Drugging the Switch-II Pocket of K-Ras

描述（由申请人提供）：小GTPase K-Ras是癌症中最常见的突变癌基因。直接抑制其他癌基因，如融合蛋白BCR-Abl、B-Raf V600E等，已经为携带相应激酶靶点的患者带来了突破性的治疗方法。尽管K-Ras突变在癌症中普遍存在，但这种致癌基因的直接抑制剂在很大程度上是不可用的，直到最近的几篇报道。我们最近使用基于二硫化物的系留筛选确定了肺癌中最常见的K-Ras突变的抑制剂，甘氨酸-12到半胱氨酸（G12C）。这些抑制剂结合到Ras的两个可移动结构域之一的开关ii后面的一个新口袋。这个口袋，我们称之为开关ii口袋（S-IIP），可以利用变结构控制核苷酸亲和力

DESCRIPTION (provided by applicant): The small GTPase K-Ras is the most frequently mutated oncogene in cancer. Direct inhibition of other oncogenes such as the fusion protein BCR-Abl, B-Raf V600E, and others, has resulted in breakthrough therapies for patients harboring the respective kinase target. Despite the prevalence of K-Ras mutations in cancer, direct inhibitors of this oncogene have been largely unavailable until several recent reports. We recently identified inhibitors of the most common K-Ras mutation in lung cancer, glycine-12 to cysteine (G12C), using a disulfide-based tethering screen. These inhibitors bind to a novel pocket behind switch-II, one of the two mobile domains of Ras. This pocket, which we have termed the switch-II pocket (S-IIP), can be exploited to allosterically control nucleotide affinity and effector interactions and lock Ras in an inactive state. Our current compounds depend on covalent attachment of the inhibitor to the mutant cysteine-12. However, across cancers of all tissues, non-cysteine substitutions account for a majority of K-Ras mutations. In order to develop inhibitors capable of targeting these mutants (including the most frequent mutants, G12D and G12V), we aim to develop small molecules that non-covalently target the K-Ras S-IIP and do not depend on the presence of a mutant cysteine at position-12. In the course of our covalent inhibitor studies we characterized a hydrophobic region within the S-IIP that accounts for a high proportion of inhibitor binding affinity, which we refer to as the high affinity sub-pocet. A survey of the original tethering screen library suggests that the majority of fragments were too short to reach this region. We propose to introduce unnatural cysteine residues in close proximity to the high affinity sub-pocket of the S-IIP to use as temporary covalent handles for screening an expanded library of tethering fragments. Using this approach to maximize the chemical space we scan, we aim to identify tight-binding tethering fragments that display high ligand efficiency (high affinity relative to their mass) to serve as starting points for the ultimae goal of developing non-covalent inhibitors of the S-IIP. Structural analysis of the S-IIP suggests that mutation of methionine-72 (M72) or valine-9 (V9) should afford optimal cysteine positioning. Preliminary screening of a library of disulfide-containing fragments against K-Ras M72C and K-Ras V9C using intact protein mass spectrometry uncovered several fragments that bind to M72C with high ligand efficiency. Initial chemical optimization of these reversible covalent hits i conjunction with structural characterization using X-ray crystallography in Aim 1 will be imperative for understanding the basis for binding in the S-IIP. These data will help guide the progression from fragments that require reversible covalent attachment through disulfide bonds (M72C) to lead compounds binding non-covalently to K-Ras G12D and G12V in Aim 2. Finally, we will evaluate the biochemical and cellular effects of these compounds in Aim 3.