Understanding the role of KRAS nanoclustering in signalling and disease

丝裂原活化蛋白激酶（MAPK）途径参与由多种细胞内和细胞外刺激激活的多种基本细胞过程。MAPK通路的失调与包括癌症在内的许多人类疾病的发展有关。RAS是MAPK通路中一个重要的GDP/GTP调节分子开关，在大约25%的人类癌症中发现RAS内的功能获得性错义突变。因此，直接或间接地抑制RAS信号传导是药物发现活动的主要焦点。尽管最近在共价靶向G12 C突变方面取得了一些进展，但仍然存在靶向其他突变和耐药性的问题。因此，抑制RAS功能和识别RAS介质的新方法需要进一步的研究和新方法。Affimer试剂是可以分离的工具，其结合靶蛋白以调节功能。Affimers（一种具有由两个可变区组成的小探针表面的生物制剂）已被分离出来，它们与KRAS结合，其中一些抑制重要的生物过程，如纳米簇。该项目将利用Affimer试剂来充分了解纳米簇在信号传导和癌症进展过程中的重要性，以及确定RAS信号传导的新型可药用介质。使用各种过程，包括X射线晶体学（以解决Affimer-RAS蛋白质的结构），EM研究（以成像细胞膜中的RAS二聚化），细胞生物学（以了解抑制RAS纳米簇如何调节细胞信号传导和癌细胞存活）和使用Affimer介导的蛋白质干扰的细胞表型筛选测定（以识别KRAS突变的效应子）。

The mitogen activated protein kinase (MAPK) pathway is involved in a variety of fundamental cellular processes activated by a diverse set of intracellular and extracellular stimuli. Dysregulation of the MAPK pathway has been implicated in the development of many human diseases, including cancer. RAS is an important GDP/GTP regulated molecular switch within the MAPK pathway and gain-of-function missense mutations within RAS are found in approx 25% of all human cancers. Therefore, the inhibition of RAS signalling, either directly or indirectly are major focuses in drug discovery campaigns. Although there had been some recent advances in covalently targeting the G12C mutation there remains the issue of targeting other mutations and drug resistance. As a result, new ways of inhibiting RAS function and identifying mediators of RAS requires further research and novel approaches. Affimer reagents are tools that can be isolated that bind to target proteins to regulate function. Affimers (a biologic with a small probe surface comprised of two variable regions) have been isolated that bind to KRAS, some of which inhibit important biological processes like nanoclustering. This project will utilise Affimer reagents to fully understand the importance of nanoclustering during signalling and cancer progression, as well as, identifying novel druggable mediators of RAS signalling. Using a variety of processes including X-ray crystallography (to solve the structure of Affimer-RAS proteins), EM studies (to image RAS dimerization in cellular membranes), cell biology (to understand how inhibiting RAS nanoclustering regulates cellular signalling and cancer cell survival) and cellular phenotypic screening assays using Affimer-mediate protein interference (to identify effectors of KRAS mutations).