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

摘要 总的来说，三种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是最常突变的亚型（72%），占所有RAS的≥90% 胰腺、肺和结肠直肠肿瘤中的突变。因此，有强烈的兴趣，在发展反- RAS癌症疗法。RAS在GDP结合的“非活性”和GTP结合的“活性”状态之间循环，并结合 鸟嘌呤核苷酸通过由蛋白质的动态“开关”区域包围的大的中心口袋。 RAS亚型中的癌症相关突变总是使RAS与GTP共存，从而使它们成为 组成性激活。几乎所有旨在发现RAS直接抑制剂的当前策略都被设计为 与效应物如RAF和PI 3 K的结合竞争。不幸的是，RAS上的效应子结合位点 缺乏可靶向的口袋和产生以足够的亲和力结合（使它们有用）的分子 作为潜在的RAS化疗）已被证明是困难的。然而，两个不同的团体最近 成功地开发了RASG 12 C的变构抑制剂，其不可逆地结合取代的半胱氨酸侧 链这种特异性靶向RAS突变形式的策略可能更有利，因为抑制RAS突变形式的发生。 直接使用致癌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一个概念的证明，但它不能扩展到其他缺乏RAS的蛋白质。 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- 竞争性抑制剂阻断GTP结合以使RAS失活。这种做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， 细胞中的核苷酸。然而，我们最近发现，一些RAS突变体表现出降低的能力， 与GTP结合，这反而使它们致癌。其中包括RASG 13 D、RASA 146 T和RASK 117 N， 占结直肠癌中所有突变KRAS的约30%。对GTP亲和力的降低使得这些RAS 易受小分子抑制的突变体，具有超过正常RAS的潜在选择性。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测定法， 通量筛选（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.