A Fragment-Based Strategy for K-RAS Covalent Inhibitors

摘要 KRAS是胰腺导管腺癌中最常见的突变癌基因，突变率为95 在结肠直肠癌中为45%，在肺腺癌中为30%。最常见的K-RAS突变 存在于密码子12处，即G12 D、G12 V、G12 C和G12 R。K-RAS被认为是不可用的，因为缺乏 明确的药物结合口袋。但最近的一项突破是利用共价抑制剂， 与K-RAS G12 C半胱氨酸结合。这些化合物中有几种正在进行临床试验，其中一种被给予FastTrack FDA的地位。不幸的是，只有一小部分K-RAS癌基因突变体携带G12 C突变， 并且G12 D、G12 V和其它突变不提供可接近的半胱氨酸亲核试剂。在最近的工作与 我们通过高分辨率结构和广泛的生化研究表明， 与Tyr-82形成的键在Switch II和Switch之间产生了一个明确的新结合位点 I/II口袋。最近进行的额外片段筛选鉴定了形成共价结合的片段。 K-RAS开关II Tyr-64处的键，以抑制七子鸟嘌呤（SOS）对GTdR的激活 交换因子在这里，我们假设与酪氨酸或其他氨基酸在K- RAS将抑制癌细胞中的活化或效应物结合并阻断K-RAS信号传导。我们的初步数据和 我们在这方面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， 采用基于配体和结构的方法从大型商业化的 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 光谱分析、核苷酸交换和效应物结合研究，以筛选命中化合物的片段文库， 并表征从片段生长策略中出现的小分子。在具体目标3中，我们 使用X射线晶体学来解决命中碎片和碎片衍生物的结构 越来越努力。我们还进行了细胞生物学研究，以证实K-RAS的直接参与，K-RAS的抑制， RAS信号传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 癌基因突变体的活性，并抑制PDAC和肺腺癌癌细胞的生存能力。 这些化合物将作为出发点，追求领先的优化努力，以发展 用于治疗K-RAS驱动的肿瘤的治疗剂。

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.