Bromodomain (BRD) containing proteins are essential for acetylated lysine (Kac) recognition on histone proteins during transcriptional activation. BRDT is a testis-specific BRD protein required for male germ cell differentiation has been validated as a target for non-hormonal male contraception. Differential scanning fluorimetry (DSF), a thermal shift assay, was used to detect protein binding in a fragment library screen, with a hit identified as a fragment shifting the transition temperature (Tm) of the protein ≥ 1 °C as compared to the DMSO control. From this screen, 16 were chosen for confirmation via dose response using the DSF assay. Ten positive hits were repurchased and retested in dose response format before moving on to a protein-observed 19F-NMR (PrOF-NMR) confirmation assay using 19F-labeled BRDT. The fragments that showed BRDT binding in the PrOF-NMR assay were then taken into crystallographic studies to provide data on binding interactions. A number of crystal structures have been solved, allowing for the prioritization of potential lead compounds. Selected hits will undergo rounds of hit-to-lead optimization and structure activity relation manipulations to refine lead compounds to create potent and selective BRDT inhibitors. In addition to the fragment screen, we have also been pursuing a number of known kinase inhibitors as possible starting points for the development of a BRDT inhibitor, as several kinase inhibitors have been shown to bind to the BET family. PLK1 inhibitor BI-2536 has been crystalized with BRD4, a member of the BET family closely related to BRDT. Analog fragments of this kinase inhibitor are currently being synthesized in our lab and tested against BRDT using DSF and PrOF-NMR. Fragments that show good binding will be taken forward to crystallographic studies to help guide further SAR optimization.

Identification and characterization of potential BRDT inhibitors by fragment-based screening using differential scanning fluorimetry, PrOF-NMR, and protein crystallography

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.