Reagents and conditions: a) i) (E)-4-Bromocrotonic acid, oxalyl chloride, DMF, THF, 0 °C to rt, 45 min, ii) 43, NMP, 0 °C, 75 min, 94% (based on a 40% Cl : 60% Br ratio determined using To ensure that the addition of the alkyne handle did not hinder covalent labelling of BRD4(1) L94C , all of the clickable probes were evaluated using 'labelling assay 1' (Table ). Compounds 28-30 showed a similar or increased percentage of labeling compared to the parent fragments 12 and 14. Like its parent fragment 12, compound 30 displayed low levels of non-specific labelling, leading to a second labelling event accounting for 3% of total labeled protein (Figure ). Compound 41 showed only 2% of BRD4(1) L94C labeling after 4 hours, demonstrating that modification of the acrylamide moiety was not tolerated. Compound 42, however, showed 37% of BRD4(1) L94C labeling after 1 hour, and 82% after 4 hours. This result shows that attachment of the alkyne moiety on the phenolic oxygen is well tolerated. To evaluate proteome-wide selectivity of the clickable probes, HEK293T cells transiently expressing BRD4(1) L94C or BRD4(1) WT were incubated with 10 µM of probes 28-30 and 42 for 1 hour before the cells were lysed and the CuAAC reaction with TAMRA-PEG3-azide was conducted (Figure ). SDS-PAGE separation and analysis using in-gel fluorescence and Coomassie blue staining revealed high levels of off-target binding to endogenous proteins for all three of the isoxazole-derived probes 28-30 (Figure and). Of these three probes, 30 exhibited the highest level of BRD4(1) L94C engagement, with a stronger fluorescent band at the expected molecular weight (~49 kDa) in cells expressing BRD4(1) L94C , which was not visible in the BRD4(1) WT expressing cells (Figure ). In contrast, the γ-lactam-based probe 42, displayed exquisite proteome-wide selectivity towards BRD4(1) L94C with little evidence of it binding to any other proteins. Treatment of HEK293T cells transiently expressing BRD4(1) L94C with 42 at concentrations ranging from 1-10 μM showed that covalent labeling of this protein is dose dependent (Figure ). A time course of 0.5-4 h revealed that labeling occurs in a time-dependent manner, with maximum labeling observed at the longer time point (Figure ). BRD4(1) L94C target engagement of compound 30 was investigated using a competition experiment with the BRD4(1) L94C ligand OXFBD04 (3). Increasing concentrations of OXFBD04 (3) resulted in a concomitant reduction in the level of BRD4(1) L94C labelling by compound 30 (Figure and).