Drugging the Switch-II Pocket of K-Ras

对 K-Ras 的 Switch-II 口袋进行麻醉

• 批准号: 9337416
• 负责人: KEVAN M. SHOKAT
• 金额: $ 29.85万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-12 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9337416
• 关键词: Address; Affinity; Applications Grants; Binding; Binding Sites; Biochemical; Biological Assay; Cells; Cellular Assay; Chemicals; Clinical Trials; Colon Carcinoma; Cysteine; Data; Development; Disulfides; Evaluation; Glycine; Goals; Guanosine Triphosphate; Hydrophobicity; In Vitro; K-ras Oncogene; KRAS2 gene; Lead; Legal patent; Lesion; Libraries; Ligand Binding; Ligands; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Mass Spectrum Analysis; Measures; Methionine; Monomeric GTP-Binding Proteins; Mutate; Mutation; Nature; Nucleotides; Oncogenes; Oncogenic; Patients; Pharmaceutical Preparations; Phosphotransferases; Positioning Attribute; Prevalence; Proteins; Ras Inhibitor; Reporting; Roentgen Rays; Scanning; Site; Structure-Activity Relationship; Sulfhydryl Compounds; Surveys; Tissues; Valine; Work; X-Ray Crystallography; base; bcr-abl Fusion Proteins; chemical synthesis; disulfide bond; inhibitor/antagonist; insight; mutant; novel; public health relevance; scaffold; screening; small molecule

DESCRIPTION (provided by applicant): The small GTPase K-Ras is the most frequently mutated oncogene in cancer. Direct inhibition of other oncogenes such as the fusion protein BCR-Abl, B-Raf V600E, and others, has resulted in breakthrough therapies for patients harboring the respective kinase target. Despite the prevalence of K-Ras mutations in cancer, direct inhibitors of this oncogene have been largely unavailable until several recent reports. We recently identified inhibitors of the most common K-Ras mutation in lung cancer, glycine-12 to cysteine (G12C), using a disulfide-based tethering screen. These inhibitors bind to a novel pocket behind switch-II, one of the two mobile domains of Ras. This pocket, which we have termed the switch-II pocket (S-IIP), can be exploited to allosterically control nucleotide affinity and effector interactions and lock Ras in an inactive state. Our current compounds depend on covalent attachment of the inhibitor to the mutant cysteine-12. However, across cancers of all tissues, non-cysteine substitutions account for a majority of K-Ras mutations. In order to develop inhibitors capable of targeting these mutants (including the most frequent mutants, G12D and G12V), we aim to develop small molecules that non-covalently target the K-Ras S-IIP and do not depend on the presence of a mutant cysteine at position-12. In the course of our covalent inhibitor studies we characterized a hydrophobic region within the S-IIP that accounts for a high proportion of inhibitor binding affinity, which we refer to as the high affinity sub-pocet. A survey of the original tethering screen library suggests that the majority of fragments were too short to reach this region. We propose to introduce unnatural cysteine residues in close proximity to the high affinity sub-pocket of the S-IIP to use as temporary covalent handles for screening an expanded library of tethering fragments. Using this approach to maximize the chemical space we scan, we aim to identify tight-binding tethering fragments that display high ligand efficiency (high affinity relative to their mass) to serve as starting points for the ultimae goal of developing non-covalent inhibitors of the S-IIP. Structural analysis of the S-IIP suggests that mutation of methionine-72 (M72) or valine-9 (V9) should afford optimal cysteine positioning. Preliminary screening of a library of disulfide-containing fragments against K-Ras M72C and K-Ras V9C using intact protein mass spectrometry uncovered several fragments that bind to M72C with high ligand efficiency. Initial chemical optimization of these reversible covalent hits i conjunction with structural characterization using X-ray crystallography in Aim 1 will be imperative for understanding the basis for binding in the S-IIP. These data will help guide the progression from fragments that require reversible covalent attachment through disulfide bonds (M72C) to lead compounds binding non-covalently to K-Ras G12D and G12V in Aim 2. Finally, we will evaluate the biochemical and cellular effects of these compounds in Aim 3.

描述（由申请人提供）：小 GTPase K-Ras 是癌症中最常见的突变癌基因。直接抑制融合蛋白 BCR-Abl、B-Raf V600E 等其他癌基因，为携带相应激酶靶标的患者带来了突破性疗法。尽管 K-Ras 突变在癌症中普遍存在，但直到最近的几份报告之前，这种癌基因的直接抑制剂基本上还无法获得。我们最近使用基于二硫键的束缚筛选，鉴定了肺癌中最常见的 K-Ras 突变（甘氨酸 12 到半胱氨酸 (G12C)）的抑制剂。这些抑制剂与 switch-II 后面的一个新口袋结合，switch-II 是 Ras 的两个移动结构域之一。我们将这个口袋称为 switch-II 口袋 (S-IIP)，可用于变构控制核苷酸亲和力 和效应器相互作用并将 Ras 锁定在非活动状态。我们目前的化合物依赖于抑制剂与突变型半胱氨酸 12 的共价连接。然而，在所有组织的癌症中，非半胱氨酸取代占 K-Ras 突变的大部分。为了开发能够靶向这些突变体（包括最常见的突变体 G12D 和 G12V）的抑制剂，我们的目标是开发非共价靶向 K-Ras S-IIP 并且不依赖于 12 位突变半胱氨酸的存在的小分子。在我们的共价抑制剂研究过程中，我们表征了 S-IIP 内的疏水区域，该区域占抑制剂结合亲和力的很大比例，我们将其称为高亲和力子池。对原始系留屏幕库的调查表明，大多数碎片都太短而无法到达该区域。我们建议在 S-IIP 的高亲和力子口袋附近引入非天然半胱氨酸残基，用作临时共价手柄来筛选扩展的束缚片段文库。使用这种方法来最大化我们扫描的化学空间，我们的目标是识别具有高配体效率（相对于其质量的高亲和力）的紧密结合束缚片段，作为开发 S-IIP 非共价抑制剂的最终目标的起点。 S-IIP 的结构分析表明，蛋氨酸 72 (M72) 或缬氨酸 9 (V9) 的突变应提供最佳的半胱氨酸定位。使用完整蛋白质质谱法对 K-Ras M72C 和 K-Ras V9C 的含二硫键片段文库进行初步筛选，发现了几个以高配体效率与 M72C 结合的片段。这些可逆共价命中的初始化学优化以及目标 1 中使用 X 射线晶体学的结构表征对于理解 S-IIP 中结合的基础至关重要。这些数据将有助于指导目标 2 中从需要通过二硫键 (M72C) 可逆共价连接的片段发展到与 K-Ras G12D 和 G12V 非共价结合的先导化合物。最后，我们将在目标 3 中评估这些化合物的生化和细胞效应。