Maturation of Ral targeting peptides into covalent inhibitors or PROTACs: inhibitors for Ras-driven cancers

小分子GTP酶调控真核细胞中的信号转导途径。原型小G蛋白RAS是人类癌症中突变最多的癌基因。另一种小G蛋白，Ral，参与RAS信号转导，在人类癌症中，Ral通路在RAS下游起主要作用。这打开了一种靶向RAS驱动的癌症的方法，通过抑制RAR调节的信号通路和开发细胞探针来剖析RAS信号。欧文实验室已经设计出一组能以150 nM的亲和力和必需的特异性结合到ral上的装订多肽。修饰后的版本进入细胞，并抑制Ral-效应器的相互作用。尽管它们具有良好的亲和力，但由于天然效应器的亲和力较高，它们不太可能成为有效的治疗药物。然而，它们为进一步开发两种模式提供了一个很好的起点：靶向Ral Tyr82的共价抑制剂和降解Ral的PROTACs。我们将与阿斯利康发现科学公司的Jefferson Revell合作，将这些多肽转化为共价缩醛抑制剂和靶向RAL的PROTAC。阿斯利康目前正在通过合理设计、基于片段的文库扫描、Del文库整合和AI/ML努力，扩大其获得新型E3连接酶的机会。这些努力已经导致了几个新一代的小分子E3连接酶，它们具有高亲和力结合和低体内毒性。再加上开发新型无毒CPP的努力，AZ处于与欧文集团合作生产高度具体和有效的PROTAC的绝佳地位。简而言之，我们计划通过一种短的柔性连接物(例如，聚乙二醇、Psar等)，通过化学偶联(例如点击化学)将RAL结合肽与合适的E3连接酶连接，从而构建针对RAL的新型PROTAC。还将研究一种生成ral共价抑制剂的补充方法，需要通过短接头将ral结合到反应性小分子实体，其中几个例子是商业上可用的，适合于与ral中存在的Tyr82的芳香羟基功能的邻近诱导反应。下一代多肽抑制剂将在DO实验室中使用结合试验进行表征，以确定RAR的亲和力和与效应器的竞争。我们将监测Ral Tyr82的共价修饰，并监测PROTAC多肽对Ral的降解。所有多肽的结合将在细胞培养中得到验证，以确定它们是否有能力逆转RAS驱动的特征，例如细胞增殖、非锚定生长和病灶形成。如果时间允许，我们将在老鼠模型上进行试验。我们将在K-RasG12D驱动的非小细胞肺癌(NSCLC)和胰腺导管腺癌(PDAC)的KPC模型中确定我们的多肽对K-RAS驱动的信号转导的影响。这两个模型都可以在CR-UK剑桥研究所(CI)获得，在那里可以使用小动物成像来纵向分析肿瘤的发展和消退。

Small GTPases regulate signal transduction pathways in eukaryotic cells. The prototype small G protein, Ras, is the most mutated oncogene in human cancers. A second small G protein, Ral, is involved in Ras signalling and the Ral pathway has been shown to be of primary importance downstream of Ras in human cancers. This opens a way to target Ras-driven cancers via inhibition of Ral-regulated signalling pathways and developing cellular probes to dissect Ras signalling. The Owen lab have engineered a panel of stapled peptides that bind to Ral with 150 nM affinity and exquisit specificity. Modified versions enter cells and inhibit Ral-effector interactions. Despite their good affinity they are unlikely to be efficacious therapeutics due to the higher affinity of the native effectors. However they provide an excellent starting point to develop two further modalities: covalent inhibitors targeting Ral Tyr82 and Ral degrading PROTACs. Conversion of these peptides into covalent Ral inhibitors and Ral-targeting PROTACs will be taken forward in collaboration with Jefferson Revell, Discovery Science, AstraZeneca. AstraZeneca is currently expanding its access to novel E3 ligases through rational design, fragment-based library scanning, DEL library interogation and AI/ML efforts. Such efforts have resulted in several next generation small molecule E3 ligases with high affinity binding and low in vivo toxicity. Coupled with efforts in developing novel non-toxic CPPs AZ are in an excellent position to generate highly specific and potent PROTACs in collaboration with the Owen group. Briefly, we plan to construct the novel PROTACs targeting Ral through chemical conjugation (e.g. click chemistry) of Ral binding peptides to suitable E3 ligases via a short flexible linker (e.g. PEG, pSAR etc.). A complementary approach to generation of covalent inhibitors of Ral will also be investigated, requiring conjugation of Ral binders through a short linker to a reactive small molecule entity, several examples of which are commercially available and suitable for proximity-induced reaction with the aromatic hydroxyl function of Tyr82 present in Ral. The next-generation peptide inhibitors will be characterized in the DO lab using binding assays, to determine affinities for Ral and competition with effectors. We will monitor covalent modification of Ral Tyr82 and monitor degradation of Ral by PROTAC peptides. Binding of all peptides will be validated in cell culture for their ability to reverse Ras-driven characteristics e.g. cell proliferation, anchorage-independent growth and foci formation. If time permits, we will undertake trials in mouse models. We will determine the effect of our peptides on K-Ras-driven signalling in two well-characterized mouse models: K-RasG12D-driven non-small cell lung cancer (NSCLC) and the KPC model of pancreatic ductal adenocarcinoma (PDAC). Both models are available at the CR-UK Cambridge Institute (CI) where small animal imaging can be employed for longitudinal analysis of tumour development and regression.