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 总的来说，三种 RAS 基因（HRAS、NRAS 和 KRAS）是人类突变最多的癌基因 其中，KRAS 是最常突变的亚型（72%），占所有 RAS 的 ≥90% 因此，人们对开发抗肿瘤药物产生了浓厚的兴趣。 RAS 癌症疗法。RAS 在 GDP 结合的“非活性”状态和 GTP 结合的“活性”状态之间循环，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 异构体中与癌症相关的突变总是用 GTP 填充 RAS，从而使它们 事实上，目前所有旨在寻找 RAS 直接抑制剂的策略都是为了实现这一目的。 与效应子的结合竞争，例如 RAF 和 PI3K 不幸的是，效应子结合位点位于 RAS 上。 缺乏可靶向的口袋并产生具有足够亲和力的分子（使它们有用） 作为潜在的 RAS 化疗）已被证明是困难的，然而，最近两个不同的研究小组已经证明了这一点。 成功开发出 RASG12C 的变构抑制剂，该抑制剂不可逆地与取代的半胱氨酸侧结合 这种专门针对 RAS 突变形式的策略可能更有利于抑制。 直接致癌 RAS 似乎更有效，同时可能提供更少的正常细胞毒性。 虽然这一发现代表了概念的证明，但它不能扩展到缺乏 适当取代的反应性侧链。 抑制 RAS 信号传导的一种看似合乎逻辑的方法是开发可逆的 GTP- 阻断 GTP 结合从而使 RAS 失活的竞争性抑制剂这种方法被认为是不可能的。 由于 RAS 对 GTP 的高亲和力（皮摩尔）和高浓度的鸟嘌呤，许多人认为 然而，我们最近发现一些 RAS 突变体表现出降低的能力。 结合 GTP，这反而使它们致癌。这些包括 RASG13D、RASA146T 和 RASK117N。 约占结直肠癌中所有突变 KRAS 的 30%。 对 GTP 的亲和力降低导致这些 RAS 突变。 突变体易受小分子抑制，具有比正常 RAS 潜在的选择性。 使用我们新开发的基于荧光的鸟嘌呤核苷酸置换测定在高 通量筛选 (HTS) 程序，用于寻找致癌 RAS 的抑制剂。