Novel Peptides that Block the Interaction between Wnt and Frizzled

Wnt/Frizzled signaling is involved in many developmental processes but is mostly silent in healthy adult organs. However, a reactivation of this signaling pathway is generally observed during pathological conditions, which makes it a promising therapeutic target. The recently presented crystal structure of Xenopus Wnt8 in complex with the cysteine rich domain (CRD) of Frizzled8 demonstrates that the shape of the Wnt protein resembles the outline of a hand with a thumb and index finger grasping the CRD at two opposing sites. The thumb contains a palmitic acid lipid at the tip that engages a groove on the CRD whereas the index finger forms a strong hydrophobic contact with a groove on the opposite side of the CRD. Here we investigated the antagonistic properties of peptides that resemble the fragments of Wnts that interact with the CRD of Frizzled receptors on canonical Wnt/Frizzled signaling in a cell-based assay. Peptides resembling the thumb and index finger of Wnt3a and Wnt5a were synthesized and tested in mouse 3T3 cells expressing the TOPFlash reporter. Canonical Wnt signaling was induced by addition of Wnt3a conditioned medium (CM). Peptide fragments of both thumb and index finger were able to inhibited Wnt3a induced signaling at micromolar concentrations. Furthermore, we demonstrated that palmitoylation on the thumb fragment and length of the peptides are critical for antagonistic activity. Therefore, we can conclude that peptide fragments of Wnts can serve as new antagonists for Wnt/Frizzled signaling by interacting with the Frizzled CRD. Keyword(s): Wnt, Frizzled, Peptides