Direct inhibition of mutant K-Ras for the treatment of cancer

描述(申请人提供)：抑制K-RAS癌基因的药物的开发代表了人类癌症治疗中最大的未得到满足的需求之一。RAS基因是癌症中突变最频繁的癌基因，在所有癌症类型中累积突变频率超过30%。带有RAS突变的癌症具有侵袭性，对标准治疗的反应很差。以前的靶向K-RAS的尝试都失败了，这是因为很难与活性SIT的微克分子核苷酸亲和力竞争，而且大多数GTP酶都有很高的相似性。我们的科学联合创始人凯文·肖卡特教授发现了一种新颖的小分子方法来瞄准化学上最容易处理的K-RAS突变体，该突变体包含甘氨酸-12到半胱氨酸的突变。G12C突变是肺癌中最常见的K-RAS突变。事实上，在所有K-RAS突变的肺癌中，有43%含有G12C突变。这种突变在K-RAS的表面定位了一个化学反应的巯基。我们对与K-rasG12C、H-rasG12C共价结合的分子进行了基于质谱学的初步500化合物文库筛选，并与WT K-RAS进行了反筛选。从筛选文库中鉴定出17个匹配，对初始匹配的第一轮优化导致发现了一种有效的抑制剂JO-01-18。我们现在已经解决了与K-RAS G12C结合的JO-01-148的晶体结构，并在半胱氨酸部分附近的蛋白质表面鉴定了一个先前未描述的变构口袋。这个口袋可以识别结合在口袋中的不可逆转的抑制剂，并选择性地将半胱氨酸定位在12位。重要的是，这些小分子只抑制 突变的K-RAS，而不是正常的蛋白质。我们现在已经解决了10多个结合在这个变构口袋上的不可逆缓蚀剂的X射线晶体结构，合成了120多个化合物。一个明确的特区已经建立。我们现在建议在生化和细胞分析中进一步验证我们的G12C先导化合物。第一阶段的具体目标是：(1)开发评估K-RAS效应器结合的方法；(2)证明G12C不可逆结合物可以破坏K-RAS效应器结合；(3)证明与野生型K-RAS和其他K-RAS突变的细胞相比，G12C不可逆结合物可以不同于野生型K-RAS和其他K-RAS突变的细胞影响G12C突变的肿瘤细胞。第一阶段的里程碑是确定K-ras G12C抑制剂，它可以抑制带有G12C突变的肿瘤细胞的增殖(以IC50值衡量)，比具有其他K-ras突变的肿瘤细胞或野生型RAS强五倍。总而言之，我们希望I期结果能证明我们可以产生一种小分子抑制物，它将特异性地抑制带有K-RAS G12C突变的肿瘤细胞的生长。如果我们的方法成功，我们的第二阶段研究将更全面地检查用于IND应用的铅配方的安全性、有效性和PK/生物分布。

DESCRIPTION (provided by applicant): The development of drugs that inhibit the K-Ras oncogene represents one of the greatest unmet needs in the treatment of human cancer. The Ras gene is the most frequently mutated oncogene in cancer, with a greater than 30% cumulative mutation frequency across all cancer types. Cancers with Ras mutations are aggressive and respond poorly to standard therapies. Previous attempts to target K-Ras have failed due to the difficulty of competing with the picomolar nucleotide affinity for the active sit and due to the high similarity of most GTPases. Our scientific co-founder, Professor Kevan Shokat, has discovered a novel, small molecule approach to target the most chemically tractable K-Ras mutant that contains a glycine-12 to cysteine mutation. The G12C mutation is the most common K-Ras mutation in lung cancer. Indeed, 43% of all lung cancers with K-Ras mutations contain the G12C mutation. This mutation positions a chemically reactive sulfhydryl group on the surface of K-Ras. We have carried out a preliminary 500 compound library screen based on mass spectrometry for molecules which bind covalently to K-rasG12C, H-rasG12C and counterscreened against WT K-Ras. 17 hits were identified from the screening library, and the first round optimization of the initial hits led to the discovery of a potent inhibitor JO-01-18. We have now solved the crystal structure of JO-01-148 bound to K-Ras G12C and identified a previously undescribed allosteric pocket on the surface of the protein adjacent to the cysteine moiety. This pocket makes it possible to identify irreversible inhibitors that bind in the pocket and selectively target the cysteine at position 12. Importantly, these small molecules inhibit only mutant K-Ras and not the normal protein. We have now solved more than 10 X-ray crystal structures of irreversible inhibitors bound to this allosteric pocket and synthesized more than 120 compounds. A clear SAR has been established. We are now proposing to further validate our lead G12C compounds in biochemical and cellular assays. The Phase I specific aims are: (1) Develop assays to evaluate K-Ras effector binding; (2) Demonstrate that the G12C irreversible binders can disrupt K-Ras effector binding; and (3) Demonstrate that the G12C irreversible binders can differentially affect tumor cells with G12C mutation compared to cells with wild type K-Ras and other K-Ras mutations. The Phase I milestone is the identification of K-ras G12C inhibitors that suppress proliferation of tumor cells with G12C K-ras mutation five-fold more potently (as measured by IC50 values) relative to tumor cells with other K-Ras mutations or wild type Ras. Collectively, we expect the Phase I results to demonstrate that we can generate a small molecule inhibitor that will specifically inhibit the growth of tumor cells wih K-Ras G12C mutation. If our approach is successful, our Phase II studies will more fully examine the safety, efficacy, and PK/biodistribution of a lead formulation for advancement to an IND application.