4. Microarray analysis of the transcriptional

Change -1 Log Fold effect following 4-h treatment of HepG2 cells with 342 -2 5 μM RVX-208 or 0.5 μM(+)-JQ1. (A) Venn diagram RVX-208 of statistically significant (P < 0.05) differential 14 TNS4 MYBL1 KRCC1 3 DHRS2 Down CYP1A1 expression of genes that are up- or down-regu- ZNF280C ST3GAL3 MAB21L3 HIST2H4A RVX-208 LINC00467 lated with a 1.5-fold change (or greater) when cells C -3 0 3 D (+)-JQ1 RVX-208 (+)-JQ1 RVX-208 Down-regulated are treated with compound. The transcriptional Up-regulated + HIST2H4A HIST2H4A qRTPCR effect of ( )-JQ1 is 10x higher than that of RVX- SLFN5 RABL2A AREG MYBL1 208. (B) Log-fold change for the top 10 statistically ARRDC4 CYP1A1 KRCC1 KRCC1 NR1H4 significant (+)-JQ1 (Upper) or RVX-208 (Lower) ZSWIM6 RFC1 MYLIP MYBL1 EREG BRD2 10 CNNM4 DHRS2 10 regulated genes, highlighting the weaker effect of AREG ADARB1 SRP9 INHBE ZNF117 -log (p) TXNIP RPL23AP82 -log (p) INHBE ZNF117 RVX-208 on transcription. (C) Top 45 up-/down- EREG WDR47 ZNF737 BRD2 + HEXIM1 MYBL1 regulated genes in the case of ( )-JQ1 (Left)and MYBL1 SLFN5 MYLIP RVX-208 (Right) treated cells. The color scale in the C1orf63 HIST1H2BK NR1H4 DHRS2 ZNF43 ApoA1 ApoA1 Inset represents log-fold change of expression 0246810 SERPINB9 KLRAP1 0246810 BTG1 STOX1 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 compared with the untreated control. (D) Volcano VAMP1 HIST1H2AM Log Fold Change Log Fold Change STXBP1 ST3GAL3 plot of the top 1,000 genes that are up-/down- GTF2B HIST1H2BD + KLHL28 C6orf203 E Not-treated F3 (+)-JQ1 regulated in the case of ( )-JQ1 (Left) and RVX-208 HSPA1A NPIP 2 (Right) after 4 h of treatment with 0.5 μM(+)-JQ1 HNF4A DNAJC28 2 ADAT2 PNPLA8 1 or 5 μM RVX-208. Top genes are sorted by their ARSI LINC00467 KLHL14 ZNF280C 1 fold-change and are highlighted and colored when ARID5B SNORD59B 0 ZBTB38 HIST1H2BC (+)-JQ1 they are up-regulated (red) or down-regulated DHRS3 TST 0 fi DUSP16 ZNF117 2 (blue). Genes veri ed by qPCR are highlighted. (E) CCR6 RPS3A 3 RVX-208 Quantitative real-time PCR on eight representative NAV2 TNS4 1 DKK1 LIF genes identified in A using gene-specific primers IRS1 CACNB4 Relative Quantity 2 ZNF114 LOC151009 0 (Table S5). Cells were treated with 0.5 μM(+)-JQ1

GCNT3 EREG Relative Quantity RVX-208 1 μ KANK1 FLJ11710 2 or 5 M RVX-208. Gene expression was monitored SLC16A6 DGKK for 48 h, as indicated in the Inset.(F) Dose-dependent C3orf33 FAM209B 0 GPAM SH3TC2 1 gene expression on the same set of eight genes tested TNS4 PCDHA13 HNF1A TBX4 BRD2 in E, measured at 4 h. Even at the highest concen- AREG EREG

0 MYLIP INHBE NR1H4 LOC151009 CYP4Z1 MYBL1 PALMD MAB21L3 [(+)-JQ1]/(µM) [RVX-208]/(µM) ZNF117 tration, RVX-208 had only a modest transcriptional MAB21L3 ZNF208 BRD2 AREG EREG + MYLIP

INHBE effect compared with ( )-JQ1. Error bars in E and F NR1H4 MYBL1 25.0 6.25 12.5 0.78 1.56 3.12 0.00 0.00 0.50 1.00 0.06 0.03 0.13 ART4 VPS52 0.25 AREG LINC00652 Time (h) 2 4 8 243048 ZNF117 represent SD from triplicate experiments.

rearrangement that unmasks BD2 and directs it toward acetylated against corresponding DMSO controls. Assays were performed as previously chromatin (42). This observation suggests that BET transcrip- described (7, 45), with minor modifications from the manufacturer’sprotocol. tional regulators are controlled posttranslationally and that specific targeting, such as the one highlighted here toward BD2, may Isothermal Titration Calorimetry. Experiments were carried out on an ITC200 microcalorimeter from MicroCal at 15 °C in 50 mM Hepes, pH 7.5 (at 25 °C), be able to perturb and modulate BET function in a context- 150 mM NaCl by titrating protein into ligand solutions (reverse titrations), dependent manner. However, further development of domain and and data were corrected for protein heats of dilution and deconvoluted fi isoform speci c inhibitors will be necessary to unravel the exact using the MicroCal Origin software as previously described (1, 7). Dissocia- role of BET bromodomains in gene transcription. tion constants and thermodynamic parameters are listed in Tables S2 and S3.

Materials and Methods Fluorescent Recovery After Photobleaching. Fluorescent recovery after pho- Cloning, Protein Expression, and Purification. The BRD regions of human BRD3 tobleaching (FRAP) studies were performed in U2OS cells transfected with and BRD4 were cloned/amplified as previously described (7). The full-length mammalian overexpression constructs encoding GFP chimeras of BRD3, using mouse BRD3 ortholog was used to reconstruct the human clone, by first a Zeiss LSM 710 scanhead coupled to an inverted Zeiss Axio Observer.Z1 mutating the C-terminal region and then cloning into pDONR223-hBRD3 microscope equipped with a high-numerical-aperture (N.A. 1.3) 40× oil im- according to Tillett and Neilan (43) and confirmed by sequencing. Protein mersion objective equipped with a heated chamber set to 37 °C, using expression and purification were carried out as previously described (7). a protocol modified from previous studies (7).

RVX-H, RVX-OH, and RVX-208 Synthesis. The synthesis and characterization of Crystallization, Data Collection, and Structure Reﬁnement. Cocrystallization these compounds were carried out following the synthetic route previously and structure determination for complexes of BET BRDs with RVX-OH and described (44) with minor changes, using the synthetic scheme highlighted in RVX-208 were carried out following published procedures (7). Data collection Fig. S1C. and reﬁnement statistics can be found in Table S4.

Protein Stability Shift Assay (Tm Assay). Thermal melting experiments were Cell Culture and RNA Extraction. HepG2 cells were treated so that a ﬁnal carried out using an Mx3005p Real-Time PCR machine as previously described concentration of 0.1% DMSO was achieved. Cells were harvested, washed, Δ obs (7). Temperature shifts ( Tm ) for three independent measurements per and lysed in situ using standard protocols. Total RNA was extracted and protein/compound are summarized in Table S1. prepared using RNeasy columns, and RNA was quantiﬁed and quality controlled using a Nanodrop spectrophotometer. Competitive Histone Displacement Assay (AlphaScreen Assay). Experiments were run on a PHERAstar FS plate reader using an AlphaScreen 680 excitation/570 DNA Microarray Analysis. An Affymetrix GeneChip WT Terminal Labeling and

emission ﬁlter set. IC50 values were calculated in Prism 5 after normalization Controls Kit was used according to the manufacturer’s instructions and processed

19758 | www.pnas.org/cgi/doi/10.1073/pnas.1310658110 Picaud et al. Downloaded by guest on October 1, 2021 on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000. Data were ACKNOWLEDGMENTS. We are grateful for support received by the Struc- processed in R using Bioconductor (46). Background correction and normaliza- tural Genomics Consortium, a registered charity (number 1097737) that tion were carried out using the Robust Multichip Array (RMA) (47). A linear receives funds from the Canadian Institutes for Health Research, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Pﬁzer, Eli model was applied (limma) followed by empirical Bayesian analysis, and genes Lilly, Takeda, AbbVie, the Novartis Research Foundation, the Ontario were considered differentially expressed if the adjusted P value, calculated using Ministry of Research and Innovation, and the Wellcome Trust (092809/Z/ the Benjamini–Hochberg method (48) to minimize false discovery rate, was less 10/Z). P.F. and S.P. are supported by Wellcome Trust Career Development than 0.05 and the mean level of expression was greater than 1.5-fold. Fellowship (095751/Z/11/Z).

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