Finally, as initially suggested by the X-ray structure (vide supra), only one of the 5-methyl analogues, 11, displayed selective labelling of BRD4(1) L94C . However, only 10% of labeled BRD4(1) L94C was observed, substantially lower than the 56% labeling observed for the corresponding 3-methyl regioisomer 10 (Table ). Other 5-methylisoxazole derivatives possessing reactive enone and ynone electrophiles were observed to label both BRD4(1) L94C and BRD4(1) WT unselectively (Table and Figure ). To investigate whether screening of an unbiased set of covalent fragments could identify ligands for BRD4(1) L94C , we evaluated 250 previously published cysteine-reactive compounds, comprising 138 acrylamide-and 112 chloroacetamide-functionalized reactive fragments with molecular weights ranging from 152-300 Da. Due to the higher throughput of this screening assay, the conditions varied compared to 'labelling assay 1'. In this case, 1 μM of BRD4(1) L94C was incubated with either a chloroacetamide fragment (50 μM) or an acrylamide fragment (200 μM) at 4 °C for 24 hours (referred to as 'labelling assay 2'). Samples were then analyzed using protein LCMS, where a positive hit was classified as a single protein labelling event with a mass shift corresponding to the mass of the fragment (± 2 Da), and a labelled peak of >20% intensity. Of the 250 diverse reactive compounds screened against BRD4(1) L94C , only one chloroacetamide and one acrylamide fragment were classified as hits (Figure and). The chloroacetamide hit (26, Figure ) displayed 27% labeling of BRD4(1) L94C (Figure ) and the acrylamide hit (27, Figure ) showed 89% labeling of BRD4(1) L94C (Figure ). Given the difference in the two screening conditions, a selection of isoxazole fragments identified by 'labelling assay 1' were included as positive (10, 13, and 21) and negative (17) controls. All of these control compounds showed protein labelling trends comparable to those obtained from labelling assay 1, confirming the overlap between the two assay conditions (Figure ).