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

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.