Development of Ligands and Degraders Targeting MAGE-A3.

ABSTRACT

Type I melanoma antigen (MAGE) family members are detected in numerous tumor types, and expression is correlated with poor prognosis, high tumor grade, and increased metastasis. Type I MAGE proteins are typically restricted to reproductive tissues, but expression can recur during tumorigenesis. Several biochemical functions have been elucidated for them, and notably, MAGEs regulate proteostasis by serving as substrate recognition modules for E3 ligase complexes. The repertoire of E3 ligase complexes that can be hijacked for targeted protein degradation continues to expand, and MAGE-E3 complexes are an especially attractive platform given their cancer-selective expression. Additionally, type I MAGE-derived peptides are presented on cancer cell surfaces, so targeted MAGE degradation may increase antigen presentation and improve immunotherapy outcomes. Motivated by these applications, we developed novel, small-molecule ligands for MAGE-A3, a type I MAGE that is widely expressed in tumors and associates with TRIM28, a RING E3 ligase. Chemical matter was identified through DNA-encoded library (DEL) screening, and hit compounds were validated for in vitro binding to MAGE-A3. We obtained a cocrystal structure with a DEL analog and hypothesize that the small molecule binds at a dimer interface. We utilized this ligand to develop PROTAC molecules that induce MAGE-A3 degradation through VHL recruitment and inhibit the proliferation of MAGE-A3 positive cell lines. These ligands and degraders may serve as valuable probes for investigating MAGE-A3 biology and as foundations for the ongoing development of tumor-specific PROTACs.