Discovery of Arginine-Reactive Covalent Kinase Inhibitors Overcoming Clinical Drug Resistance

Covalent kinase inhibitors (CKIs) serve as potent therapeutics, yet their development largely depends on conserved cysteine residues. Many kinases lack accessible cysteines or develop resistance mutations at these sites, limiting clinical utility. To diversify targetable nucleophilic amino acids, we developed a modular strategy for arginine reactive CKIs using two warhead types: O-cyanobenzaldehyde (CNBA) and phenylglyoxal (PGO). We first synthesized RA2, a CNBA-based AURKA inhibitor targeting arginine (R220), which showed weak cellular activity (GI50 > 2 μM). Using PGO warhead, we generated covalent inhibitors C1 against ALK-G1202R and L1a against TRKA-G595R; both strongly inhibited arginine-mutant kinases but showed unsatisfactory cellular potency. Further optimization of PGO electrophilicity via methoxy substitution yielded L1c, which exhibited potent cellular activity (GI50 = 0.39 μM) against drug-resistant Ba/F3-CD74-TRKA-G595R cells and sustained target engagement. Collectively, this work expands the CKI toolbox to include arginine-targeting warheads and provides a modular framework to overcome point mutation-driven resistance.