In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C
In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C
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DOI：
10.1073/pnas.1404639111
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发表时间：
2014-06-17
影响因子：
11.1
通讯作者：
Westover, Kenneth D.
中科院分区：
文献类型：
--
作者：
Hunter, John C.;Gurbani, Deepak;Westover, Kenneth D.
用小分子抑制剂直接靶向致癌V-Ki-ras 2 Kirsten大鼠肉瘤病毒癌基因同源物（K-Ras）历来被认为是极具挑战性的。最近关于直接与K-Ras G12 C突变体结合的化合物的报道表明了克服阻碍开发此类化合物的关键障碍的途径。我们的目标是靶向鸟嘌呤核苷酸（GN）结合口袋，因为这个口袋的天然内容物决定了K-Ras的信号状态。在这里，我们表征了不可逆抑制剂SML-8-73-1（SML），其靶向K-Ras G12 C的GN结合口袋。我们报告了与SML结合的G12 C K-Ras的高分辨率X射线晶体结构，揭示了该化合物以类似于GDP的方式结合，与Cys-12形成共价键。所得到的构象使K-Ras处于开放的非活性构象，预计其不会有效地与下游效应物相关联或激活下游效应物。Ras家族GN结合口袋的保守性分析揭示了活性位点和邻近区域周围的侧链的变异性，特别是在开关I区域。这种变异性可能使Ras和其他GT3抑制剂的新迭代具有特异性。SML的高分辨率原位化学蛋白质组学分析证实，SML有效地区分K-Ras G12 C和其他细胞GTP结合蛋白。生化分析提供了额外的证据，SML是能够竞争的GTP和GDP的GN结合位点的毫摩尔浓度。
Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.