Mitogen-activated protein kinases (MAPK) are important therapeutic targets, and yet no inhibitors have advanced to the market. Here we applied the GPU-accelerated continuous constant pH molecular dynamics (CpHMD) to calculate the pK a's and profile the cysteine reactivities of all 14 MAPKs for assisting the targeted covalent inhibitor design. The simulations not only recapitulated but also rationalized the reactive cysteines in the front pocket of JNK1/2/3 and the extended front pocket of p38α. Interestingly, the DFG - 1 cysteine in the DFG-in conformation of ERK1/ERK2 was found somewhat reactive or unreactive; however, simulations of MKK7 showed that switching to the DFG-out conformation makes the DFG - 1 cysteine reactive, suggesting the advantage of type II covalent inhibitors. Additionally, the simulations prospectively predicted several druggable cysteine and lysine sites, including the αH head cysteine in JNK1/3 and DFG + 6 cysteine in JNK2, corroborating the chemical proteomic screening data. Given the low cost and the ability to offer physics-based rationales, we envision CpHMD simulations to complement the chemo-proteomic platform for systematic profiling cysteine reactivities for targeted covalent drug discovery.
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