Design of Class I/IV Bromodomain-Targeting Degraders for Chromatin Remodeling Complexes.

Targeted protein degradation is an emerging technology that can be used for modulating the activity of epigenetic protein targets. Among bromodomain-containing proteins, a number of degraders for the BET family have been developed, while non-BET bromodomains remain underexplored. Several of these proteins are subunits in chromatin remodeling complexes often associated with oncogenic roles. Here, we describe the design of class I (BPTF and CECR2) and IV (BRD9) bromodomain-targeting degraders based on two scaffolds derived from pyridazinone and pyrimidine-based heterocycles. We evaluate various exit vectors and linkers to identify analogues that demonstrate selectivity within these families. We further use an in-cell NanoBRET assay to demonstrate that these heterobifunctional molecules are cell-permeable, form ternary complexes, and can degrade nanoluciferase-bromodomain fusions. As a first example of a CECR2 degrader, we observe that our pyrimidine-based analogues degrade endogenous CECR2 while showing a smaller effect on BPTF levels. The pyridazinone-based compounds did not degrade BPTF when observed through Western blotting, further supporting a more challenging target for degradation and a goal for future optimization.

Development of an N-Terminal BRD4 Bromodomain-Targeted Degrader.

Targeted protein degradation is a powerful induced-proximity tool to control cellular protein concentrations using small molecules. However, the design of selective degraders remains empirical. Among bromodomain and extra-terminal (BET) family proteins, BRD4 is the primary therapeutic target over family members BRD2/3/T. Existing strategies for selective BRD4 degradation use pan-BET inhibitors optimized for BRD4:E3 ubiquitin ligase (E3) ternary complex formation, but these result in residual inhibition of undegraded BET-bromodomains by the pan-BET ligand, obscuring BRD4-degradation phenotypes. Using our selective inhibitor of the first BRD4 bromodomain, iBRD4-BD1 (IC50 = 12 nM, 23- to 6200-fold intra-BET selectivity), we developed dBRD4-BD1 to selectively degrade BRD4 (DC50 = 280 nM). Notably, dBRD4-BD1 upregulates BRD2/3, a result not observed with degraders using pan-BET ligands. Designing BRD4 selectivity up front enables analysis of BRD4 biology without wider BET-inhibition and simplifies designing BRD4-selective heterobifunctional molecules, such as degraders with new E3 recruiting ligands or for additional probes beyond degraders.

Alternative Mechanisms for DNA Engagement by BET Bromodomain-Containing Proteins.

Epigenetic reader domains regulate chromatin structure and modulate gene expression through the recognition of post-translational modifications on histones. Recently, reader domains have also been found to harbor double-stranded (ds) DNA-binding activity, which is as functionally critical as histone association. Here, we explore the dsDNA recognition of the N-terminal bromodomain of the bromodomain and extra-terminal (BET) protein, BRD4. Using protein-observed 19F NMR, 1H-15N HSQC NMR, electrophoretic mobility shift assays (EMSA), and competitive-inhibition assays, we establish the binding surface of dsDNA and find it to be largely overlapping with the acetylated histone (KAc)-binding site. Rather than engaging in electrostatic contacts, we find dsDNA to interact competitively within the KAc-binding pocket. These interactions are distinct from the highly homologous BET bromodomain, BRDT. Nine additional bromodomains have also been characterized for interacting with dsDNA, including tandem BET bromodomains. Together, these studies help establish a binding model for dsDNA interactions with BRD4 bromodomains and elucidate the chromatin-recognition mechanisms of the BRD4 protein for regulating gene expression.

A Structure-based Design Approach for Generating High Affinity BRD4 D1-Selective Chemical Probes.

Chemical probes for epigenetic proteins are essential tools for dissecting the molecular mechanisms for gene regulation and therapeutic development. The bromodomain and extra-terminal (BET) proteins are master transcriptional regulators. Despite promising therapeutic targets, selective small molecule inhibitors for a single bromodomain remain an unmet goal due to their high sequence similarity. Here, we address this challenge via a structure-activity relationship study using 1,4,5-trisubstituted imidazoles against the BRD4 N-terminal bromodomain (D1). Leading compounds 26 and 30 have 15 and 18 nM affinity against BRD4 D1 and over 500-fold selectivity against BRD2 D1 and BRD4 D2 via ITC. Broader BET selectivity was confirmed by fluorescence anisotropy, thermal shift, and CETSA. Despite BRD4 engagement, BRD4 D1 inhibition was unable to reduce c-Myc expression at low concentration in multiple myeloma cells. Conversely, for inflammation, IL-8 and chemokine downregulation were observed. These results provide new design rules for selective inhibitors of an individual BET bromodomain.

New Design Rules for Developing Potent Cell-Active Inhibitors of the Nucleosome Remodeling Factor (NURF) via BPTF Bromodomain Inhibition.

The nucleosome remodeling factor (NURF) alters chromatin accessibility through interactions with its largest subunit,the bromodomain PHD finger transcription factor BPTF. BPTF is overexpressed in several cancers and is an emerging anticancer target. Targeting the BPTF bromodomain presents a potential strategy for its inhibition and the evaluation of its functional significance; however, inhibitor development for BPTF has lagged behind those of other bromodomains. Here we describe the development of pyridazinone-based BPTF inhibitors. The lead compound, BZ1, possesses a high potency (Kd = 6.3 nM) and >350-fold selectivity over BET bromodomains. We identify an acidic triad in the binding pocket to guide future designs. We show that our inhibitors sensitize 4T1 breast cancer cells to doxorubicin but not BPTF knockdown cells, suggesting a specificity to BPTF. Given the high potency and good physicochemical properties of these inhibitors, we anticipate that they will be useful starting points for chemical tool development to explore the biological roles of BPTF.

4-Methyl-1,2,3-Triazoles as N-Acetyl-Lysine Mimics Afford Potent BET Bromodomain Inhibitors with Improved Selectivity.

The bromodomain and extra terminal (BET) protein family recognizes acetylated lysines within histones and transcription factors using two N-terminal bromodomains, D1 and D2. The protein-protein interactions between BET bromodomains, acetylated histones, and transcription factors are therapeutic targets for BET-related diseases, including inflammatory disease and cancer. Prior work demonstrated that methylated-1,2,3-triazoles are suitable N-acetyl lysine mimetics for BET inhibition. Here we describe a structure-activity relationship study of triazole-based inhibitors that improve affinity, D1 selectivity, and microsomal stability. These outcomes were accomplished by targeting a nonconserved residue, Asp144 and a conserved residue, Met149, on BRD4 D1. The lead inhibitors DW34 and 26 have a BRD4 D1 Kd of 12 and 6.4 nM, respectively. Cellular activity was demonstrated through suppression of c-Myc expression in MM.1S cells and downregulation of IL-8 in TNF-α-stimulated A549 cells. These data indicate that DW34 and 26 are new leads to investigate the anticancer and anti-inflammatory activity of BET proteins.

Opportunity knocks for uncovering the new function of an understudied nucleosome remodeling complex member, the bromodomain PHD finger transcription factor, BPTF.

Nucleosome remodeling provides access to genomic DNA for recruitment of the transcriptional machinery to mediate gene expression. The aberrant function of nucleosome remodeling complexes has been correlated to human cancer, making them emerging therapeutic targets. The bromodomain PHD finger transcription factor, BPTF, is the largest member of the human nucleosome remodeling factor NURF. Over the last five years, BPTF has become increasingly identified as a protumorigenic factor, prompting investigations into the molecular mechanisms associated with BPTF function. Despite a druggable bromodomain, small molecule discovery is at an early stage. Here we highlight recent investigations into the biology being discovered for BPTF, chemical biology approaches used to study its function, and small molecule inhibitors being designed as future chemical probes and therapeutics.

Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement*.

Bromodomain and extra-terminal (BET) family proteins, BRD2-4 and T, are important drug targets; however, the biological functions of each bromodomain remain ill-defined. Chemical probes that selectively inhibit a single BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here, we develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays, we show that our lead compound is 9-33 fold selective for BRD4 D1 over the other BET bromodomains. X-ray crystallography supports a role for the selectivity based on reorganization of a non-conserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers, BRD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation may shed light on differential BET bromodomain functions.

New inhibitors for the BPTF bromodomain enabled by structural biology and biophysical assay development.

Bromodomain-containing proteins regulate transcription through protein-protein interactions with chromatin and serve as scaffolding proteins for recruiting essential members of the transcriptional machinery. One such protein is the bromodomain and PHD-containing transcription factor (BPTF), the largest member of the nucleosome remodeling complex, NURF. Despite an emerging role for BPTF in regulating a diverse set of cancers, small molecule development for inhibiting the BPTF bromodomain has been lacking. Here we cross-validate three complementary biophysical assays to further the discovery of BPTF bromodomain inhibitors for chemical probe development: two direct binding assays (protein-observed 19F (PrOF) NMR and surface plasmon resonance (SPR)) and a competitive inhibition assay (AlphaScreen). We first compare the assays using three small molecules and acetylated histone peptides with reported affinity for the BPTF bromodomain. Using SPR with both unlabeled and fluorinated BPTF, we further determine that there is a minimal effect of 19F incorporation on ligand binding for future PrOF NMR experiments. To guide medicinal chemistry efforts towards chemical probe development, we subsequently evaluate two new BPTF inhibitor scaffolds with our suite of biophysical assays and rank-order compound affinities which could not otherwise be determined by PrOF NMR. Finally, we cocrystallize a subset of small molecule inhibitors and present the first published small molecule-protein structures with the BPTF bromodomain. We envision the biophysical assays described here and the structural insights from the crystallography will guide researchers towards developing selective and potent BPTF bromodomain inhibitors.

NMR Analyses of Acetylated H2A.Z Isoforms Identify Differential Binding Interactions with the Bromodomain of the NURF Nucleosome Remodeling Complex.

Gene specific recruitment of bromodomain-containing proteins to chromatin is affected by post-translational acetylation of lysine on histones. Whereas interactions of the bromodomain with acetylation patterns of native histones (H2A, H2B, H3, and H4) have been well characterized, the motif for recognition for histone variants H2A.Z I and H2A.Z II by bromodomains has yet to be fully investigated. Elucidating these molecular mechanisms is crucial for understanding transcriptional regulation in cellular processes involved in both development and disease. Here, we have used protein-observed fluorine NMR to fully characterize the affinities of H2A.Z I and II acetylation patterns for BPTF's bromodomain and found the diacetylated mark of lysine 7 and 13 on H2A.Z II to have the strongest interaction with K7ac preferentially engaging the binding site. We further examined the selectivity of H2A.Z histones against a variety of bromodomains, revealing that the bromodomain of CECR2 binds with the highest affinity and specificity for acetylated H2A.Z I over isoform II. These results support a possible role for different H2A.Z transcriptional activation mechanisms that involve recruitment of chromatin remodeling complexes.

Selectivity, ligand deconstruction, and cellular activity analysis of a BPTF bromodomain inhibitor.

Bromodomain and PHD finger containing protein transcription factor (BPTF) is an epigenetic protein involved in chromatin remodelling and is a potential anticancer target. The BPTF bromodomain has one reported small molecule inhibitor (AU1, rac-1). Here, advances made on the structure-activity relationship of a BPTF bromodomain ligand are reported using a combination of experimental and molecular dynamics simulations leading to the active enatiomer (S)-1. Additionally, a ligand deconstruction analysis was conducted to characterize important pharmacophores for engaging the BPTF bromodomain. These studies have been enabled by a protein-based fluorine NMR approach, highlighting the versatility of the method for selectivity, ligand deconstruction, and ligand binding. To enable future analysis of biological activity, cell growth analyses in a panel of cancer cell lines were carried out using CRISPR-Cas9 and (S)-1 to identify cell-based model systems that are sensitive to BPTF inhibition.