Straightforward hit identification approach in fragment-based discovery of bromodomain-containing protein 4 (BRD4)...

A combination approach of a fragment screening and "SAR by catalog" was used for the discovery of bromodomain-containing protein 4 (BRD4) inhibitors. Initial screening of 3695-fragment library against bromodomain 1 of BRD4 using thermal shift assay (TSA), followed by initial hit validation, resulted in 73 fragment hits, which were used to construct a follow-up library selected from available screening collection. Additionally, analogs of inactive fragments, as well as a set of randomly selected compounds were also prepared (3 × 3200 compounds in total). Screening of the resulting sets using TSA, followed by re-testing at several concentrations, counter-screen, and TR-FRET assay resulted in 18 confirmed hits. Compounds derived from the initial fragment set showed better hit rate as compared to the other two sets. Finally, building dose-response curves revealed three compounds with IC₅₀ = 1.9-7.4 μM. For these compounds, binding sites and conformations in the BRD4 (4UYD) have been determined by docking.