Drugging the Switch-II Pocket of K-Ras

描述(申请人提供)：小的GTP酶K-RAS是癌症中最常见的突变癌基因。对其他癌基因的直接抑制，如融合蛋白BCR-Abl、B-Raf V600E等，已经为携带各自的激酶靶点的患者带来了突破性的治疗。尽管K-RAS基因突变在癌症中普遍存在，但直到最近的几篇报道之前，这种癌基因的直接抑制剂在很大程度上是不可用的。最近，我们使用基于二硫化物的系链筛选技术，确定了肺癌中最常见的K-RAS突变的抑制物，甘氨酸-12到半胱氨酸(G12C)。这些抑制剂结合到Switch-II后面的一个新口袋上，Switch-II是RAS的两个移动结构域之一。这种口袋，我们称之为Switch-II口袋(S-IIP)，可以用来通过变构来控制核苷酸亲和力 和效应器相互作用，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的半胱氨酸-12的共价结合。然而，在所有组织的癌症中，非半胱氨酸替代是K-RAS突变的主要原因。为了开发能够靶向这些突变体(包括最常见的突变体G12D和G12V)的抑制剂，我们的目标是开发非共价靶向K-RAS S-IIP的小分子，并且不依赖于-12位突变半胱氨酸的存在。在我们的共价抑制剂研究过程中，我们表征了S-IIP内的一个疏水区，它占抑制剂结合亲和力的很高比例，我们称之为高亲和力亚区。对最初的系留筛选文库的调查表明，大多数片段太短，无法到达这个区域。我们建议在S-IIP的高亲和力亚袋附近引入非天然的半胱氨酸残基，作为临时的共价柄用于筛选扩大的系链片段文库。使用这种方法最大化我们扫描的化学空间，我们的目标是识别显示出高配体效率(相对于其质量的高亲和力)的紧密结合的拴系片段，作为开发S-IIP的非共价抑制剂的最终目标的起点。对S-IIP的结构分析表明，蛋氨酸-72(M72)或缬氨酸-9(V9)的突变应该提供半胱氨酸的最佳定位。用完整蛋白质谱仪初步筛选抗K-RAS M72C和K-RAS V9C的二硫键片段文库，发现了几个与M72C结合的高配基效率的片段。对这些可逆共价HITI进行初步的化学优化，结合目标1中的X射线结晶学结构表征，将是理解S-IIP中结合基础的关键。这些数据将有助于指导从需要通过二硫键(M72C)可逆共价连接的片段到导致目标2中的K-RAS G12D和G12V非共价结合的化合物的进展。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.