A Fragment-Based Strategy for K-RAS Covalent Inhibitors

摘要 KRAS是胰腺癌中突变最多的癌基因，突变率高达95% (PDAC)，结直肠癌为45%，肺腺癌为30%。最常见的K-RAS突变 发生在第12密码子，即G12D、G12V、G12C和G12R。K-RAS被认为是无法下药的，因为缺乏 定义明确的毒品捆绑口袋。但最近在共价抑制剂方面取得了突破，这种抑制剂形成了一种 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，其中一种已经服用了FastTrack 食品和药物管理局的地位。不幸的是，只有一小部分K-RAS癌基因突变体存在G12C突变， 而G12D、G12V和其他突变不提供可获得的半胱氨酸亲核试剂。在最近与 我们通过高分辨结构和广泛的生化研究表明，Ras GTPase re 与Tyr-82的成键形成了位于开关II和开关之间的明确定义的新结合部位 I/II口袋。最近进行的附加片段筛选确定了形成共价的片段 K-RAS开关II的酪氨酸-键抑制SOS鸟嘌呤激活的GTP酶 交换系数。这里，我们假设与酪氨酸或其他氨基酸在K-上形成共价键 RAS将抑制激活或效应器结合，并阻断癌细胞中的K-RAS信号。我们的初步数据和 在该领域的丰富经验使我们在实现我们的目标方面处于有利地位。在具体目标1中，我们 使用基于配基和结构的方法从大型商业数据库中生成片段电泳库 集合，我们遵循一种基于结构的方法将命中片段增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组分 光谱、核苷酸交换和效应器结合研究以筛选命中化合物的片段文库， 并对从碎片生长策略中出现的小分子进行表征。在具体目标3中，我们将 用X射线结晶学研究碎片及其衍生产物的结构 越来越多的努力。我们还进行了细胞生物学研究，以确认K-RAS的直接结合，K-RAS的抑制- RAS信号转导和抑制癌细胞增殖。我们希望识别出高质量的碎片和小的 在野生型和突变型K-RAS癌基因上形成共价键的分子，抑制K-RAS野生型或 癌基因突变在癌细胞株中的活性，并抑制PDAC和肺腺癌癌细胞的活力。 这些化合物将作为推动优化工作的起点，以开发 治疗K-RAS驱动的肿瘤的治疗药物。

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.