In the validation of protein targets of interest, rapid development of fragment hits towards more potent tool molecules is essential. It was anticipated that hits from the PhABit screen could be used as reporters for site-specific displacement assays to enable immediate screening. Compounds that compete with the reporter PhABit would induce a concentration-dependent reduction in crosslinking, enabling calculation of pIC50 values (Figure ). A proof-of-concept study was performed using PhABit 2 to develop a binding assay for BRD4-BD1 (Figure ). A library of ~1000 compounds was selected based on a similarity search (Tanimoto, >0.7, MW<400 Da) from the hit PhABits identified previously (1-9). The compounds were incubated with PhABit 2 and BRD4-BD1 before irradiation and analysis by LC-MS to determine crosslinking yield. A range of crosslinking inhibition was observed from 1-100%, with >50% observed for 208 compounds (21% of the total screen) (Figure ). The majority of these hits were derived from dimethylisoxazole PhABits 3 and 4, consistent with the presence of this motif in a number of BET targeting series in the GSK compound collection. The remaining 40 hit compounds (20-59) were derived from PhABits 2, 7, 8 and 9 and grouped into four chemical series (Figure ). Compounds 22 and 28 are structurally related to the pan-bromodomain binder bromosporin and had previously been identified as binding to BRD4-BD1. Compound 58 mimics the acetylated native ligand and relates to other reported acetyl lysine mimetics, while compound 40 does not resemble known BRD4 binders.