Confirmation and characterisation of BRD4 PhABit hits BRD4-BD1 was selected for in-depth analysis of the screening results and follow-up studies on the hits. BRD4 is a member of the bromodomain and extra-terminal domain (BET) family of proteins, which are involved in epigenetic regulation and are potential therapeutic targets for the treatment of disease, including cancer and inflammation. The BRD4-BD1 PhABit screen afforded 68 hits (12%) that were displaced by JQ1, which gave strong (13), and moderate (55) crosslinking. Hits included the aforementioned dimethyl isoxazoles 3 and 4, other 5-membered heterocycles (e.g. 6 and 7), acetyl lysine mimetics (1 and 9) and less typical structures (5 and 8), which could represent novel binding motifs for the acetyl lysine pocket (Figure ). Orthogonal confirmation of non-covalent binding to the acetyl lysine binding site was performed using a BRD4-BD1 TR-FRET competition assay. Six of the nine hits (2-4, 6 and 8-9) were found to inhibit bromodomain-substrate binding with weak to moderate affinity (pIC50 values 4.0-5.7, Figure ).