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）：小的GTPase K-RAS是癌症中最常见的癌基因。直接抑制其他肿瘤基因，例如融合蛋白BCR-ABL，B-RAF V600E等，为具有各自激酶靶的患者带来了突破性的疗法。尽管癌症中K-RAS突变的普遍存在，但直到最近的几份报道，这种癌基因的直接抑制剂在很大程度上尚无。我们最近使用基于二硫键的链球菌筛查筛选了肺癌中最常见的K-RAS突变（G12C）中最常见的K-RAS突变的抑制剂。这些抑制剂结合了Switch-II后面的新型口袋，这是RAS的两个移动域之一。我们称之为开关II口袋（S-IIP）的口袋可以被利用为变构控制核苷酸亲和力 以及效应子的相互作用和锁定不活跃状态的RAS。我们当前的化合物取决于抑制剂与突变体半胱氨酸12的共价附着。但是，在所有组织的癌症中，非半胱氨酸取代占大多数K-RAS突变。为了开发能够靶向这些突变体的抑制剂（包括最常见的突变体，G12D和G12V），我们的目标是开发出非共价靶向K-RAS S-IIP的小分子，并且不取决于位置12处突变的半胱天素体。在我们的共价抑制剂研究过程中，我们表征了S-IIP中一个疏水区域，该区域占抑制剂结合亲和力的高比例，我们称为高亲和力子位点。对原始束缚屏幕库的调查表明，大多数碎片太短而无法到达该地区。我们建议将不自然的半胱氨酸残基与S-IIP的高亲和力子口袋近距离引入，以用作临时的共价手柄，以筛选扩展的束缚片段。使用这种方法来最大化我们扫描的化学空间，我们旨在确定表现出高配体效率（相对于其质量的高亲和力）的紧密结合的绑扎片段，以作为开发S-IIP非共价抑制剂的最后一个目标的起点。 S-IIP的结构分析表明，蛋氨酸72（M72）或Valine-9（V9）的突变应提供最佳的半胱氨酸定位。使用完整的蛋白质质谱法对二硫键片段的初步筛选，对K-RAS M72C和K-RAS V9C进行了初步筛选，发现了几个以高配体效率与M72C结合的片段。在AIM 1中使用X射线晶体学的结构表征对这些可逆的共价命中的初始化学优化对于理解在S-IIP中结合的基础至关重要。这些数据将有助于指导从二硫键（M72C）中需要可逆共价附着的碎片的进展，从而在AIM 2中铅化合物无共价与K-RAS G12D和G12V结合。