Construction of Covalent Bisubstrate Inhibitor of Protein Kinase Reacting with Cysteine Residue at Substrate-Binding...

Recent clinical success with targeted covalent inhibitors points to new possibilities for development of protein kinase (PK)-targeted drugs by exploiting reactive cysteine residues in and around the ATP-binding site. However, more than 300 human PKs lack cysteine residues in the ATP-binding site. Here, we report the first covalent bisubstrate PK inhibitor whose electrophilic warhead reaches outside the ATP-binding site and reacts with a distant cysteine residue. A series of covalent inhibitors and their reversible counterparts were synthesized and characterized. The most potent reversible inhibitor possessed picomolar affinity and its cysteine-reactive counterpart revealed high value of k_inact/K_I ratio (6.2 × 10⁷ M^-1 s^-1) for the reaction with the catalytic subunit of cAMP-dependent PK (PKAc). Under optimized conditions, fluorescent dye-labeled covalent inhibitors demonstrated PKA-selectivity in the cell lysate and reacted with several proteins inside live cells, including PKAc. The disclosed compounds serve as leads for targeting PKs possessing an analogously positioned cysteine residue.