Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state. Biochemical studies show that 2C07 alters nucleotide preference and inhibits SOS binding and catalyzed nucleotide exchange. 2C07 was converted to irreversible covalent analogs, which target both nucleotide states, inhibit PI3K activationin vitro, and function as occupancy probes to detect reversible engagement in competition assays. Targeting both nucleotide states opens the possibility of inhibiting oncogenic mutants of Ras, which exist predominantly in the GTP state in cells.

中文翻译：

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Ras Binder 在 GTP 和 GDP 状态中引入了改进的 Switch-II Pocket

据报道，K-Ras(G12C) 共价抑制剂专门识别 GDP 状态。在这里，我们利用非天然半胱氨酸 (K-Ras(M72C)) 的二硫键束缚来识别新的 switch-II 口袋 (S-IIP) 结合配体 (2C07)，该配体参与活性 G​​TP 状态。2C07 与 H-Ras(M72C) 结合的共晶结构揭示了在我们称为 S-IIG 的隐秘凹槽中的结合。在 GppNHp 状态下，2C07 与修饰的 S-IIP 结合将开关 I 推离核苷酸，从而破坏了采用规范 GTP 状态所必需的极性接触网络。生化研究表明，2C07 会改变核苷酸偏好并抑制 SOS 结合和催化核苷酸交换。2C07 被转化为不可逆的共价类似物，其靶向两种核苷酸状态，在体外抑制 PI3K 激活，并作为占据探针来检测竞争测定中的可逆参与。靶向两种核苷酸状态开启了抑制 Ras 致癌突变体的可能性，Ras 突变体主要以 GTP 状态存在于细胞中。