Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression.

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.

Selective and Cell-Active PBRM1 Bromodomain Inhibitors Discovered through NMR Fragment Screening.

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 Kd 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.

Covalent-Fragment Screening of BRD4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites.

BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to nonhomologous cysteine residues within the C-terminal BRD4 bromodomain (BRD4-BD2), we performed a midthroughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify BRD4. Subsequent mass spectrometry, NMR, and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to BRD4 among human bromodomains. This site is orthogonal to the BRD4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays nor an acetylated histone peptide in AlphaScreen assays. Finally, we tethered our top-performing covalent fragment to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace BRD4 from chromatin.

Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.

The sirtuin family of proteins catalyze the NAD+-dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD+ and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn2+ release from the conserved sirtuin Zn2+-tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD+ and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn2+, consistent with S-nitrosation of the Zn2+-tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment.