Dissecting KRAS oncoprotein signaling with small molecule inhibitors

项目摘要/摘要 小分子GTP酶调节多种细胞功能，其异常激活在疾病中起着关键作用。 也许最重要的fi不能是它们与癌症的联系，一种疾病，KRAS基因突变在~1/3 病人。考虑到这一点，癌症热点突变激活KRAS的机制是一个核心 癌症生物学中的概念。在生理条件下，KRAS在活跃的(GTP结合的)和 不活跃的(受GDP限制的)构象。其缓慢的内源性GTP水解酶是由GTP酶激活催化的 蛋白质(GAP)。KRAS的常见突变阻止了水解过渡态的稳定 导致癌蛋白被认为在水解液中缺乏fi，对间隙不敏感，并且具有结构性 在癌细胞中处于活跃状态(即处于活跃状态或GTP结合状态)。新兴疗法具有挑战性 KRAS癌蛋白激活的传统模型。也许最有力的证据来自于 选择性靶向KRAS G12C的抑制剂，KRAS是肺癌中最常见的KRAS突变。G12C抑制剂 只与GDP结合(或水解)构象结合，并通过以下方式捕获处于非活性状态的癌蛋白 防止以GDP换取GTP。为了有效，这些非活性状态的选择性药物需要完整的ff 突变KRAS对GTP的水解性研究以类似的方式，抑制核苷酸交换(通过 KRAS上游的靶向因子)已被报道抑制突变的KRAS激活和/或肿瘤 成长。在本申请的初步数据中提出的这种和其他新兴的治疗效果可能 如果突变的KRAS GTP酶处于激活状态，则是不可能的。我们的原则证明 实验表明，细胞蛋白的存在可以增强突变的KRAS和 KRAS癌蛋白对非活性状态的选择性抑制具有广泛的敏感性。我们现在建议(I) 在癌细胞中分离突变的KRAS GTPase活性增强子，(II)确定其三级结构 常见KRAS突变体及其增强子的复合体和(III)表征新的失活基因的ff效应 抑制在癌症中发现的常见KRAS突变的国家选择性药物。这项工作将解释 突变体KRAS生理失活的机制基础和Refi 解释突变如何在癌症中激活KRAS的模型。拟议的研究将为KEY 癌症生物学的进展，对患者的治疗和转化有很大的影响。

PROJECT SUMMARY/ABSTRACT Small GTPases regulate diverse cellular functions and their aberrant activation plays a key role in disease. Perhaps most significant is their association with cancer, a disease where KRAS is mutated in ~1/3 of patients. With this in mind, the mechanism by which cancer hotspot mutations activate KRAS is a central concept in cancer biology. Under physiologic conditions, KRAS cycles between an active (GTP-bound) and an inactive (GDP-bound) conformation. Its slow intrinsic GTP hydrolysis is catalyzed by GTPase-activating proteins (GAPs). Common mutations in KRAS prevent the stabilization of the hydrolysis transition-state leading to oncoproteins that are thought to be deficient in hydrolysis, insensitive to GAPs, and constitutively active in cancer cells (i.e., `locked' in their active or GTP-bound, state). Emerging therapies are challenging the conventional model of KRAS oncoprotein activation. Perhaps the strongest evidence is provided by inhibitors selectively targeting KRAS G12C, the most common KRAS mutation in lung cancer. G12C inhibitors bind only to the GDP-bound (or hydrolyzed) conformation and trap the oncoprotein in an inactive state by preventing the exchange of GDP for GTP. To be effective, these inactive state selective drugs require intact GTP hydrolysis by mutant KRAS. In a similar fashion, inhibition of nucleotide-exchange (as achieved by targeting factors upstream of KRAS) has been reported to suppress mutant KRAS activation and/or tumor growth. This and other emerging therapeutic effects presented in the preliminary data of this application could not be possible if mutant KRAS GTPases were `locked' in their active state. Our proof-of-principle experiments suggest the presence of cellular proteins that enhance the GTPase activity of mutant KRAS and that KRAS oncoproteins are broadly susceptible to inactive state selective inhibition. We now propose (i) to isolate enhancers of mutant KRAS GTPase activity in cancer cells, (ii) to determine the tertiary structure of common KRAS mutants in complex with their enhancer and (iii) to characterize the effects of novel inactive state selective drugs that suppress common KRAS mutants found in cancer. This work will explain the mechanistic basis responsible for the physiologic inactivation of mutant KRAS and refine the conceptual model explaining how mutations activate KRAS in cancer. The proposed study will pave the way for key advances in cancer biology with a large potential for therapeutic and translational impact in patients.