Comparing HTS with Fragment-Based Design for B-Raf and the Ras-Raf Interaction

描述(由申请人提供)：我们建议使用计算建模、生物物理核磁共振研究和生化分析来比较传统高通量筛选(HTS)方法和新的综合配体设计策略的有效性。我们将比较这两种方法的有效性与两个靶点--一个传统的激酶(B-Raf)和一个具有挑战性的K-Ras和B-Raf之间的蛋白质-蛋白质相互作用。K-RAS是一种在癌症生物学中具有重要意义的癌蛋白。RAS癌蛋白(K-RAS、H-RAS、N-RAS)是30多年前发现的，但对小分子药物的直接靶向一直具有抵抗力。因此，尽管KRAS基因在20%的肿瘤中发生突变，在60%的胰腺癌中发生突变，但没有治疗突变的KRAS肿瘤的方法。K-RAS激活效应蛋白(Raf蛋白、PI3K蛋白和Ral-GDS等)是通过K-RAS上的Switch I区介导的，该区域与B-Raf和其他效应蛋白的RAS结合域形成扩展的β片层。破坏这种蛋白质-蛋白质相互作用的抗体可以逆转RAS诱导的动物肿瘤形成。因此，这种相互作用的小分子抑制剂很可能对含有突变KRAS的肿瘤是有用的治疗方法。如果我们证明这种新的设计方法比标准的HTS更有效，它将对发现小分子抑制剂针对目前无法用药的靶蛋白的可能性产生深远的影响。因此，该项目可能导致第一个针对突变K-RAS靶向肿瘤的小分子疗法，并可能打开大量无法药物抑制的小分子靶点。 与公共健康相关：我们的目标是开发一种新的方法来设计不可药物蛋白的抑制剂。这种方法可能最终导致新的治疗剂的发现。

DESCRIPTION (provided by applicant): We propose to compare the effectiveness of the conventional high-throughput screening (HTS) approach with a new integrated ligand design strategy using computational modeling, biophysical NMR studies and biochemical assays. We will compare the effectiveness of these two approaches against two targets-a conventional kinase (B-Raf) and a challenging protein-protein interaction between K-Ras and B-Raf. K-Ras is an oncoprotein of paramount importance of cancer biology. The Ras oncoproteins (K-Ras, H-Ras, N-Ras) were discovered over 30 years ago, but have been resistant to direct targeting with small molecule drugs. Thus, despite the fact that the KRAS gene is mutated in ~20% of all tumors, and >60% of pancreatic cancers, there is no therapy for treating mutant KRAS tumors. K-Ras activation of effector proteins (Raf proteins, PI3K proteins, and Ral-GDS, among others) is mediated through the switch I region on K-Ras, which forms an extended beta sheet with the Ras-binding domain of B- Raf and other effector proteins. An antibody that disrupts this protein-protein interaction reverses Ras-induced tumor formation in animals. Thus, a small molecule inhibitor of this interaction is likely to be a useful therapeutic for tumors containing mutant KRAS. If we demonstrate increased effectiveness of this new design approach compared to standard HTS, it will have a profound effect on the likelihood of small molecule inhibitor discovery against currently undruggable target proteins. This project could thus lead to the first small molecule therapeutics for targeting tumors with mutant K-Ras and may open up a large number of undruggable targets to small molecule inhibition. PUBLIC HEALTH RELEVANCE: Our goal is to develop a new approach to designing inhibitors of undruggable proteins. This approach may ultimately result in the discovery of new therapeutic agents.