Dissecting KRAS oncoprotein signaling with small molecule inhibitors

项目总结/摘要 小GTP酶调节多种细胞功能，其异常激活在疾病中起关键作用。 也许最重要的是它们与癌症的关系，癌症是一种KRAS突变的疾病，约1/3的 患者考虑到这一点，癌症热点突变激活KRAS的机制是一个核心问题。 癌症生物学中的概念。在生理条件下，KRAS在活性（GTP结合的）和非活性（ATP结合的）之间循环。 非活性（GDP结合）构象。其缓慢的内在GTP水解是由GTP酶激活催化的 蛋白质（GAP）。KRAS中的常见突变阻止水解过渡态的稳定 导致被认为缺乏水解，对GAP不敏感，并且组成性 在癌细胞中有活性（即，“锁定”在它们的活性或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。也许最有力的证据是 选择性靶向KRAS G12 C（肺癌中最常见的KRAS突变）的抑制剂。G12 C抑制剂 仅与GDP结合（或水解）构象结合，并通过以下方式将癌蛋白捕获在非活性状态 防止GDP与GTP的交换。为了有效，这些非活性状态选择性药物需要完整的 突变型KRAS的GTP水解。以类似的方式，核苷酸交换的抑制（如通过抑制核苷酸交换所实现的）也可以被抑制。 KRAS上游的靶向因子）抑制突变型KRAS活化和/或肿瘤生长。 增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以 如果突变型KRAS GTP酶被“锁定”在其活性状态，则不可能。我们的原理证明 实验表明存在增强突变型KRAS的GT3活性的细胞蛋白， KRAS癌蛋白广泛易受非活性状态选择性抑制。我们现建议（i） 分离癌细胞中突变型KRAS GT3活性的增强子，（ii）确定 常见的KRAS突变体与它们的增强子复合，以及（iii）表征新的失活的KRAS突变体的效应。 国家选择性药物，抑制常见的KRAS突变体发现的癌症。这项工作将解释 负责突变KRAS的生理失活的机制基础，并完善概念 解释突变如何激活癌症中的KRAS的模型。拟议的研究将为关键的 癌症生物学的进展，对患者的治疗和转化影响具有巨大潜力。

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.