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

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 kinact/KI 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.