PBRM1 is a subunit of the PBAF chromatin remodeling complex that uniquely contains six bromodomains. PBRM1 can operate as a tumor suppressor or tumor promoter. PBRM1 is a tumor promoter in prostate cancer, contributing to migratory and immunosuppressive phenotypes. Selective chemical probes targeting PBRM1 bromodomains are desired to elucidate the association between aberrant PBRM1 chromatin binding and cancer pathogenesis and the contributions of PBRM1 to immunotherapy. Previous PBRM1 inhibitors unselectively bind SMARCA2 and SMARCA4 bromodomains with nanomolar potency. We used our protein-detected NMR screening pipeline to screen 1968 fragments against the second PBRM1 bromodomain, identifying 17 hits with K d values from 45 μM to >2 mM. Structure–activity relationship studies on the tightest-binding hit resulted in nanomolar inhibitors with selectivity for PBRM1 over SMARCA2 and SMARCA4. These chemical probes inhibit the association of full-length PBRM1 to acetylated histone peptides and selectively inhibit growth of a PBRM1-dependent prostate cancer cell line.

The islets of Langerhans are a heterogeneous mixture of endocrine (hormone‐secreting) cells in the mammalian pancreas. Of particular interest are the β‐cells responsible for maintaining glucose homeostasis by secreting insulin in response to increased blood glucose. Type 1 diabetes (T1D) is characterized by autoimmune‐mediated destruction of β‐cells, resulting in an inability to produce and release insulin in response to changing glucose levels and subsequent reliance on daily insulin injections. There is currently no cure for T1D. The bromodomain and extra‐terminal domain (BET) family of proteins, comprised of BRD2, BRD3, BRD4 and BRDT, are a druggable family of proteins that can be targeted by pan‐BET bromodomain inhibitors (BETi), including the small molecule (+)‐JQ1. Recent studies have shown that targeting the BET family of proteins with BETi may prevent and treat T1D. However, the systemic use of current generation BETi is associated with off‐target, adverse effects including fatigue, gastrointestinal toxicity and memory loss. Since any treatment for T1D would be widely used in a pediatric patient population, avoiding off‐target effects is imperative. Here, we propose to ameliorate the off‐target effects of BET bromodomain inhibition by targeting BETi directly to pancreatic β‐cells. To achieve this, we exploited the high concentration of Zn2+ ions (~20 mM) present in β‐cells relative to other cells in the body. We report the synthesis of a novel, zinc‐chelating analogue of (+)‐JQ1, [(+)‐JQ1‐DPA], in which the BETi (+)‐JQ1 was conjugated, via an ethylenediamine linker, to dipicolyl amine (DPA). DPA chelates Zn2+ and thus enables (+)‐JQ1 to accumulate in β‐cells. As control compounds we also synthesized (+)‐JQ1‐DBA, a non‐zinc‐chelating analogue of (+)‐JQ1‐DPA, in which (+)‐JQ1 was conjugated to dibenzyl amine (DBA) and (‐)‐JQ1‐DPA, a zinc‐chelating compound that is inactive as a BETi. Molecular modeling and biophysical assays showed that (+)‐JQ1‐DPA and (+)‐JQ1‐DBA, but not (‐)‐JQ1‐DPA, retain binding to both bromodomains of BRD4 in vitro. Cellular assays demonstrated that (+)‐JQ1‐DPA retains efficacy; in particular, (+)‐JQ1‐DPA, attenuated the expression of NF‐ĸB target genes in β‐cells stimulated with the pro‐inflammatory cytokine, IL‐1β, a model for β‐cell destruction. Finally, using islets isolated from the INS1Cre; ROSA26mTmG mouse, an established mouse model that expresses enhanced green fluorescent protein in insulin‐positive cells and mTomato in insulin‐negative cells, we demonstrated that (+)‐JQ1‐DPA affects gene transcription in pancreatic β‐cells but not in other pancreatic cell types. In conclusion, we have synthesized a zinc‐chelating analogue of (+)‐JQ1 that is selectively active in pancreatic β‐cells over other islet cell types, including macrophages and α‐cells.

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Chromatin structure is important in modulating gene expression. A central component of chromatin structure is the covalent post‐translational modification (PTM) of histones. Distinct histone PTMs can determine the accessibility of chromatin and recruit different effector proteins and components of the transcriptional machinery. Histone are modified by “writer” and “eraser” enzymes that can add and remove PTMs, which are recognized by evolutionarily conserved reader domains. Bromodomain is one type of evolutionarily conserved “reader” domain composed of ~110 amino acids, including a conserved asparagine that specifically binds acetylated lysine. There are approximately 80 bromodomains in humans, primarily in chromatin‐associated proteins. Most proteins contain one or two bromodomains. The exception is Polybromo‐1, which contains six tandem bromodomains in addition to two bromo‐adjacent homolog (BAH) domains involved in protein‐protein interactions and a high mobility group (HMG) for interacting with DNA. PBRM1 is a subunit of the polybromo‐associated BAF (PBAF) chromatin remodeling complex, one of three major SWI/SNF subcomplexes found humans. These chromatin remodeling complexes alter chromatin accessibility by evicting, sliding and exchanging nucleosomes on DNA. In clear cell renal cell cancer carcinoma (ccRCC), the most common subtype of kidney cancer, PBRM1is the second most mutated gene after VHL, with PBRM1mutations found in approximately 40% of ccRCC patients. Mouse models indicate that PBRM1 deletion paired with VHL deletion in the kidney results in tumors, establishing PBRM1 as a bona fide tumor suppressor. While most PBRM1 mutations in patients are also loss of function mutations, ~15‐20% of PBRM1 mutations are missense mutations, with a large portion found at highly conserved residues in the fourth bromodomain (BD4). Using recombinant PBRM1 BD4 mutants, we have assessed how the most common missense mutations found in patients affect the stability and acetyllysine binding of PBRM1 BD4, revealing new insight into bromodomain structure and function. In addition we have expressed a subset of these PBRM1 missense mutations in renal cancer cell lines and assessed how they affect PBRM1 binding to acetyllysine substrates, and subsequent renal cancer growth.