The SWI/SNF multi-subunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF mutant tumors, including SMARCA4-deficient lung cancer, however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstruct the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic and pharmacological tools. We evaluate a selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterize its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF complex (e.g. Lung, Synovial Sarcoma, Leukemia, and Rhabdoid tumors). We demonstrate that PFI-3 is a potent, cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor fails to display an antiproliferative phenotype. Mechanistically, the lack of pharmacological efficacy is reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation and target gene expression studies. Further, using inducible RNAi and cDNA complementation (bromodomain- and ATPase-dead constructs), we unequivocally identify the ATPase domain, and not the bromodomain of SMARCA2, as the relevant therapeutic target with the catalytic activity suppressing defined transcriptional programs. Taken together, our complementary genetic and pharmacological studies exemplify a general strategy for multi-domain protein drug-target validation and in case of SMARCA2/4 highlight the potential for drugging the more challenging helicase/ATPase domain to deliver on the promise of synthetic-lethality therapy.

The bromodomain containing proteins TRIM24 (Tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethyl benzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor, 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.

Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2A in cancer cells is associated with poor prognosis making the bromodomain of ATAD2A a promising epigenetic therapeutic target. In the development of an invitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2A bromodomain. Structural studies on apo-, peptide and smallmolecule-ATAD2A complexes (by co-crystalization) revealed the bromodomain adopts a “closed”, histone-compatible conformation, and a more “open” ligand-compatible conformation of the binding-site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2A binders which aided in the validation of the invitro screen and in the analysis of these conformational studies.

Highlights d PGD is a top hit in a loss-of-function genetics screen in OXPHOS-deficient cancer d OXPHOS-deficient cells depend on PGD in vitro and in vivo d PGD inhibition affects glycolysis, reductive carboxylation, and redox homeostasis d Pharmacological inhibition of OXPHOS renders PGD dependent

Palladium-catalyzed reactions of cis-1,1-di-tert-butyl-2,3-dimethylsilirane with disubstituted alkynes produced thermally stable silirenes in high yields (81−86%). Alkyl-, aryl-, trimethylsilyl-, and heteroatom-substituted alkynes were employed. The silirenes reacted with phenylacetylene and <3 mol % of PdCl2(PPh3)2 to produce trisubstituted siloles. A common catalytic cycle involving palladasilacyclobutene accounts for both the formation of silirene and the trisubstituted silole. The palladium-catalyzed extrusion of silylene effectively transfers silylene from one strained ring (a silirane) to form another strained ring (a silirene) and represents an unusual metal-mediated process.