Design principles and therapeutic applications of novel synthetic WNT signaling agonists

Abstract

Wingless-related integration site or Wingless and Int-1 or Wingless-Int (WNT) signaling is crucial for embryonic development, and adult tissue homeostasis and regeneration, through its essential roles in cell fate, patterning, and stem cell regulation. The biophysical characteristics of WNT ligands have hindered efforts to interrogate ligand activity in vivo and prevented their development as therapeutics. Recent breakthroughs have enabled the generation of synthetic WNT signaling molecules that possess characteristics of natural ligands and potently activate the pathway, while also providing distinct advantages for therapeutic development and manufacturing. This review provides a detailed discussion of the protein engineering of these molecular platforms for WNT signaling agonism. We discuss the importance of WNT signaling in several organs and share insights from the initial application of these new classes of molecules in vitro and in vivo. These molecules offer a unique opportunity to enhance our understanding of how WNT signaling agonism promotes tissue repair, enabling targeted development of tailored therapeutics.

Keywords: cell biology; molecular medicine.

Graphical abstract

Figure 1

The canonical WNT signaling pathway WNT off: in the absence of WNT binding to FZD and LRP, the destruction complex, consisting of AXIN, APC, CK1⍺, and GSK3β, targets cytoplasmic β-catenin for degradation. Consequently, GROUCHO binds TCF/LEF in the nucleus and blocks transcription of target genes. WNT on: after binding of WNT to FZD and LRP, the destruction complex is recruited to, and anchored at, the membrane via DVL. β-catenin is free to accumulate in the cytoplasm and translocates to the nucleus, where it binds TCF/LEF and initiates transcription of target genes.

Figure 2

Discovery and development of WNT mimetics (A) Endogenous WNT ligand binding FZD and LRP, resulting in downstream signaling events. (B) WNT surrogates consisting of an FZD binder and an LRP binder elicit similar signaling cascades. (C) Design principles and one optimized WNT mimetic. (D) Linker region after the CRD of FZD. Yellow stars denote activation of the WNT signaling pathway.

Figure 3

Cell-targeted WNT mimetics (A) A bispecific tetravalent WNT mimetic. (B) A cell targeting receptor fused to a WNT mimetic. (C) A WNT mimetic split into two inactive molecules: one with two FZD binding arms and the other with two LRP binding arms. Each molecule is connected to a binder that binds a different epitope of the common bridging receptor βKlotho (KLB). Yellow stars denote activation of WNT signaling.

Figure 4

Cell-targeted RSPO mimetics and WNT superagonists (A) An E3 ligase targets FZD for receptor degradation, thus inhibiting the cellular response to WNT. (B) RSPO enhances the cellular response to WNT by interfering with E3 ligase-mediated FZD degradation. RSPO alone does not activate WNT signaling in the absence of endogenous WNTs. (C) An ASGR1-targeted RSPO molecule specifically enhances WNT signaling in ASGR1-expressing hepatocytes. (D) A WNT superagonist consisting of a WNT mimetic and an RSPO mimetic can replace both WNTs and RSPOs. Yellow stars denote activation of WNT signaling.

Figure 5

Applications of WNT mimetics Different WNT mimetics targeting different FZDs and cell types have been applied and have shown regenerative potential in bone, liver, cornea, intestine, lung, retina vessel, BBB, and salivary gland. WNT mimetics can also be applied to enhance patient-derived or allogeneic iPSC or organoid expansion ex vivo that can be used as cell therapy.