Project Summary The current cancer treatment bottleneck showcases the need for more innovative approaches in drug discovery efforts. As of recent, immune-checkpoint inhibitors (ICHs) have shown promise in cancer therapeutics, and work by manipulating immunoregulatory pathways involved in tumor detection and elimination. Many cancer types overexpress the PD-L1 receptor to inhibit T cell activation and subsequently evade the immune response. Though proven effective, the PD-1/PD-L1 pathway targeting ICHs are pharmacologically limited by occupancy- based inhibition. A new drug paradigm has emerged through the development of proteolysis targeting chimeras (PROTACs) that are not limited by such a mechanism. PROTACs are heterobifunctional molecules consisting of an E3 ubiquitin (Ub) ligase ligand, a target protein warhead, and a linker connecting the two and work by bringing an E3 Ub ligase and the target protein together through bivalent binding. The formation of a ternary structure through this bivalent interaction drives the polyubiquitination and subsequent degradation of the target via the ubiquitin proteasome system (UPS). Although this drug design has shown great medicinal promise through research findings and clinical trials, there is seldom literature on the use of transmembrane E3 Ub ligase recruiting PROTACs to degrade cancer-promoting membrane proteins such as PD-L1. The proposed project is aimed at investigating the applicability of such PD-L1 PROTACs by recruitment of the transmembrane E3 Ub ligase ZNRF3. In doing so, three specific aims will be investigated: Aim 1. Identifying cyclic peptide binders of the ZNRF3 extracellular domain. Previous research in the Liu lab utilized a cyanobenzothiazole based linker that allows for cyclization of peptides on a phage surface with applications in phage selections against CD44 and SARS-CoV-2 spike protein epitopes. Recombinant bacterial expression of biotinylated ZNRF3 ECD has been developed, and the binding of selected cyclic peptides will be validated through BLI and flow cytometry. Aim 2. Synthesis and in-vitro characterization of PD-L1 PROTACs. Reported binders of PD-L1 will be incorporated in the PROTAC design via polyethylene glycol (PEG) and polyether linkages of varying lengths. The optimal positioning of linkers will be determined through structure-activity relationships (SAR) analysis, binding assays, co-cocrystal structures, and molecular docking studies of ligand-protein interactions of the ZNRF3 ECD and PD-L1. Aim 3. In-vivo characterization of PD-L1 PROTACs. Degradation potency will be evaluated with western-blotting of residual PD-L1 and ZNRF3 dependency on the induced degradation of PD-L1 and mode of action (proteosome vs lysosome) will be assessed. In addition, whole-cell proteomics studies will be conducted to investigate any consequences on normal cellular activities due to PROTAC activity. By accomplishing these aims, new and important conclusions can be made about the efficacy and mode of action of our designed PROTACs that we hope will have a lasting impact on the cancer therapeutics bottleneck and general drug discovery efforts.

Project Narrative With the development of proteolysis targeting chimeras (PROTACs), a new dimension in drug discovery has emerged. This project proposes to develop and test novel cancer therapeutic PROTACs aimed at targeting the PD-L1 receptor protein, thus infringing upon tumor inhibition of T-cell activation which is a common immune evasion strategy of many cancer types. Conclusions drawn from this study will provide important preliminary insight as to whether PROTACs that recruit extracellular E3 ubiquitin ligases can be utilized to induce degradation of cancer-associated membrane proteins, thereby showcasing a new frontier in oncological drug discovery research.