Chemoproteomics-Enabled De Novo Discovery of Metallothionein Degraders Facilitates Probing Its Role in Cancer

Proteolysis targeting chimeras (PROTACs) represent powerful tools to modulate the activity of classically “undruggable” proteins, but their application has been limited to known ligands and a few select protein classes. Herein, we present our chemoproteomic strategy for de novo discovery of PROTAC degraders. Using comparative PROTAC versus ligand global proteomics analyses, we rapidly identify proteins selectively downregulated by “untargeted” PROTAC probes containing an E3 ligase recruiter and various cysteine-reactive protein-of-interest (POI) ligands. In this manuscript, we showcase our approach by identifying a first-in-class acrylamide- and VHL-based PROTAC for metallothionein 2A (MT2A) – a small, cysteine-rich, metal-binding protein implicated in heavy metal detoxification, zinc homeostasis, and cellular metastasis. Notably, isoform-specific MT overexpression has been shown to augment cellular migration and invasion across several cancer cell lines, though precise mechanisms are unknown due to insufficient tools to study MTs. We show that optimized PROTAC AA-BR-157 covalently binds conserved C44, degrades overexpressed MT2A with nanomolar potency, and reduces migration and invasion of triple negative breast cancer MDA-MB-231 cells. We further demonstrate a time-dependent increase in intracellular zinc levels following MT2A degradation as well as downregulation of protein diaphanous homolog 3 (DIAPH3), a positive regulator of actin and cell motility. Super-resolution imaging of MDA-MB-231 cells shows that downregulation of MT2A and DIAPH3 inhibits cell polarization and thereby migration, suggesting that MT2A regulates motility via DIAPH3-dependent cytoskeletal remodeling. In summary, our strategy enables the discovery of PROTACs for novel disease-related targets and lays the groundwork for expansion of the druggable proteome.