Having demonstrated that the WT protein is not susceptible to reaction with electrophilic fragments, we next determined suitable sites to incorporate a Cys mutation. Analysis of the X-ray crystal structure of BRD4(1) WT bound to OXFBD04 (3), a 3,5-dimethylisoxazole-based BRD ligand that we previously developed (PDB ID: 6FSY) (Figure ), indicated that the ZA loop region might be a suitable area for mutagenesis as it is located above the KAc binding pocket and is therefore unlikely to negatively impact on native KAc binding. The L94 residue, which is conserved across the BET family (Figure ), is part of the ZA loop and is located above the 5-position methyl group of the OXFBD04 isoxazole. This placement suggests that attachment of a 5-position electrophile could locate the L94C cysteine and the electrophile proximal, allowing them to react. Interestingly, Baud suggesting that modifications at this position are tolerated by the protein. An L94V mutation led to a two-fold decrease in tetra-acetylated H4-mimicking peptide (H41-20(KAc)4) binding affinity, while the L94A mutation decreased the affinity by ten-fold. To further investigate the impact of L94C on protein stability, molecular dynamics (MD) simulations using GROMACS 2016.4 were employed, which compared the backbone root mean square deviation (RMSD) and backbone root mean square fluctuation (RMSF) of BRD4(1) WT and BRD4(1) L94C .