Development of covalent docking to identify new cysteine-targeted K-Ras inhibitors to address allelic variation in cancer

项目总结/摘要 肿瘤中的等位基因变异是癌症中大量研究的主题。一些致癌Ras突变体出现在 比其他基因更具致癌性，导致这些突变等位基因的频率过高。 然而，癌症生物学家还不能推导出等位基因变异的分子机制。我会 我想用分子学的方法来解决这个遗传问题。K-Ras（G12 C）通常在 40%的K-Ras驱动的非小细胞肺腺癌肿瘤，而K-Ras（G13 C），一种据称类似的肿瘤， 突变体仅占肿瘤群体1%。通过设计K-Ras的化学探针， 希望阐明K-Ras作为致癌驱动因子的基本机制。我想调查一下 使用化学探针分析K-Ras（G12 C）和K-Ras（G13 C）之间的生物化学和结构差异。我 假设共价对接是一种可行策略，用于开发针对先前靶点的共价探针 标记为化学上难处理的，例如小GTP酶，例如Ras。DOCKovalent是一种分子对接 由Shoichet和汤顿小组共同努力开发的软件。目标1涉及 在K-Ras（G12 C）上测试DOCKovalent，K-Ras是一种临床相关的致癌基因，我们的团队已经积累了 化学和结构数据。在Shoichet实验室，我获得了初步数据， DOCKovalent可用于预测K-Ras（G12 C）的有效和特异性小分子探针。我们将 尝试使用结构技术（如X射线晶体学）验证共价对接结果， 氢氘交换质谱法。目标2涵盖了共价对接的应用 新型且未表征的癌基因K-Ras（G13 C），迄今为止还没有抑制剂。在这里，我将使用一个 多管齐下的方法，包括基于结构的接头设计，分子对接筛选，和片段， 基于二硫键的束缚，寻找新的K-Ras（G13 C）的有效抑制剂。初步数据显示，K- Ras（G13 C）是反应性的，并且可以被共价亲电试剂标记。目标3涉及调查 肺腺癌中K-Ras（G12 C）和K-Ras（G13 C）等位基因变异的机制。生化 对K-Ras的相似突变等位基因的研究已经确定K-Ras突变体在它们的 生化功能和结构构象。我会用功能分析结合X光 晶体学以研究K-Ras（G12 C）和K-Ras（G13 C）是否也是如此。我也会用我们的小 分子，以检查可比较的K-Ras（G12 C）和K-Ras（G13 C）肺腺癌细胞系是否具有 对下游Ras驱动途径抑制的不同敏感性。该项目的完成将阐明 这两个重要的致癌突变体之间的差异，有可能在未来的应用，以其他 K-Ras突变体该项目将进一步验证DOCKovalent方法作为突变体的策略， 小GTP酶的特异性抑制剂开发。

Project Summary / Abstract Allelic variation in tumors is a heavily investigated topic in cancer. Some mutants in oncogenic Ras appear more oncogenic than others, leading to an overrepresentation in the frequency of those mutant alleles. However, cancer biologists have not been able to derive the molecular mechanism of allelic variation. I would like to take a molecular approach to addressing this genetic question. K-Ras(G12C) is commonly mutated in 40% of K-Ras-driven non-small cell lung adenocarcinoma tumors, and yet K-Ras(G13C), a supposedly similar mutant, only represents 1% of that tumor population. By designing chemical probes for K-Ras, we would like to elucidate fundamental mechanisms of K-Ras as an oncogenic driver. I would like to investigate the biochemical and structural differences between K-Ras(G12C) and K-Ras(G13C) using chemical probes. I hypothesize that covalent docking is a viable strategy for developing covalent probes for targets previously labeled as chemically intractable such as small GTPases such as Ras. DOCKovalent is a molecular docking software developed at UCSF by the combined efforts of the Shoichet and Taunton groups. Aim 1 involves testing DOCKovalent on K-Ras(G12C), a clinically relevant oncogene for which our group has amassed chemical and structural data. With the Shoichet lab, I have obtained preliminary data that suggests that DOCKovalent can be used to predict potent and specific small molecule probes for K-Ras(G12C). We will attempt to validate the covalent docking results using structural techniques such as X-ray crystallography and Hydrogen-Deuterium Exchange Mass Spectrometry. Aim 2 covers the application of covalent docking to the novel and uncharacterized oncogene, K-Ras(G13C), which has no inhibitors to date. Here, I will use a multipronged approach including structure-based linker design, molecular docking screening, and fragment- based disulfide tethering to find new potent inhibitors for K-Ras(G13C). Preliminary data reveals that K- Ras(G13C) is reactive and can be labeled by covalent electrophiles. Aim 3 involves investigating the mechanism of allelic variation in lung adenocarcinoma for K-Ras(G12C) and K-Ras(G13C). Biochemical investigations of similar mutant alleles of K-Ras have determined that K-Ras mutants differ slightly in their biochemical function and structural conformation. I will use functional assays coupled with X-ray crystallography to investigate if the same is true for K-Ras(G12C) and K-Ras(G13C). I will also use our small molecules to examine if comparable K-Ras(G12C) and K-Ras(G13C) lung adenocarcinoma cell lines have differential sensitivity to inhibition of downstream Ras-driven pathways. Completion of this project will elucidate the differences between these two important oncogenic mutants with the potential for future application to other K-Ras mutants. This project will furthermore validate the DOCKovalent method as a strategy for mutant- specific inhibitor development for small GTPases.