Abstract 1324 A disulfide constrained peptide platform to identify ZNRF3 antagonists for Wnt signaling activation

Naturally occurring disulfide constrained peptides (DCPs) demonstrate a wide range of pharmacological activities. They show great potential as templates for drug discovery. These peptides are characterized by conserved cysteine residues that form intramolecular disulfide bonds. Taking advantage of phage display technology, we designed DCP phage libraries with enriched molecular diversity to enable the discovery of ligands against disease-causing proteins of interest. Focusing on the Wnt pathway, we developed DCPs that promote Wnt signaling activity via interfering with ZNRF3, an E3 ubiquitin ligase that controls the cell surface abundance of the Wnt receptor complex FZD/LRP. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we offer insights on the ground rules governing optimal ZNRF3 clearance through engineering multimeric DCP formats that induce expansive growth of human intestinal organoids. This work describes a strategy for the rapid development of potent, biologically active Wnt signaling pathway agonists.