Abstract
Misregulation of Wnt signaling is common in human cancer. The development of small molecule inhibitors against the Wnt receptor, frizzled (FZD), may have potential in cancer therapy. During small molecule screens, we observed binding of carbamazepine to the cysteine-rich domain (CRD) of the Wnt receptor FZD8 using surface plasmon resonance (SPR). Cellular functional assays demonstrated that carbamazepine can suppress FZD8-mediated Wnt/β-catenin signaling. We determined the crystal structure of the complex at 1.7 Å resolution, which reveals that carbamazepine binds at a novel pocket on the FZD8 CRD. The unique residue Tyr52 discriminates FZD8 from the closely related FZD5 and other FZDs for carbamazepine binding. The first small molecule-bound FZD structure provides a basis for anti-FZD drug development. Furthermore, the observed carbamazepine-mediated Wnt signaling inhibition may help to explain the phenomenon of bone loss and increased adipogenesis in some patients during long-term carbamazepine treatment.
Highlights
Ligands belonging to the Wnt family of secreted lipoproteins play central roles in tissue morphogenesis and homeostasis through binding to members of the frizzled (FZD) family of cell surface receptors.[1]
The small molecule FZM1 has been identified as an FZD4 misfolding chaperone, which is likely to bind to the intracellular loops of FZD4,9 and its derivatives can act as allosteric agonists of noncanonical Wnt signaling.[10]
Small molecule antagonists that bind to the FZD cysteine-rich domain (CRD) could have therapeutic potential in cancers with upregulated Wnt signaling.[1]
Summary
Ligands belonging to the Wnt family of secreted lipoproteins play central roles in tissue morphogenesis and homeostasis through binding to members of the frizzled (FZD) family of cell surface receptors.[1]. The overall structure of FZD8CRD, either apo or in complex with carbamazepine, is almost identical to those of previously reported apo or Wnt-bound FZD8CRD14−16 except at the CRD C-terminus, which forms a β-hairpin with the remaining residues of a Rhinovirus 3C protease cleavage site (used to remove purification tags; Figure 2C and the Experimental Section). In both our structures, the ASUs contain dimers resulting from two-fold noncrystallographic symmetry (Figure 2A,B). We found that carbamazepine can only partially suppress Wnt3a-induced luciferase activity and even at the high concentration of 64 μM, luciferase activity remained around 60% (Figure 7)
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