Abstract 2617 Structural studies of Polybromo-1 bromodomain inhibitor selectivity

Polybromo-1 (PBRM1) is a unique subunit of the Polybromo BRG1/BRM-associated factors (PBAF) complex, a member of the SWItch/Sucrose Non-Fermentable (SWI/SNF) family of ATP-dependent chromatin remodeling complexes.Through its six tandem bromodomains, PBRM1 links epigenetic histone lysine acetylation (Kac) to PBAF-mediated transcriptional regulation and chromatin remodeling.PBRM1 is a tumor promoter in prostate cancer, contributing to migratory and immunosuppressive phenotypes.Loss of PBRM1 function shows enhanced response to anti-PD1 immunotherapy in human and murine studies of clear cell renal cell carcinoma (ccRCC).This highlights the importance of PBRM1 in cancer biology and indicates that targeting its bromodomains may provide novel therapeutic opportunities.Previous attempts to develop PBRM1 inhibitors have shown a lack of selectivity, as these compounds also bind the bromodomains of SMARCA2 and SMARCA4, which are subunits of other SWI/SNF complexes, leading to off-target effects.We employed our proteindetected NMR screening pipeline to develop selective PBRM1 bromodomain inhibitors.Screening 1,968 fragments against the second bromodomain of PBRM1 revealed a high-affinity hit, which was further optimized through structure-activity relationship (SAR) studies to generate a selective PBRM1 inhibitor, PB16.Notably, PB16 does not bind the bromodomains of SMARCA2 or SMARCA4 as determined by differential scanning fluorimetry and isothermal titration calorimetry (ITC).To elucidate the mechanism underlying the selectivity of PB16, we determined the crystal structure of PB16 bound to the second PBRM1 bromodomain.We used this structure, combined with NMR chemical shift perturbation mapping and sequence alignment, to identify potential residues in the second PBRM1 bromodomain that confer selectivity.Sitedirected mutagenesis and ITC revealed that a unique tyrosine residue in PBRM1 is vital for its ability to bind PB16.This tyrosine creates an altered binding pocket in the second PBRM1 bromodomain compared to the SMARCA2 and SMARCA4 bromodomains, which possess a leucine residue in the same position.Additionally, we compared PB16 to another recently-reported selective PBRM1 inhibitor, GNE-235, and found both inhibitors share the same selectivity mechanism.However, PB16 demonstrates cell activity while GNE-235 does not, making PB16 a more promising candidate for therapeutic applications.In summary, our findings reveal a novel, selective, and cell-active PBRM1 bromodomain inhibitor with potential for targeted therapeutic intervention.This work provides insights into the structural basis of PBRM1 bromodomain selectivity and lays the foundation for further exploration of its role in cancer immunotherapy.