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

摘要 这三个RAS基因(HRAS、NRAS和KRAS)是人类中突变最多的癌基因 在这些癌症中，KRAS是突变最频繁的亚型(72%)，占所有RAS的90%的≥ 胰腺、肺和结直肠肿瘤的突变。因此，人们对开发反病毒药物非常感兴趣。 RAS癌症疗法。RAS在GDP绑定的“非活动”状态和GTP绑定的“活动”状态之间循环，并绑定 鸟嘌呤核苷酸通过一个大的中央口袋被蛋白质的动态“开关”区域包围。 RAS亚型中的癌症相关突变总是以GTP填充RAS，从而使它们 构成激活的。几乎所有当前旨在寻找RAS直接抑制剂的策略都旨在 与效应器的绑定竞争，如RAF和PI3K。不幸的是，RAS上的效应器结合部位 没有靶向的口袋和产生的分子以足够的亲和力结合(使它们有用 作为潜在的RAS化疗药物)已被证明是困难的。然而，最近有两个不同的团体 成功开发出与取代半胱氨酸侧不可逆结合的RASG12C变构抑制剂 链条。这种专门针对RAS突变形式的策略可能更有利，因为它可以抑制 直接致癌RAS似乎更有效，同时潜在地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，但它不能扩展到其他缺乏 适当取代活性侧链。 一种似乎合乎逻辑的抑制RAS信号的方法是开发可逆的GTP- 竞争性抑制剂，阻断GTP结合，使RAS失活。这种方法被认为是不可能的。 许多是因为RAS对GTP的高亲和力(皮摩尔)和高浓度的鸟嘌呤 细胞中的核苷酸。然而，我们最近表明，一些RAS突变体表现出降低的能力 绑定GTP，这自相矛盾地使它们致癌。这些产品包括RASG13D、RASA146T和RASK117N以及 约占结直肠癌所有突变KRAS的30%。对GTP的亲和力降低导致这些RAS 易受小分子抑制的突变体比正常RAS具有潜在的选择性。因此，我们建议 使用我们新开发的基于荧光的鸟嘌呤核苷酸置换分析在高密度的 吞吐量筛选(HTS)计划，以寻找致癌RAS的抑制剂。

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.