Fig. 4. Covalent cysteine-based inhibition strategies. (A) Electrophilic covalent inhibitors inactivate their target through covalent attachment to the cysteine thiol functional group. However, the electrophilic center (e.g., acrylamide, haloacetamide, and vinyl sulfonamide) can also react with other cellular nucleophiles such as glutathione as well as the amino and imidazole groups of amino acids. (B) Nucleophilic covalent strategy as an alternative or complementary inhibition mechanism. According to this approach, active site-directed small-molecule inhibitors containing a reactive nucleophilic center form a covalent bond with a cysteine side chain that has oxidized to sulfenic acid. Such modifications form transiently in specific proteins during H₂O₂-mediated signal transduction in normal cells, but form constitutively in diseases associated with chronically elevated levels of H₂O₂, including cancer. In the sulfenic acid oxidation state, the electron deficient sulfur exhibits enhanced electrophilic character that can be selectively targeted by certain nucleophilic compounds. Because sulfenic acid is a unique chemical moiety in biochemistry, this strategy could decrease the potential for off-target activity while retaining the advantages gained by covalent targeting.