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: 10290524
• 负责人: Samy Meroueh
• 金额: $ 46.92万
• 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/10290524
• 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; base; cancer cell; cheminformatics; covalent bond; design; experience; inhibitor/antagonist; 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是最常见的癌基因，突变率为95％，胰腺导管腺癌中的突变率为95％ （PDAC），结直肠癌45％，肺腺癌30％。最常见的K-RAS突变 发生在密码子12，即G12D，G12V，G12C和G12R。由于缺乏 定义明确的药物结合口袋。但是最近通过形成A的共价抑制剂实现了突破 与K-RAS G12C半胱氨酸结合。这些化合物中的几种是在临床试验中，并且给出了一种。 FDA的状态。不幸的是，只有一小部分K-Ras癌基因突变体藏有G12C突变， G12D，G12V和其他突变不能提供可访问的半胱氨酸亲核试剂。在最近与 RAS GTPase RAL我们通过高分辨率结构和广泛的生化研究展示了共价 用Tyr-82形成的债券形成创建了一个定义明确的新颖绑定位点，位于开关II和开关之间 I/II口袋。最近进行了其他片段筛选，这是形成共价的片段 在K-Ras Switch II Tyr-64上的键以抑制七儿子（SOS）鸟嘌呤的GTPase激活 交换因子。在这里，我们假设在K-上与酪氨酸或其他氨基酸的共价形成 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处于实现目标的强大位置。在特定目标1中，我们 采用配体和基于结构的方法来生成来自大型商业的碎片电力库 收集，我们遵循一种基于结构的方法，将命中片段发展到相邻的口袋中以增强 它们的结合亲和力和反应速率。在特定的目标2中，我们将执行完整的完整蛋白质质量 光谱，核苷酸交换和效应子的结合研究，用于筛选碎片化合物的碎片库， 并表征从碎片增长策略中产生的小分子。在特定的目标3中，我们将 使用X射线晶体学来解决从片段中出现的命中片段和衍生物的结构 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信号传导和抑制癌细胞增殖。我们希望确定高质量的碎片和小 在野生型和突变k-ras Oncogenes上形成共价键的分子，抑制K-Ras野生型或 癌细胞系中的癌基突变活性，并抑制PDAC和肺腺癌癌细胞活力。 这些化合物将作为为开发领先优化努力而追求的起点 治疗K-RAS驱动肿瘤的治疗剂。