Drugging the Switch-II Pocket of K-Ras

描述（由申请人提供）：小GTK-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 V600 E等，已经为携带相应激酶靶标的患者带来了突破性疗法。尽管K-Ras突变在癌症中普遍存在，但直到最近的几篇报道，这种癌基因的直接抑制剂才基本上可用。我们最近使用基于二硫化物的系链筛选确定了肺癌中最常见的K-Ras突变的抑制剂，甘氨酸-12变为半胱氨酸（G12 C）。这些抑制剂结合到开关II后面的一个新口袋，开关II是Ras的两个移动的结构域之一。这个口袋，我们称之为开关-II口袋（S-IIP），可用于变构控制核苷酸亲和力 和效应子相互作用并将Ras锁定在非活动状态。我们目前的化合物依赖于抑制剂与突变型半胱氨酸-12的共价连接。然而，在所有组织的癌症中，非半胱氨酸取代占大多数K-Ras突变。为了开发能够靶向这些突变体（包括最常见的突变体G12 D和G12 V）的抑制剂，我们的目标是开发非共价靶向K-Ras S-IIP并且不依赖于在位置-12处突变半胱氨酸的存在的小分子。在我们的共价抑制剂研究过程中，我们表征了S-IIP内的疏水区域，该疏水区域占抑制剂结合亲和力的高比例，我们将其称为高亲和力子袋。对原始系链筛选文库的调查表明，大多数片段太短，无法到达该区域。我们建议在S-IIP的高亲和力子口袋附近引入非天然半胱氨酸残基，以用作临时共价手柄，用于筛选扩展的系链片段文库。使用这种方法来最大限度地提高我们扫描的化学空间，我们的目标是确定紧密结合的拴系片段，显示高配体效率（相对于其质量的高亲和力），作为开发非共价抑制剂的S-IIP的最终目标的起点。S-IIP的结构分析表明，甲硫氨酸-72（M72）或缬氨酸-9（V9）的突变应提供最佳的半胱氨酸定位。使用完整蛋白质质谱法初步筛选针对K-Ras M72 C和K-Ras V9 C的含二硫键片段的文库，发现了几个以高配体效率结合M72 C的片段。这些可逆共价命中的初始化学优化结合目标1中使用X射线晶体学的结构表征对于理解S-IIP中结合的基础将是必要的。这些数据将有助于指导从需要通过二硫键可逆共价连接的片段（M72 C）到与Aim 2中的K-Ras G12 D和G12 V非共价结合的先导化合物的进展。最后，我们将在目标3中评估这些化合物的生化和细胞效应。

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.