Abstract
Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N terminus of Frizzled contains a conserved cysteine-rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC50 values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine-rich domain with submicromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine-rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled.
Highlights
Our work demonstrates that interrupting the Wnt-FZD cysteine-rich domain (CRD) interaction by small molecules from structure based drug discovery is of highly promising potential for the development of new therapeutics against diseases influenced by abnormal Wnt signaling
It was shown that Xenopus Wnt8 (XWnt8) binds to a cavity formed by residues Ile46, Gly47, Tyr48, Ile95, Leu97, Gln141, Gly142, Asn143, Pro144, Asp145, Thr146, Leu147, Met149, Asp150, and Tyr151 of the mouse FZD8 CRD, which is located at the FZD8 CRD dimerization interface in the crystal structure (Protein Data Bank code 1IJY)
A UNITY three-dimensional query was built by defining the hydrophobic region formed by residues Tyr48, Ile95, Leu97, Leu147, and Met149 (Fig. 1), all of which are critical for Wnt-FZD8 CRD binding
Summary
It was shown that a monoclonal antibody that targets the CRD domains of FZDs can block canonical Wnt signaling induced by Wnt ligands and is able to reduce the growth of different types of tumor [9]. A recent x-ray structure of Xenopus Wnt (XWnt8) in complex with the mouse FZD8 CRD confirmed that this site directly interacts with the XWnt ligand and plays a key role in mediating Wnt signaling [20, 21]. Based on the structural information, several hierarchical virtual screening (VS) simulations were conducted to select small molecule compounds that can target the Wnt binding site on the FZD8 CRD. Our work demonstrates that interrupting the Wnt-FZD CRD interaction by small molecules from structure based drug discovery is of highly promising potential for the development of new therapeutics against diseases influenced by abnormal Wnt signaling
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