A Fragment-Based Strategy for K-RAS Covalent Inhibitors

基于片段的 K-RAS 共价抑制剂策略

• 批准号: 10443837
• 负责人: Samy Meroueh
• 金额: $ 45.98万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-02 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10443837
• 关键词: Affinity; Amino Acids; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cancer cell line; Cell Proliferation; Cell Survival; Cells; Cellular biology; Clinical Trials; Codon Nucleotides; Collection; Colon Carcinoma; Colorectal; Colorectal Cancer; Communities; Computing Methodologies; Crystallization; Cysteine; Data; Development; Event; Fluorescence; Fluorides; GTP Binding; Goals; Guanine; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; KRAS2 gene; Lead; Libraries; Ligands; Lung Adenocarcinoma; Lysine; Malignant Neoplasms; Malignant neoplasm of lung; Mass Spectrum Analysis; Methods; Mission; Mutate; Mutation; Non-Small-Cell Lung Carcinoma; Nucleotides; Oncogenes; Pancreatic Ductal Adenocarcinoma; Pharmaceutical Preparations; Phosphorylation; Positioning Attribute; Proteins; Public Health; RAS inhibition; Ras Inhibitor; Reaction; Reporting; Resolution; Roentgen Rays; Serine; Signal Transduction; Site; Solubility; Son; Structure; Testing; Therapeutic; Therapeutic Agents; Threonine; Time; Tyrosine; United States National Institutes of Health; Western Blotting; Work; X-Ray Crystallography; adduct; antagonist; base; cancer cell; cheminformatics; covalent bond; design; experience; inhibitor; lead optimization; liquid chromatography mass spectrometry; mortality; mutant; non-oncogenic; novel; pancreatic cancer patients; pancreatic ductal adenocarcinoma cell; reaction rate; screening; small molecule; small molecule inhibitor; structural biology; therapeutic development; three dimensional structure; tumor; tumor growth

ABSTRACT KRAS is the most frequently mutated oncogene with mutation rates of 95% in pancreatic ductal adenocarcinoma (PDAC), 45% in colorectal cancer, and 30% in lung adenocarcinomas. The most common K-RAS mutations occur at codon 12, namely G12D, G12V, G12C, and G12R. K-RAS was considered undruggable due to lack of well-defined drug-binding pockets. But a recent breakthrough was achieved with covalent inhibitors that form a bond with K-RAS G12C cysteine. Several of these compounds are in clinical trials, and one was given FastTrack status by the FDA. Unfortunately, only a small fraction of K-RAS oncogene mutants harbor the G12C mutation, and G12D, G12V and other mutations do not provide an accessible cysteine nucleophile. In recent work with the RAS GTPase Ral we showed through high-resolution structures and extensive biochemical studies that covalent bond formation with Tyr-82 created a well-defined novel binding site located between the Switch II and the Switch I/II pockets. Additional fragment screening carried out more recently identified a fragment that forms a covalent bond at K-RAS Switch II Tyr-64 to inhibit activation of the GTPase by the Son-of-Sevenless (SOS) guanine exchange factor. Here, we hypothesize that covalent bond formation with tyrosine or other amino acids on K- RAS will inhibit activation or effector binding and block K-RAS signaling in cancer cells. Our preliminary data and extensive experience in the field puts us in a strong position to accomplish our objectives. In Specific Aim 1, we employ ligand- and structure-based methods to generate fragment electrophile libraries from large commercial collections, and we follow a structure-based method to grow hit fragments into neighboring pockets to enhance their binding affinity and reaction rates. In Specific Aim 2, we will carry out well-established intact protein mass spectrometry, nucleotide exchange, and effector binding studies to screen fragment libraries for hit compounds, and to characterize small molecules that emerge from fragment growing strategies. In Specific Aim 3, we will use X-ray crystallography to solve the structure of hit fragments and derivatives that emerge from fragment growing efforts. We also carry out cell biological studies to confirm direct engagement of K-RAS, inhibition of K- RAS signaling, and inhibition of cancer cell proliferation. We expect to identify high quality fragments and small molecules that form a covalent bond at wild-type and mutant K-RAS oncogenes, inhibit K-RAS wild-type or oncogene mutant activity in cancer cell lines, and inhibit PDAC and lung adenocarcinoma cancer cell viability. These compounds will serve as starting points to pursue in lead optimization efforts towards the development of therapeutic agents for the treatment of K-RAS-driven tumors.

抽象的 KRAS是胰腺导管腺癌中最常突变的癌基因，突变率为95% (PDAC)，结直肠癌中 45%，肺腺癌中 30%。最常见的 K-RAS 突变 发生在密码子 12 处，即 G12D、G12V、G12C 和 G12R。由于缺乏 K-RAS，K-RAS 被认为是不可成药的 明确的药物结合袋。但最近在共价抑制剂方面取得了突破，该抑制剂形成了 与 K-RAS G12C 半胱氨酸结合。其中几种化合物正在进行临床试验，其中一种获得了 FastTrack FDA 的地位。不幸的是，只有一小部分 K-RAS 癌基因突变体带有 G12C 突变， G12D、G12V 和其他突变不提供可接近的半胱氨酸亲核试剂。在最近与 我们通过高分辨率结构和广泛的生化研究表明，RAS GTPase Ral 共价 与 Tyr-82 形成的键在 Switch II 和 Switch 之间创建了一个明确的新型结合位点 I/II 口袋。最近进行的额外片段筛选鉴定了形成共价键的片段 结合在 K-RAS Switch II Tyr-64 上，抑制七子之子 (SOS) 鸟嘌呤对 GTP 酶的激活 交换因子。在这里，我们假设与 K-上的酪氨酸或其他氨基酸形成共价键 RAS 将抑制癌细胞中的激活或效应子结合并阻断 K-RAS 信号传导。我们的初步数据和 该领域的丰富经验使我们有能力实现我们的目标。在具体目标 1 中，我们 采用基于配体和结构的方法从大型商业中生成片段亲电子库 集合，我们遵循基于结构的方法将命中片段生长到相邻的口袋中以增强 它们的结合亲和力和反应速率。在具体目标 2 中，我们将进行完善的完整蛋白质质量分析 光谱分析、核苷酸交换和效应子结合研究，以筛选片段库中的命中化合物， 并表征片段生长策略中出现的小分子。在具体目标 3 中，我们将 使用X射线晶体学来解析命中碎片的结构以及从碎片中产生的衍生物 不断增长的努力。我们还进行细胞生物学研究，以确认 K-RAS 的直接参与，抑制 K- RAS 信号转导和抑制癌细胞增殖。我们希望能够识别出高质量的碎片和小碎片 在野生型和突变型 K-​​RAS 癌基因上形成共价键的分子，抑制野生型或突变型 K-​​RAS 癌基因突变在癌细胞系中的活性，并抑制PDAC和肺腺癌细胞的活力。 这些化合物将作为先导化合物优化工作的起点，以开发 用于治疗 K-RAS 驱动的肿瘤的治疗剂。