Potent and selective binders of the E3 ubiquitin ligase ZNRF3 stimulate Wnt signaling and intestinal organoid growth

Selective and precise activation of signaling transduction cascades is key for cellular reprogramming and tissue regeneration. However, the development of small or large molecule agonists for many signaling pathways has remained elusive and is rate limiting to realize the full clinical potential of regenerative medicine. Focusing on the Wnt pathway, here we describe a series of disulfide-constrained peptides (DCPs) that promote Wnt signaling activity by modulating the cell surface levels of ZNRF3, an E3 ubiquitin ligase that controls the abundance of the Wnt receptor complex FZD/LRP at the plasma membrane. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we engineered multimeric DCPs that induce expansive growth of human intestinal organoids, revealing a dependence between valency and ZNRF3 clearance. Our work highlights a strategy for the development of potent, biologically active Wnt signaling pathway agonists via targeting of ZNRF3. To study the functional relevance of ZNRF3 domains required for DCP activity unbiasely, we generated a SW48 Cas9 cell line and delivered a CRISPR/Cas9 tiling library of gRNA spanning the entire ZNRF3. We treated the cells with DMSO or DCP for 1 h and subjected the cells to FACS sorting. To rule out gRNAs that resulted in premature truncation and loss of ZNRF3 expression, we first filtered out all hits that altered the cell surface abundance of ZNRF3 in DMSO control treated cells. Next, we evaluated the selective enrichment of gRNAs in the top 10% of ZNRF3 expressing cells in the MK1-2.A2.B9.1 DCP treated cells compared to DMSO treatment .