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

小GTP酶调节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。第二种小G蛋白Ral参与Ras信号传导，并且Ral途径已被证明在人类癌症中Ras的下游具有首要重要性。这开辟了一种通过抑制Ral调节的信号传导途径和开发细胞探针来剖析Ras信号传导来靶向Ras驱动的癌症的方法。 Owen实验室已经设计了一组与Ral结合的钉合肽，其具有150 nM的亲和力和精确的特异性。修饰的版本进入细胞并抑制Ral-效应物相互作用。尽管它们具有良好的亲和力，但由于天然效应物的亲和力较高，它们不太可能是有效的治疗剂。然而，它们提供了一个很好的起点，以开发两个进一步的模式：共价抑制剂靶向Ral Tyr 82和Ral降解PROTAC。 将这些肽转化为共价Ral抑制剂和靶向Ral的PROTAC将与Jefferson Revell，Discovery Science，AstraZeneca合作进行。 阿斯利康目前正在通过合理设计、基于片段的文库扫描、DEL文库查询和AI/ML努力扩大其对新型E3连接酶的获取。这些努力已经产生了几种具有高亲和力结合和低体内毒性的下一代小分子E3连接酶。再加上开发新型无毒CPP的努力，AZ在与Owen集团合作生产高度特异性和有效的PROTAC方面处于非常有利的地位。简言之，我们计划通过Ral结合肽与合适的E3连接酶的化学缀合（例如点击化学）经由短的柔性接头（例如PEG、pSAR等）构建靶向Ral的新型PROTAC。还将研究产生Ral的共价抑制剂的补充方法，其需要通过短接头将Ral结合剂缀合至反应性小分子实体，其几个实例是市售的并且适合于与Ral中存在的Tyr 82的芳族羟基官能团的邻近诱导反应。 下一代肽抑制剂将在DO实验室中使用结合测定法进行表征，以确定对Ral的亲和力和与效应物的竞争。我们将监测Ral Tyr 82的共价修饰并监测Ral被PROTAC肽降解。所有肽的结合将在细胞培养物中验证其逆转Ras驱动特征（例如细胞增殖、非贴壁依赖性生长和病灶形成）的能力。如果时间允许，我们将在小鼠模型中进行试验。我们将在两种充分表征的小鼠模型中确定我们的肽对K-Ras驱动的信号传导的影响：K-RasG 12 D驱动的非小细胞肺癌（NSCLC）和胰腺导管腺癌（PDAC）的KPC模型。这两种模型都可在CR-UK剑桥研究所（CI）获得，其中小动物成像可用于肿瘤发展和消退的纵向分析。

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.