A HTS Approach to Discover Guanine Nucleotide-Competitive Inhibitors of Oncogenic KRAS

发现致癌 KRAS 鸟嘌呤核苷酸竞争性抑制剂的 HTS 方法

• 批准号: 10007623
• 负责人: KENT ROSSMAN
• 金额: $ 38.95万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-13 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10007623
• 关键词: Accounting; Advanced Malignant Neoplasm; Affinity; Amino Acid Substitution; BRAF gene; Binding; Binding Sites; Biochemical; Biological Assay; Bypass; Cell Proliferation; Cells; Cessation of life; Chemicals; Colorectal; Colorectal Cancer; Colorectal Neoplasms; Crystallization; Cysteine; Data; Development; Diagnosis; Diversity Library; Exhibits; Fluorescence; GTP Binding; Guanine; Guanine Nucleotides; Guanosine Triphosphate; Human; KRAS2 gene; Lead; Lung; Lung Neoplasms; MAP Kinase Gene; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Molecular Conformation; Mutate; Mutation; Normal Cell; Oncogenes; Oncogenic; Oncoproteins; Pancreas; Pathway interactions; Patients; Property; Protein Isoforms; Protein Region; Proteins; RAS genes; Ras Inhibitor; Reporting; Resistance development; Roentgen Rays; Series; Side; Signal Transduction; Site; Specificity; Structure; Surface; Therapeutic; Toxic effect; Validation; Work; X-Ray Crystallography; anticancer research; base; biological systems; cancer therapy; cancer type; chemotherapy; design; effective therapy; high throughput screening; inhibitor/antagonist; interest; metaplastic cell transformation; mutant; nanomolar; novel; pancreatic neoplasm; ras Guanine Nucleotide Exchange Factors; ras Proteins; screening program; small molecule; success; tumor; virtual

Abstract Collectively, the three RAS genes (HRAS, NRAS and KRAS) are the most mutated oncogenes in human cancers, and of these, KRAS is the isoform most frequently mutated (72%), accounting for ≥90% of all RAS mutations in pancreatic, lung and colorectal tumors. Accordingly, there is intense interest in developing anti- RAS cancer therapies. RAS cycles between GDP-bound “inactive” and GTP-bound “active” states, and binds guanine nucleotides via a large central pocket surrounded by the dynamic “switch” regions of the protein. Cancer-associated mutations in RAS isoforms invariably populate RAS with GTP thus rendering them constitutively activated. Virtually all current strategies which aim to find direct inhibitors of RAS are designed to compete with the binding of effectors, such as RAF and PI3K. Unfortunately, the effector binding site on RAS is devoid of targetable pockets and generating molecules that bind with sufficient affinity (to make them useful as potential RAS chemotherapies) has proven difficult. However, two different groups have recently succeeded in developing allosteric inhibitors of RASG12C which irreversibly bind to the substituted cysteine side chain. This strategy of specifically targeting mutant forms of RAS may be more advantageous as inhibiting oncogenic RAS directly would seemingly be more efficacious while potentially offering less normal cell toxicity. While this discovery represents a proof of concept, it cannot be extended to other RAS proteins lacking the appropriately substituted reactive sidechains. One seemingly logical approach to inhibiting RAS signaling would be to develop reversible GTP- competitive inhibitors that block GTP binding to render RAS inactive. This approach is considered not possible by many because of the high affinity (picomolar) of RAS for GTP and the high concentration of guanine nucleotides in cells. However, we have recently shown that some RAS mutants exhibit a reduced ability to bind GTP, which paradoxically makes them oncogenic. These include RASG13D, RASA146T and RASK117N and account for ~30% of all mutant KRAS in colorectal cancers. Reduced affinity for GTP renders these RAS mutants vulnerable to small molecule inhibition with potential selectivity over normal RAS. Thus, we propose using our novel, newly developed fluorescence-based guanine nucleotide displacement assay in a high- throughput screening (HTS) program to search for inhibitors of oncogenic RAS.

抽象的 总体而言，三个RAS基因（HRA，NRA和KRAS）是人类中最突变的肿瘤基因 癌症，其中KRA是最常见的同工型（72％），占所有RA的≥90％ 胰腺，肺和结直肠肿瘤的突变。彼此之间，人们对发展反对有浓厚的兴趣 RAS癌症疗法。 GDP结合的“非活动”和GTP结合的“活动”状态之间的RAS循环，并结合 鸟嘌呤核苷酸通过一个大的中央口袋，周围是蛋白质的动态“开关”区域。 RAS同工型中与癌症相关的突变总是用GTP填充RAS 组成性激活。几乎所有旨在查找RA的直接抑制剂的策略旨在 竞争效果的结合，例如RAF和PI3K。不幸的是，RAS上的效应子结合位点 没有可靶向的口袋和产生具有足够亲和力结合的分子（使其有用 作为潜在的RAS化学疗法）已被证明很困难。但是，最近有两个不同的组 成功地开发了RASG12C的变构抑制剂，该抑制剂不可逆地与替代的半胱氨酸侧结合 链。这种专门针对Ras突变形式的策略可能会更有利地有利 致癌性RA直接似乎更有效，同时可能提供较少的正常细胞毒性。 尽管该发现代表了概念证明，但它不能扩展到其他缺乏的RAS蛋白 适当取代的反应性侧链。 一种抑制RAS信号传导的逻辑方法似乎是发展可逆的GTP- 阻断GTP结合到使RAS无活性的竞争性抑制剂。这种方法被认为是不可能的 由于GTP的Ras的高亲和力（picomolar）和高浓度的鸟嘌呤 细胞中的核苷酸。但是，我们最近表明，一些RAS突变体暴露了降低的能力 结合GTP，矛盾地使它们致癌。其中包括rasg13d，rasa146t和rask117n以及 占大肠癌中所有突变KRA的约30％。降低对GTP的亲和力使这些RAS 突变体容易受到小分子抑制作用，并且对正常RA的潜在选择性。那我们建议 使用我们的小说，新开发的基于荧光的基于荧光的鸟嘌呤核丁基位移测定法 吞吐量筛选（HTS）程序以搜索致癌性RAS的抑制剂。