Multiscale calculations reveal new insights into the reaction mechanism between KRAS and α, β-unsaturated carbonyl of covalent inhibitors.

Utilizing α,β-unsaturated carbonyl group as Michael acceptors to react with thiols represents a successful strategy for developing KRAS inhibitors. Despite this, the precise reaction mechanism between KRAS and covalent inhibitors remains a subject of debate, primarily due to the absence of an appropriate residue capable of deprotonating the cysteine thiol as a base. To uncover this reaction mechanism, we first discussed the chemical reaction mechanism in solvent conditions via density functional theory (DFT) calculation. Based on this, we then proposed and validated the enzymatic reaction mechanism by employing quantum mechanics/molecular mechanics (QM/MM) calculation. Our QM/MM analysis suggests that, in biological conditions, proton transfer and nucleophilic addition may proceed through a concerted process to form an enolate intermediate, bypassing the need for a base catalyst. This proposed mechanism differs from previous findings. Following the formation of the enolate intermediate, solvent-assisted tautomerization results in the final product. Our calculations indicate that solvent-assisted tautomerization is the rate-limiting step in the catalytic cycle under biological conditions. On the basis of this reaction mechanism, the calculated / for two inhibitors is consistent well with the experimental results. Our findings provide new insights into the reaction mechanism between the cysteine of KRAS and the covalent inhibitors and may provide valuable information for designing effective covalent inhibitors targeting KRAS and other similar targets.