The recent development of mutant-selective inhibitors for the oncogenic KRAS^G12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS^G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK_a of the C12 thiol is depressed (pK_a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS^Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS^G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K_d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK_a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS^G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS^G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS^G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS^G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

最近针对致癌KRAS ^G12C等位基因的突变选择性抑制剂的开发引起了相当大的兴奋。这些抑制剂与位于 RAS 磷酰基结合环内的突变 C12 硫醇共价结合，将 KRAS ^G12C蛋白锁定在非活性状态。虽然这些抑制剂的临床试验前景乐观，但有关这种硫醇反应性的机制问题仍然存在。在这里，我们通过 NMR 和独立的生化测定表明，C12 硫醇的 pK _a降低（pK _a ∼7.6），与化学连接的敏感性一致。使用经验证的适合停流光谱的荧光 KRAS ^Y137W变体，我们表征了添加 ARS-853 或 AMG 510 后KRAS ^G12C荧光变化的动力学，注意到在低温下，ARS-853 的添加引发了快速的第一阶段荧光变化（归因于结合，K _d  = 36.0 ± 0.7 μM）和第二个较慢的 pH 依赖性阶段，代表共价连接。与 C12 硫醇的较低 pK _a一致，我们发现 KRAS ^G12C在体外和细胞环境中都很容易发生可逆和不可逆氧化，从而阻止了 ARS-853 的共价结合。此外，我们发现，通过分子动力学模拟评估，与未修饰的蛋白质相比，KRAS ^{G12C Cys12 氧化为亚磺酸盐改变了 RAS 构象和动力学，使其与 KRAS G12D}更相似。总而言之，这些发现为未来 KRAS ^G12C药物发现工作提供了见解，并确定了 G12C 氧化的发生与目前未知的生物学后果。