As fragment 27 displayed a single covalent labeling of BRD4(1) L94C at a level of 89% , this compound represented a promising candidate for further investigation (Figure ). When the labeling was assessed under 'labeling assay 1' conditions, used for the structure-guided fragment screening, compound 27 labeled 61% of BRD4(1) L94C with no covalent binding to BRD4(1) WT detected (Figure and S12). The shorter timepoint used in 'labeling assay 1' (4 hours instead of 24 hours used in 'labeling assay 2') likely accounts for the lower level of labeling displayed by the fragment (61% of labeling versus 89%). Overall, two compounds from the structure-guided approach (12 and 14, Table ) and one compound from the diverse fragment-based screening (27, Table ) were selected for further evaluation. Using an AlphaScreen™ assay, the three identified fragments were evaluated for their ability to displace the H41-20(KAc)4 peptide from BRD4(1) L94C and BRD4(1) WT . The compounds were incubated at room temperature for 1 or 4 hours in the presence of the biotinylated H41-20(KAc)4 peptide, and either BRD4(1) L94C or BRD4(1) WT . Under these conditions, all three fragments displaced the peptide from BRD4(1) L94C , which is consistent with the fragments binding to the BRD4(1) L94C in the expected location (Table ). In all cases the IC50 values at 4 hours were lower than those seen at 1 hour, which is a characteristic of covalent inhibition (Figure ). The longer the mutant protein and electrophile are incubated together, the more time there is for a covalent interaction to occur.