Recent progress and structural analyses of domain‐selective BET inhibitors

Epigenetic mechanisms for controlling gene expression through heritable modifications to DNA, RNA, and proteins, are essential processes in maintaining cellular homeostasis. As a result of their central role in human diseases, the proteins responsible for adding, removing, or recognizing epigenetic modifications have emerged as viable drug targets. In the case of lysine‐ε‐N‐acetylation (Kac), bromodomains serve as recognition modules (“readers”) of this activating epigenetic mark and competition of the bromodomain‐Kac interaction with small‐molecule inhibitors is an attractive strategy to control aberrant bromodomain‐mediated gene expression. The bromodomain and extra‐terminal (BET) family proteins contain eight similar bromodomains. These BET bromodomains are among the more commonly studied bromodomain classes with numerous pan‐BET inhibitors showing promising anticancer and anti‐inflammatory efficacy. However, these results have yet to translate into Food and Drug Administration‐approved drugs, in part due to a high degree of on‐target toxicities associated with pan‐BET inhibition. Improved selectivity within the BET‐family has been proposed to alleviate these concerns. In this review, we analyze the reported BET‐domain selective inhibitors from a structural perspective. We highlight three essential characteristics of the reported molecules in generating domain selectivity, binding affinity, and mimicking Kac molecular recognition. In several cases, we provide insight into the design of molecules with improved specificity for individual BET‐bromodomains. This review provides a perspective on the current state of the field as this exciting class of inhibitors continue to be evaluated in the clinic.

Mitsubishi Pharmaceuticals, 10,22 and GSK. 24 In the published literature, this potent biological effect was initially applied to oncogenic BET-fusions, where the related pan-BET family bromodomain inhibitor (+)-JQ1 was used to treat a BRD4-driven NUT-midline carcinoma. 25 This rare, aggressive cancer is caused by a translocation of the BRD4 and NUT genes resulting in oncogenic transcription that is dependent on the BRD4 bromodomains. [26][27][28] Subsequent reports on the dependence of key oncogenes like MYC and BCL2 on BET proteins found additional applications for pan-BET inhibitors in oncology, 14,29 where super-enhancer regulation or expression of traditionally undruggable transcription factors were highly sensitive to BET-inhibition. 10,30 The widespread use of BET-inhibitors has since expanded beyond oncology and inflammation with the identification of new biological roles for BRD4 including diabetes, virology, and cardiovascular disease. [31][32][33][34] As BET proteins regulate a variety of transcriptional processes, the resulting cellular effects of pan-BET bromodomain inhibition are, therefore, widespread and often counterproductive for therapeutic indications. 35,36 The prototypical BET-protein, BRD4 plays important biological roles by binding histone proteins ( Figure 1A,B) to release RNAPol-II from promoter-proximal pausing and through recruitment of various transcription factors. As a result, BET proteins facilitate expression of a wide set of genes with BRD4 playing the dominant role in many cases.
This occurs through bromodomain protein-protein interactions at enhancer and super enhancer regions and additional interactions of BET protein functional domains with transcriptional complexes ( Figure 1C). Chem-SEQ results show genome occupancy profiles of BET proteins are not entirely identical. BRD2, 3 and 4 bind some overlapping areas of the genome, but do also localize to unique areas independent of other BET proteins, 37 suggesting BET proteins have both overlapping and distinct functions to specifically control expression of certain genes. In contrast to ubiquitously expressed BET proteins, the testis-specific protein BRDT plays an important role in regulating spermatogenesis and is a target for male-contraceptives. 12,38 As a result, pan-BET inhibition disrupts a broad set of biological processes, while more selective inhibition may lead to more specific biological effects.
Numerous pan-BET inhibitors have entered clinical trials for oncology and inflammation, but they have faced safety and efficacy challenges that prevented clinical progress. Pan-BET inhibitors have faced dose-limiting BETspecific toxicities and had lower efficacy than was predicted in preclinical models. Pan-BET inhibitors have high efficacy in NUT-midline carcinomas, but were unable to maintain durable responses in other oncology applications overall, and developed resistance to therapy. 36,39 Mechanisms for tumors to develop resistance to BET inhibition are through upregulation of alternate signaling pathways that restore transcription and BRD4 function, including Wnt/β−catenin, 40 FOSL1, 41 CK2, CDK9, 42 and bHLH transcription factors. 43 Toxicity concerns observed with pan-BET inhibitors across multiple trials were anemia, thrombocytopenia and a host of other GI complications (reviewed in Sun et al. 39 ). While these were reversible upon drug withdrawal-a clear benefit of an epigenetic approach-BET inhibitors did not fare well in oncology trials as a monotherapy. For example, in trials with CPI-0610 only two complete and one partial response was observed in 44 lymphoma patients 44 ; this and other trials were terminated or withdrawn due to similar concerns although CPI-0610 has progressed to phase III trials for the treatment of myelofibrosis. While efficacy may be addressed by combination therapy, improved biomarker identification, and better stratification of patients, the bigger challenge of toxicity may be addressed through improved intra-BET selectivity to specifically target the disease-relevant domains. While the high levels of on-target thrombocytopenia toxicity is thought to be ostensibly related to inhibition of BRD2 and 3, 15,45,46 pan-BD2 inhibition also appears to have weaker cellular antiproliferative effect 19 and may decrease GI-toxicities. For example, in oncology applications a response to treatment may be related to BRD4 inhibition but thrombocytopenia can result from decreasing BRD3 and BRD2 co-occupancy at GATA-1 regulatory sites, which can play compensatory roles. 46 A strategy to address these concerns requires improved selectivity relative to pan-BET family inhibition. The use of chemical biology 47,48 and genetics approaches have helped identify the differing role of individual BET bromodomains in disease and targeting these domains more specifically may provide a mechanism to decrease the associated toxicities. The first N-terminal bromodomains on BET proteins play a major role in anchoring the protein to chromatin and thus in the maintenance of basal gene expression. 49,50 In contrast, the second BET bromodomains are more likely to bind acetylated proteins such as transcription factors to facilitate gene expression in response to external stimuli. 19,50,51 These findings suggested individual domain inhibitors alone may not have adequate efficacies for all therapeutic applications, but more recent publications are providing a clearer picture of the unique biological roles each domain plays and allow more focused targeting of these domains in immunology, inflammation, and oncology. 18,19 Thus the pleiotropic nature of pan-BET inhibition and a deeper understanding of the mechanistic role individual BET proteins play in certain diseases 9,36 have led to a renewed focus on achieving selectivity within the BET family.
Although selective targeting of individual BET bromodomains was a challenge at first due to the high similarity of the 61 human bromodomains, progress has been made towards developing pan-BD1 and pan-BD2 inhibitors with preference for individual domains and a glut of new chemical matter has been reported in the last 18-24 months. From initial reports, the biology of domain-selective inhibitors appears in some cases to contrast to that of pan-inhibitors, such as the effect of Olinone on oligodendrocytes. 52 Such experiments require cautious evaluation of compound concentrations to ensure domain-selectivity is maintained at the concentrations tested. 19 Improvements in potency and precise applications of more selective molecules have shown domain-selective inhibition do yield different phenotypic effects. The reported BD1 selective inhibitors show comparable antiproliferative efficacy to pan-BET inhibitors, whereas pan-BD2 selective inhibitors are most effective as antiinflammatory agents. More importantly, domain-selective BD2 inhibitors have indeed shown lower toxicity and are tolerated at higher doses than pan-BET inhibitors. 19 Dose-escalation studies with BD1 inhibitors are yet to be reported and it remains to be seen if they will result in decreased toxicity relative to pan-BET inhibitors, but initial safety indications are promising. 53 The following sections will discuss the structure and function of the respective BET domains and the merits of selectively targeting them.

| BROMODOMAIN STRUCTURAL ANALYSIS
The bromodomain motif is an approximately 110-amino acid domain usually occurring as a substructure of larger multidomain proteins. Structurally, bromodomains are left-handed four-helix bundles (αZ, αA, αB, and αC, Figure 2A) with high sequence conservation. The substrate binding pocket is a deep hydrophobic groove between the helices flanked by two flexible loop regions (ZA and BC) with higher sequence diversity than the α-helices. 6 K ac recognition, most commonly on histone H3 or H4, and acetyl lysine mimics occur through highly conserved asparagine and tyrosine residues that form direct and water-mediated hydrogen bonds to the N-acetyl group ( Figure 2B). The water molecule interacting with bromodomain substrates is part of an ordered network of four to six waters that extends into the hydrophobic interior of the helical fold that is capped by an aromatic 'WPF-shelf' ( Figure 2C). Typical affinity ranges for native bromodomain substrates are in the low to high micromolar range. BET bromodomains have a preference for recognizing polyacetylated histone H3 or H4 peptides (BET-BD:monoacetylated K ac peptide K d~1 00-300 µM; BET-BD:diacetylated K ac peptides K d~7 -60 µM), 16 although other acyl-lysine modifications are recognized by bromodomains with weaker affinities. 6,16 BET-BD1s preferentially recognize diacetylated H4 peptides with a Gly-Gly spacer, as in K5 ac , K8 ac , and have weaker affinity for K ac residues separated by acidic residues. In contrast to BD1s, the BD2s bind K ac substrates weaker overall and are more promiscuous in their substrate recognition. 16 This conserved mode of substrate recognition and structural similarity of bromodomains makes selective inhibition a challenge.

| BET PROTEIN STRUCTURAL ANALYSIS
The BET protein family (II, Figure 2D) is the most widely studied of the bromodomain-containing proteins. The family consists of four proteins with similar structure, each containing two bromodomains, BD1 and BD2, of high sequence conservation ( Figure 3). In addition to these domains, each protein has a C-terminal effector domain (ET), and basic (BID) or phosphorylated regions (NPS & CPS) involved in protein-protein interactions with transcriptional complexes.
As a master transcriptional regulator, the protein BRD4 has a further C-terminal domain that facilitates assembly of promoter-proximal transcriptional complexes. Through CTM interactions with the p-TEFb complex of CDK9 and CyclinT, BRD4 regulates phosphorylation of heptad repeats on the C-terminal domain of RNAPol-II to release it from a paused state ( Figure 1). 54,55 Although shorter BRD4 isoforms (BRD4-S) expressed without the CTM are still able to interact with p-TEFb through BD2, 33 the CTM is primarily responsible for the p-TEFb interaction. BRDT also contains a CTM, but the testis-specific expression of BRDT limits its relevance for disease. BRD4 co-localizes and is thought to interact with the Mediator complex through an undefined mechanism. 56 In addition, BET proteins localize at enhancer and super-enhancer regions through bromodomain-acetylated histone interactions. 30 At these regions, BRD4 interacts with a number of transcription factors including p53, 57 YY1, 58 c-Jun, 59 AP2, 59 C/EBPa/b, 60 and the Myc/Max heterodimer. 9,61 Some of these interactions are thought to be acetylation-independent, 57 whereas other interactions with GATA-1, 62 TWIST, 63 and NF-κΒ 64 are known to be acetylation-dependent. Importantly, BRD4 has specificity in recognition of these transcription factors as many general transcription factors were not enriched in immunoprecipitation studies in the absence of BRD4. 65 Given the unique domain organization and the relevant role of BRD4 in disease, it is the most widely studied among BET family proteins.
Chemical biology and genetics experiments have identified a differential role between BD1 and BD2. Using a bump-and-hole approach to selectively drug individual BET bromodomains, Baud et al. 47 showed BRD4-BD1 is important for maintaining BRD4 chromatin occupancy. In a subsequent publication the same group confirmed a similar role of BD1 in BRD2 and BRD3, and showed the weakest binding of chromatin from BRDT-BD2. 48 The work of Miller et al. confirms this inability of BRDT-BD2 to engage acetylated nucleosomes and suggests this domain may be responsible for binding acetylated non-histone proteins instead. 50 In the case of BRD4, multiple studies with reporter assays have shown using domain deletions 64 or mutations 48 that both BD1 and BD2 have some effect regulating gene expression, but the combination of both domains has a much stronger effect. 48 The same findings apply to BRD4 interactions with other transcription factors, including TWIST and GATA-1. 63 Alternate approaches to study domain biology using tools like CRISPR and the dTAG system 66 will further illuminate unique features of BET bromodomain function, as will the development of small-molecule tools to probe domain function.
In addition to their common mode of substrate recognition, the similar features of BET bromodomains also contributes to the challenge of selective druggability. Overall, the BET-family of bromodomains have between 36% and 87% sequence identity ( Figure 4A). BD1s of the BET family are 73%-87% identical to other BD1 bromodomains; this number is similar among BD2s, with 64%-84% identity. In contrast to high sequence identity for BD1 or BD2 in the family, there is a lower degree of conservation across BD1 and BD2 on the same protein (40%-45%). In light of this, developing monovalent inhibitors for both bromodomains of a single BET protein with selectivity over other BET proteins is a challenging task. Instead, molecules have been reported with selectivity for either BD1s or BD2s across the family and will be the primary focus of this review.

| STRATEGIES FOR DEVELOPING DOMAIN-SELECTIVITY
The substrate binding mode through asparagine and tyrosine, as well as neighboring residues like the "WPF-shelf" motif on the ZA-loop are conserved between BD1 and BD2. But there are eight residues that differ between BD1s and BD2s further away in the area surrounding the K ac binding site ( Figure 5 and red arrows in Figure 4B). Using a central water responsible for K ac recognition as a reference point for distance measurements, these residues are 8-15 Å away from the binding site. To design domain-selective BET inhibitors, four of these divergent residues have been frequently targeted for selectivity. These are an aspartic acid/histidine (Asp144/His437, numbering based on BRD4), glycine/aspartic acid (Gly143/Asp436), isoleucine/valine (Ile146/Val439), and a lysine/proline (Lys141/Pro434) residue which can be accessed from the K ac binding site with larger molecules. Due to the ease of crystallization, numerous structures of BRD4-BD1 or BRD2-BD2 are available and are used here as representative BD1 and BD2 domains, and their respective residue identifiers are used in descriptions of selectivity below.
More commonly targeted among these residues is the Asp144/His437 difference. An analysis of their respective rotamer states in all published BRD4 bromodomain crystal structures noted the preference of Asp144 to adopt a gauche(+) chi1 rotamer oriented away from the K ac binding site whereas His437 adopts a trans conformation and points toward the binding site ( Figure 6A). 53 The preferred conformation of Asp144 is influenced by subtle differences of additional nonconserved residues in the vicinity of the BC-loop (Lys141/Pro434 and Ile146/Val439), where the side chain of Lys141 is able to rotate inwards to form a hydrogen bond with Asp144 in BD1 and restrict its mobility. The relative proximity to the binding site, large differences in their relative positions occupied, and electrostatic and steric differences between these residues has allowed the design of selective inhibitors for a number of molecules described below by targeting this "Asp/His switch," and will be referred to as such below. 67 Another structural aspect of the emerging importance in BET inhibitor discovery is the role of water molecules.
Water molecules have been identified to play three different roles in determining domain-selectivity. First, in virtually all BET crystal structures, the base of the K ac binding pocket is occupied by a series of four structured waters (orange,  that partially displaced these structured waters and resulted in BD1-selectivity. Yu et al. 77 observed contrasting selectivity for BD2 in CDD-1102 by displacing the same waters. Although the two molecules have different binding modes and target additional conserved residues for selectivity, it is clear the displacement of waters from the K ac pocket plays a role in driving selectivity and the reorganization of waters is yet to be comprehensively explored.
Second, a different water molecule with relevance for domain selectivity is located at the cap of the C-helix.
This water (green, Figure 7) plays an important role in terminating the helical hydrogen bonding pattern of the helix 78 and is often displaced in BD1-inhibitor complexes leading to selectivity, but conserved with BD2-selective inhibitors. Upon displacement of the water in BD1, the adjacent residue (Asp145) can accommodate loss of this helix cap water through hydrogen bonding via its carboxylate. In BD2, the adjacent residue is Glu438, which is longer than Asp145 and cannot form the requisite hydrogen bonds.
Finally, many of the divergent residues between BD1 and BD2 are located on the BC and ZA-loops towards the outer periphery of the binding pocket. As a result, the outer sphere waters (red, Figure 7) are frequently involved in hydrogen-bonded networks between distant non-conserved residues and engagement of these waters results in some contribution to selectivity. These waters include the engagement of BD1-specific Gln85, Lys91 and Asp145 that are exposed to solvent. Strategies for designing selectivity have involved forming long-range, through-water interactions with these nonconserved residues.
In the design of domain selective inhibitors, three areas of molecular design have emerged that summarize strategies for developing selectivity and potency. Although these are not universally applicable to all domainselective molecules, the trends are as follows: • K ac mimic: All bromodomain inhibitors have a portion of the molecule functioning as the K ac mimic to interact with conserved asparagine and tyrosine residues in the binding pocket. BET inhibitors typically employ acetamides, methyl ketones, and acetyl groups, and heterocycles such as triazoles, oxazoles, pyridones or imidazoles. These groups will be highlighted in blue in the structures below.
• WPF shelf-aromatic interaction: Usually pendant to the K ac mimicking group are aromatic groups that interact with the WPF-shelf, often with the flexible tryptophan residue. Optimization of these groups is especially F I G U R E 7 Water molecules targeted for selectivity. Clusters of water molecules surrounding the K ac binding pocket that have been targeted for selectivity (PDB ID 4lyi). Waters shown as spheres, with Asn140 and Tyr97 of BRD4 BD1 shown for clarity. [Color figure can be viewed at wileyonlinelibrary.com] important for maintaining affinity for the bromodomains. In the structures below, WPF-shelf interacting motifs are highlighted in green.
• Steric/electronic clash: portions of the molecules that can extend beyond the WPF-shelf generally make the largest contributions to domain-selectivity. These groups typically thread a narrow valley between BC-and ZAloops to interact with divergent residues that are remote from the K ac binding pocket. These groups that can discriminate between BD1 and BD2 are highlighted in red below.
For simplicity, molecules with domain-selectivity are listed in chronological order and grouped by their respective selectivity profiles. The molecules are summarized in the timeline ( Figure 8) according to their initial reports. Acetylated lysine mimics are indicated in blue and WPF-shelf interacting groups are in green. These molecules are described in more detail below: Following early success with ligand development for the PCAF bromodomain, 79 the Zhou lab designed the first molecule with BD1 selectivity using structure-based design from CBP bromodomain inhibitor, MS120-a F I G U R E 8 Timeline for reports of BET-BD1 and BD2-biased inhibitors (motif colored in blue: K ac mimic; green: interacts with WPF-shelf; red: imparts domain selectivity). BET, bromodomain and extra-terminal. [Color figure can be viewed at wileyonlinelibrary.com] diazobenzene molecule with weak off-target BRD4 binding ( Figure 9A). 80 Aromatic π−π stacking interactions of MS120 analogues were optimized to engage a tryptophan residue on the BRD4 WPF-shelf and decrease CBP binding, with the arylsulfonamide of MS435 being an early promising lead. MS435 occupies a narrow hydrophobic channel between the K ac binding site and the ZA-loop. In the following rounds of optimization on the diazobenzene and arylsulfonamide ring, MS436 was identified with 10-fold improvement in potency from MS435 and approximately 10-fold BD1 selectivity for BRD4. A different substitution pattern on the phenolic ring aided hydrogen bonding with Asn140 and shifted the orientation of the diazobenzene ring ( Figure 9B). The linear diazobenzene scaffold contacts Lys91, over 12 Å away from the K ac binding pocket, and forms a hydrogen bond through the sulfonamide oxygen ( Figure 9C). Engagement of Lys91 is one of the proposed mechanisms of BD1 selectivity as the corresponding position on BD2 is Ala380. The precise orientation of the sulfonamide moiety is important to engage Lys91 as Lys374 from the ZA-loop of BD2 occupies a similar region. MS436 does not maintain BD1 selectivity in BRD3 in an FP assay or have any effect on viability of murine macrophage cells up to 100 µM.
The molecule does disrupt NO-synthesis upon LPS-stimulation presumably by inhibiting NF-κΒ activity, although BD2 is also inhibited at the concentrations required for activity in this assay.

| Olinone
A year following the report of MS436, the same group reported a second BD1-selective inhibitor with a different scaffold. 52 Olinone was synthesized in a short SAR series varying the length of the acetamido-alkyl group from an NMR-fragment screening hit, MS7972 ( Figure 10A). The four-carbon alkyl spacer on Olinone led to a K d of 3.4 µM against BRD4-BD1 and weaker affinities were reported for three, five, and six carbon analogues and an unacetylated four-carbon chain. Pan-BD1 selectivity of Olinone was confirmed using 2D-NMR, fluorescence anisotropy, and isothermal titration calorimetry (ITC) assays. A lactone of the tetrahydropyridone-core on Olinone interacts with Asp144 on BRD4-BD1 and is the cause for selectivity. His437 at the corresponding position in BD2 presents a steric and electronic clash against this group ( Figure 10B). The biological effects of Olinone were surprisingly the opposite of pan-BET inhibition in the case of myelin and cell-cycle genes responsible for differentiation. BD1 inhibition with Olinone favored differentiation of oligodendrocyte progenitors, whereas pan-BET inhibition prevented their differentiation and BD2 inhibition had no effect in this assay. The high BD1 selectivity of Olinone provided a glimpse into the unique biology that could be addressed by domain-selective inhibition, but the lack of potency prevented strong conclusions from being made regarding the biological role of BD1.

| Compound 1
Through focused screening of a protein-protein interaction (PPI)-biased library, Raux et al. 81 identified a xanthine derivative with affinity for BRD4-BD1 (6.1, Figure 11A). The compound stood out from other hits as it had a high entropic binding component by ITC. The authors presumed a reorganization of waters in BRD4-BD1 was the cause of this energetic profile, and crystallography was used to confirm this finding of surface water restructuring around the ZA-channel. In the co-crystal structure with BRD4-BD1, the xanthine core of 6.1 functioned as a K ac mimic and the triazolo-pyrimidine was oriented towards the ZA-channel ( Figure 11B). The triazolo moiety forms hydrogen bonds with Asp88 and van der Waals interactions with Leu92 and Gln85 on either side of the ZA-channel. BD1 selectivity is attributed to the triazolo-pyrimidine core interacting with Gln85, the corresponding position of which is occupied by lysine or arginine on BD2s and BRDT-BD1. Unlike MS436, which interacts with Gln85 through a water, 6.1 forms a direct interaction with Gln85. Removal of the triazolo-pyrimidine in fragment 6.2 ( Figure 11A) resulted in a loss of BD1-selectivity and potency, confirming the mechanism of selectivity. Gln85 is 13 Å away from the K ac binding site, and the extended conformation and unique geometry of thioether spacer on 6.1 allows access to this residue through the narrow ZA-channel of BRD4-BD1. In terms of biological activity, 6.1 had a weak effect

| MS402
Following two previous reports of BD1-selective inhibitors MS436 and Olinone, a third scaffold was reported by Zhou and coworkers. 82 MS402 has ninefold selectivity for BRD4-BD1 over BD2 ( Figure 12A), and maintains a slightly lower BD1 selectivity in BRD2 and BRD3, as well as selectivity over the non-BET family bromodomain of CBP. The cyclopentenone carbonyl group functions as the K ac mimic, forming hydrogen bonds with Asn140 and to F I G U R E 11 Triazolo-pyrimidine of Compound 1 causes BRD4-BD1 selectivity. (A) Structure of Compound 1 (6.1), identified from a custom protein-protein interaction inhibitor library and fragment 6.2 to demonstrate loss of BD1 selectivity (molecule colored as defined in Figure 10 Tyr97 through a bridging water ( Figure 12B). In a similar manner to M436, MS402 passes through the ZA-channel and forms a water-mediated hydrogen bond with Gln85 interacting with the amide nitrogen of MS402. Mutation of Gln85 to alanine or lysine (as in BD2) resulted in weaker binding of MS402. As a secondary mechanism of selectivity, the authors propose van der Waals contacts of Ile146, which is a smaller Val439 in BD2, may provide additional weak contributions to selectivity. MS402 inhibited IL-17 and IFN-γ production in T-cells while it did not have any effect on cell proliferation. MS402 also decreased C-terminal RNAPol-II phosphorylation and BRD4 chromatin occupancy but did not affect BRD2 chromatin occupancy. In comparison with pan-BET inhibition, BD1 inhibition had a greater effect on immune or cytokine ontologies, and only had an effect on half as many genes (937 vs. 546 up, and 842 vs. 437 down with JQ1 and MS402, respectively, at FDR < 0.1 and fold change >1.5; compound concentrations unreported). Pan-BET inhibition also had a stronger effect on housekeeping genes than BD1 inhibition, whereas BD1 inhibition had an equal effect at super-enhancers. Finally, MS402 also showed promising efficacy in a colitis model for treatment of inflammatory bowel disease.

| 3U
In contrast to previous BD1-selective inhibitors, Ma et al. 83 took a different approach by rationally designing selectivity from a pan-BET inhibitor scaffold. The dimethyl-thiophene substituents from (+)-JQ1 are oriented towards Asp144 in BRD4-BD1 but remains >5 Å away. Large groups introduced at this position were hypothesized to clash with the equivalent His437 in BD2. The authors synthesized analogues cyclizing the vicinal dimethyl groups of (+)-JQ1 and adding bulky groups designed to access an unoccupied region near Asp144 ( Figure 13). The K ac mimic 1,2,4-triazole ring was replaced with a dimethylisoxazole, another common K ac mimicking heterocycle.
Additionally, contraction of the diazepine ring common to pan-BET inhibitors into a pyrimidinone yielded the optimal orientation of new steric bulk to clash with His437. 3u displayed >180-fold selectivity for BD1 in BRD4 by an Alphascreen assay, although domain selectivity in other BET proteins was not reported. A number of the resulting tricyclic molecules displayed antiproliferation EC 50 values comparable to pan-BET inhibitors and superior to that of BD2 inhibitor RVX208 across a panel of cancer cell lines. 3u was assayed as the racemate for its antifibrotic activity, where it decreased expression of collagen-I more effectively than pan-BD2/pan-BET inhibition.

| LT052
Jiang et al. 56  inhibitor RVX208 in these assays. Furthermore, LT052 had promising effects in vivo in a rat gout arthritis model, decreasing joint swelling and macrophage activation at sites of induced arthritis to baseline levels.

| iBET-BD1 (GSK778)
Following the initial report of the biological activity of iBET-BD1 and iBET-BD2, 19 Wellaway et al. 53 reported the development route of iBET-BD1 from a pan-BET imidazoquinolinone-based inhibitor with a slight BD1-bias, iBET151. The imidazoquinolinone 8-position of iBET151 was identified as orienting towards the Asp/His switch. In an initial round of SAR, the addition of ether-linked basic substituents at this position decreased affinity for both BRD4 bromodomains, but to a greater extent for BD2. An (S)-3-methylene pyrrolidinyl group (6.5, Figure 15A) was chosen for further development as it led to the most favorable BD1 selectivity. BRD4-BD1 affinity of this scaffold was improved by addition of a methoxymethyl on imidazoquinoline 6.6 ( Figure 15A). A combination of these two modifications yielded iBET-BD1 with 160-fold BD1 selectivity. Selectivity was rationalized by X-ray crystallography, where the pyrrolidine interacts with Asn140, Asp144, and Asp145 in BD1 via bridging waters ( Figure 15B,C).
The pyrrolidine is hindered by His437 and forced to adopt a disfavored pose in BD2 (cyan, Figure 15D). Cellular Through optimization of a BD2-selective inhibitor scaffold from RXV208, Liu et al. 84 developed chromone derivatives with BD1-selectivity and a preference for BRD4-BD1. A close analogue of RVX208 lacking a 2-hydroxyethyl group, RXV-OH, was found to bind BRD4-BD1 in a reversed orientation from that of RVX208 with BRD4-BD2 ( Figure 16B). To achieve selectivity for BRD4-BD1, compounds were synthesized targeting Asn93, a residue present in BRD4-BD1 and BRD3-BD1 but replaced with Gly, Glu, or Gln in other BET bromodomains.
Synthesized compounds were profiled by RT-qPCR at single point concentrations for expression of inflammatory response genes IL-6 and CIG5. ZL0516 contains a chiral alcohol that interacts with Asn93 to provide 9-43-fold selectivity for BRD4-BD1 against other BD1s and a 9-38-fold selectivity over BD2s. Although the mechanism for selectivity of BRD4-BD1 over BRD3-BD1 remains unclear from the proposed model, ZA-loop rigidity in other BD1s may not accommodate the large piperazine group. Upon oral or i.p. administration of ZL0516, potent effects on inflammatory response genes, IL-6 and CIG5 were observed, as well as a decrease in H3K122 acetylation levels.
This acetylation mark is described as a BRD4 biomarker, although in this context, the regulatory mechanism of this modification and its dependence on BRD4-BD1 are unclear and may be due to residual BRD4 HAT activity.
Additionally, other BET bromodomains are inhibited at the compound concentrations used in these studies and as a result the gene regulatory effects may not entirely be attributed to BRD4-BD1.

| UMN627
Using a protein-observed 19 F-NMR method, Urick et al. 85 identified a trisubstituted imidazole scaffold of published p38α kinase inhibitors with selectivity for the BET-family over a non-BET bromodomain. On further analysis of this scaffold for intra-BET selectivity, Divakaran et al. 76 identified a >55-fold preference for BET-BD1 binding. This selectivity was mediated in part by displacement of structured waters from the K ac binding site by a p-fluorophenyl group. Additional selectivity was attributed to flexibility of a BD1-conserved YNKP-motif, which is replaced by a more rigid YNPP motif in BET-BD2s. BD1 affinity was further improved by installation of alkyl amines to target an acidic region around D144 in BRD4 (e.g., UMN627, Figure 17A). Replacement of the p-fluorophenyl group with a p-trifluoro-toluyl group of this scaffold displaced additional waters from the K ac binding pocket to improve BD1 selectivity and results in an approximately 35-fold improvement in potency. The water-displacing aryl group stabilizes a BRD4-BD1 conserved Tyr98 by occupying a deeper portion of the binding pocket in comparison to other K ac mimics, which may help to explain the preference for BRD4-BD1 over other BET-BD1s ( Figure 17B,C).

UMN627 and its related analogues show a surprising upregulation of c-Myc in MM.1S multiple myeloma cells,
an effect that is in contrast with the strong effect pan-BET inhibitors have on the Myc oncogene. 86 UMN627 was able to decrease CCL2 (also called MCP-1) expression and neutrophil infiltration in LPS-stimulated mice as a potential therapy route for alcoholic hepatitis. 87 These results are similar to effects observed with pan-BET inhibitor, iBET-151, however, additional BET bromodomains are likely to be inhibited at the concentrations tested.
Alternatively, a related triazole scaffold displays pan-BD1 inhibition but is able to maintain potent Myc inhibition. 88 In subsequent reports, improved BRD4-BD1 selectivity and potency has been reported with iBRD4-BD1 89 and 26 90 (Figure 17; 23-6200-fold and >500-fold BRD4-BD1 selectivity, respectively) through modifications of the dimethyl-aryl ring. In an FP assay, the IC 50 for BRD4-BD1 was <92 nM and incomplete inhibition was observed for all other BET bromodomains at 50 μM (the highest concentration tested; ΒRDT-BD2 was not tested). 90 This selectivity was verified by ITC, in which case 26 bound to BRD4-BD1 with a K d of 15 nM. Moreover, the closest offtarget, BRD2-BD1, showed no measurable binding to 26 under the same conditions. 90 The high selectivity of these newer analogues was also used to design selective BRD4 degraders, 89 and together may provide a clearer evaluation of the biological effects from targeting BRD4-BD1 alone.

| GSK789
An orthogonal BD1-selective scaffold was reported by GSK shortly after iBET-BD1. GSK789 is derived from a naphthyridone-based ATAD2 bromodomain inhibitor 6.7 that displayed weak BET-bromodomain binding ( Figure 18A). Conversion of the ether linkage on 6.7 to an amide in compound 6.8 improved interactions in the WPF shelf (RVF shelf in ATAD2) and biased selectivity toward BET bromodomains, with a preference for BRD4-BD1. The naphthyridone core forms three hydrogen bonds with Asn140 ( Figure 18B) and introduction of the amide facilitates a through-water hydrogen-bond with Ile146 ( Figure 18C), while blocking binding in BD2 through a clash with the Asp/His switch residue His437 ( Figure 18D). This interaction is similar to an Ile146 hydrogen bond observed with Olinone ( Figure 18E).  that was able to interact with Glu151, which is located distal to the K ac binding pocket and replaced by Arg444 at the corresponding position in BRD4-BD2. In an X-ray co-crystal structure, ZL0590 occupied a region flanked by the αB and αC helices and the BC-loop and formed additional interactions with Tyr137, Gly143, Asp144, and Glu154 ( Figure 19B,C). Although PPIs are not known to occur at this shallow site, the binding mode of ZL0590 is analogous to that of typical PPI inhibitors that are able to occupy shallow, plastic surfaces.
As the reported SAR in the development of ZL0590 was based on PCR from cellular inhibition of poly(I:C)induced inflammatory genes, it remains unclear what molecular features favor binding at this newly identified binding site to drive domain-selectivity and contribute to the lack of observed binding at the orthosteric K ac pocket.
Nevertheless, in light of the strong anti-inflammatory effects observed in vivo with ZL0590 (attenuation of cytokine expression and neutrophils in orally dosed mice before airway inflammatory stimulation with poly(I:C)), occupancy of this novel site is able to affect BET-K ac recognition either directly or indirectly. The interesting binding mechanism of ZL0590 deserves to be explored further to identify if this site can be exploited for unique phenotypic effects and to understand its general druggability (Table 1).

| RVX-208
A derivative of the plant hormone resveratrol, RVX-208 was reported as a candidate from Reseverologix for the treatment of atherosclerosis. RVX-208 was initially identified from a phenotypic screen of compounds that increase the concentrations of high-density lipoprotein, ApoA1, without affecting cellular proliferation. 93 The mechanism of action was identified to be through BRD4-BD2 (K d = 130 nM), with the quinalzolinone functioning as the acetyl lysine mimic. 94 Ninefold selectivity over BD1 was partially achieved by packing of His437 against the dimethylphenyl ring of RVX-208, and additional contributions were through interactions of the 2-hydroxyethyl group. The mechanism of selectivity from the 2-hydroxyethyl group is undefined, but its removal in RVX-OH resulted in a flipped binding pose that loses interactions with His437 and a loss of selectivity ( Figure 16B). RVX-208 is able to displace BRD4 and BRD3 from chromatin in a FRAP assay, however, this effect is at concentrations where both BD1 and BD2 are inhibited and thus the role of BD2 in this process cannot be adequately inferred. 94 More importantly, the transcriptional effects of BD2 inhibition with RVX-208 is more limited than that of pan-BET inhibition; only 46 genes were affected >1.5-fold with RVX-208 compared with 754 by JQ1. A potential source for the weak antiproliferative activity observed with BD2 inhibition was the observation that RVX-208 had no effect on levels of c-Myc. Before 2016, RVX-208 was the only reported inhibitor with modest pan-BD2 selectivity. RVX-208 has progressed through clinical trials for atherosclerosis and cardiovascular disease and received breakthrough status from the FDA.
Law et al. 96 utilized a fragment-based approach to optimize BD2-selectivity for a tetrahydroquinoline scaffold, which had weak BET-binding and high-ligand efficiency. The fragment 7.2 ( Figure 21A) was optimized at two positions, and these modifications were combined onto the quinoxaline scaffold. First, addition of a toluyl group on a tetrahydroquinoxaline scaffold improved BRD4-BD1 interactions in the aromatic WPF-shelf (7.3, Figure 21A).
Expansion of the tetrahydroquinoxaline 2-methyl group into a chiral cyclopropane (7.4, Figure 21A addition of polar groups to the solvent exposed region to yield GSK268 (acetamide-substituted pyrimidine, Figure 21B) and GSK340 (tetrahydropyridine, Figure 21C). Both of these additional groups form extensive hydrogen bonding networks to solvent exposed waters and the resulting molecules have 5-50-fold BET-BD2 selectivity.

| BY27
Inspired by diazepine-containing BET-inhibitors, BY27 was designed to interact with the same BD2conserved His described above. 97 Addition of a benzylic amine spacer to pan-BET azepine-containing analogue 7.5 ( Figure 22A) resulted in compound 7.6 ( Figure 22A) with ninefold BD2 selectivity. A subsequent round of optimization to the aminopyridine ring identified a methyl-pyrazole with stronger affinity for BRD4-BD2 when tested as the racemate (K i = 15 nM). Separation of the isomers identified a more potent R-isomer (BY27, Figure 22A) and the weaker S-isomer, 7.7. Molecular docking studies with bromodomains of BRD2 identified the mode of binding and mechanism of selectivity through interaction with His433. The triazole of BY27 acts as the acetyl lysine mimic through interaction with Asn140 and Tyr97. The amine in the R-isomer of BY27 is well-positioned to interact through a network of waters and flip His433 from an "open" to a "closed" conformation ( Figure 22B). This interaction is not accessible with Asp160 occupying the corresponding position in BD1. BY27 was tested for cellular antiproliferation, effects on c-Myc and more broadly in gene expression, but due to the low degree of BD2 selectivity (5-38-fold) and the concentrations tested in these assays (150x above BRD4-BD1 K i ), the results of these experiments are more indicative of pan-BET inhibition than BD2-specific activity.

| ABBV-744
ABBV-744 was developed from a pan-BET inhibitor ABBV-075 through rational design. Addition of an ethyl amide to the pyrollo-pyridone of ABBV-075 resulted in a slight loss of BRD4-BD1 affinity (7.8, Figure 23A). 18 In crystal structures with BRD2 bromodomains, the N-methyl moiety acts as an acetyl-lysine mimic and forms three hydrogen bonds with Asn429 ( Figure 23B), whereas the additional ethyl amide occupies a channel between His433, Tyr386, and Pro430. The corresponding His433/Asp160 in BD1 is unable to form this narrow channel and likely results in the weaker BD1 binding ( Figure 23C). Extension of the methyl-sulfonate and replacement of the difluorophenyl ring with a dimethylphenolic ether maintained BD2 potency, but further impaired BD1 binding through a steric clash with Ile162. The smaller Val435 in BD2 can accommodate the additional methyl group ( Figure 23D). Finally, optimization of metabolic liabilities and for improved PK properties yielded ABBV-744 with over 325-fold BD2 selectivity in BRD4. 98

| SJ432
Using the tetrahydroquinoline pan-BET inhibitor scaffold of SJ599 ( Figure 25) 100 as a starting point, Slavish et al. 100 reported the optimization of BD2 selectivity. The furan ring on SJ599 was modified first and various metasubstituted phenyl rings were explored. A meta-acetamide was identified for improved interactions with the WPF shelf. Additional modifications to the isopropyl carbamate were explored for selectivity and a para-aminobenzoyl ester was identified to have a high degree of BD2 selectivity through interactions with His433 of the Asp/His switch. Finally, the two rings introduced for affinity and selectivity were modified due to their poor metabolic stability and high lipophilicity. Replacement of the ester with a p-nitrile, and the aryl acetamide group with a pyrazole resulted in the development of SJ432, which is currently under evaluation in N-Myc-driven neuroblastoma models.

| GSK097, GSK549, and GSK852
A further scaffold with BD2 selectivity was reported by GSK from the optimization of fragment 7.12 with sevenfold BD2 bias in BRD4 ( Figure 26A). The pyridine-containing fragment has an acetamide that functions as the K ac mimic and a benzyl group that orients towards the WPF shelf. Introduction of a second amide with substituents at the pyridone C5-position, analogous to those previously reported by Abbvie and GSK, occupied the channel between Leu383, Tyr428, and Asn429 of BRD2 to improve potency and approach His433 to provide BD2 selectivity ( Figure 26B). Replacement of the benzyl group with an indole introduced an additional hydrogen bond to Asp434 from the indole NH ( Figure 26C) to provide a fivefold improvement in BRD4-BD2 binding.
DMPK liabilities of this scaffold were optimized over three subsequent reports. First, the pyridinecarboxamide GSK097 was developed to address the low solubility of GSK549 ( Figure 26D). 101 To mimic the orientation of benzyl and indole groups in GSK620/GSK549, which are constrained by an intramolecular 7.9 | GSK217/GSK737 Upon further preclinical evaluation, GSK046 was abandoned due to the geneotoxic risk from bioactivation of the anilinecontaining ring. Aylott et al. 104 reported a scaffold hopping approach to replace the acetamido K ac mimic with a methyltriazole group, a strategy used in other BET-inhibitors ( Figure 24E). The torsional differences on the aryl ring resulting from these changes allowed replacement of the cyclohexyl group from GSK046 with cyclopropyl and ethyl amides on GSK737 and GSK217, respectively. In the case of GSK046, placement of the amide group para to the acetamide K ac mimic provides an ideal vector accessing the ZA-channel. These substituents were placed meta to the methyltriazole K ac mimic in GSK217 and GSK737 to account for the larger size of the triazole. Additionally, the methyl benzene was replaced with methyl pyridine and indole in the two molecules to decrease overall lipohilicity. Both molecules had improved metabolic stability over GSK046 while maintaining BD2-potency and selectivity.

| CDD-1102
A DNA-encoded library synthesis approach was used by Yu et al. 77 to discover novel BD2-selective molecule CDD-1102 ( Figure 27A). A split-and-pool library was synthesized with over 100 million compounds and screened against 6xHis-BRDT-BD2 alone and in competition with JQ1, then enriched by Ni-NTA affinity resin. Following additional rounds of negative selection against 6xHis-BRDT-BD1 and no-protein controls, a small panel of compounds were identified and resynthesized for testing by Alphascreen. While this manuscript was in review, the reverse of this selection experiment was also performed by the same group to identify BD1-selective molecule CDD-787 with over 500-fold selectivity (see Table 3 below). 107 The binding pose of CDD-1102 in a crystal structure with BRDT-BD1 shows unique features of molecular recognition. Whereas traditional K ac mimics directly interact with the carboxamide nitrogen of a conserved asparagine residue and through a conserved water molecule to a tyrosine residue deeper in the binding pocket (Asn351 and Try308 in BRDT), CDD-1102 does not directly interact with the carboxamide of Asn351 and displaces the conserved water ( Figure 27B). The aniline on CDD-1102 sits deeper in the binding pocket than traditional K ac mimics and is responsible for displacing the conserved water to engage Tyr308 directly, which is a unique interaction not observed in other BET inhibitors. The indazole and phenylenediamine rings of CDD-1102 stack against the WPF shelf and the extended conformation allow these rings to occupy a narrow channel between the ZA-and BC-loops ( Figure 27C). The internal amide between the indazole and phenylenediamine rings interacts with conserved Asn351 and BD2-specific His355 ( Figure 27D). Additional interactions with BD2 conserved His355 and Pro352 (Asp and Lys in BD1, respectively) provide the high degree of BD2 selectivity observed.

| GSK040
A second DNA-encoded library approach to discover BD2-selective inhibitors was reported by GSK shortly after CDD-1102. The screening strategy differed from that of Yu et al. 77 in the use of BRD4 tandem-bromodomain constructs with alanine mutations to tyrosine residues in the K ac binding pockets, an approach that may more accurately recapitulate potential inter-bromodomain interactions. Bisaryl analogues were identified to have selectivity for BRD4-BD2 via this screening strategy and DNA-free analogue 7.13 ( Figure 28) was synthesized. In a BRD4 crystal structure with the DNA-free analogue 7.13, the methoxy group was able to displace a structured water molecule while functioning as the K ac mimic. Replacement of the highly lipophilic dimethyl methoxyphenyl K ac -mimetic group with a dimethyl pyridone for a more conventional K ac -mimic, and substitution of one WPF-shelf interacting geminal diphenyl groups with a pyridine improved solubility and permeability in compound 7.14. Finally, introduction of a tetrahydropyran ring in GSK040 to target the ZA-channel improved the affinity and yielded >5000-fold selectivity towards BRD4-BD2 over BD1 ( Since the initial reports of domain-selectivity in 2013 with MS436 and RVX-208, rapid advances have been made to improve potency and broaden selectivity windows, and these molecules have been used to define the unique biology of BD1 and BD2 on BET proteins. Given its proximity to the binding site and the marked differences in steric and electrostatic properties of the Asp/His switch, it is unsurprisingly the most readily targeted residue for selectivity. However, additional subtleties between BD1 and BD2, such as a Val-Ile and water displacement, have also contributed to selectivity. And finally, residues at a greater distance away from the binding site have proven to be suitable targets for selectivity if the right vectors can be designed on scaffolds.
The development of inhibitors with enhanced selectivity within the BET-family has allowed more focused targeting of cellular processes. Inhibition of BET-BD1s appears to broadly affect cellular proliferation and simultaneously has potent anti-inflammatory effects. On the contrary, domain-selective inhibition of BET-BD2s also results in an anti-inflammatory outcome but does not affect proliferation of most cell-types. 19 These effects likely lead to the higher tolerability of domain-selective BET inhibitors, 18,100 but it remains to be seen if the cytotoxic effects of BD2 inhibitors are indeed driven by BD2 or instead caused by weaker off-target interactions.
The next frontier in domain selectivity remains the development of molecules with specificity for individual BET domains. Recent reports of molecules with a preference for BRD4-BD1 such as ZL0513 84 and trisubstituted imidazoles such as UMN627 86 and 26, 90 are indications that such molecules will be developed in due course.
Further, covalent approaches to selectively target BET bromodomains have been reported 108,109 at both the K ac recognition site and exterior surfaces. Through these combined approaches it remains to be seen how the efficacy of domain-specific inhibitors will compare with domain-selective inhibitors. Nevertheless, the promise and utility of domain-selective BET inhibition merits their design and biological evaluation.

| ADDENDUM
While this manuscript was under review, several additional domain-selective inhibitors have been reported. These are summarized in Table 3.