Reactivities of the Front Pocket N-Terminal Cap Cysteines in Human Kinases.

The front pocket (FP) N-terminal cap (Ncap) cysteine is the most popular site of covalent modification in kinases. A long-standing hypothesis associates the Ncap position with cysteine hyper-reactivity; however, traditional computational predictions suggest that the FP Ncap cysteines are predominantly unreactive. Here we applied the state-of-the-art continuous constant pH molecular dynamics (CpHMD) to test the Ncap hypothesis. Simulations found that the Ncap cysteines of BTK/BMX/TEC/ITK/TXK, JAK3, and MKK7 are reactive to varying degrees; however, those of BLK and EGFR/ERBB2/ERBB4 possessing a Ncap+3 aspartate are unreactive. Analysis suggested that hydrogen bonding and electrostatic interactions drive the reactivity, and their absence renders the Ncap cysteine unreactive. To further test the Ncap hypothesis, we examined the FP Ncap+2 cysteines in JNK1/JNK2/JNK3 and CASK. Our work offers a systematic understanding of the cysteine structure-reactivity relationship and illustrates the use of CpHMD to differentiate cysteines toward the design of targeted covalent inhibitors with reduced chemical reactivities.