In silico discovery of quinoxaline derivatives as novel LRP5/6-sclerostin interaction inhibitors

The Wnt/β-catenin signaling pathway is a key regulator of bone homeostasis. Sclerostin act as an extracellular inhibitor of canonical Wnt signaling through high-affinity binding to the Wnt co-receptor LRP5/6. Disruption of the interaction between LRP5/6 and sclerostin has been recognized as a therapeutic target for osteoporosis. We identified a quinoxaline moiety as a new small-molecule inhibitor of the LRP5/6-sclerostin interaction through pharmacophore-based virtual screening, docking simulations, and in vitro assays. Structure-activity relationship studies and binding mode hypotheses were used to optimize the scaffold and yield the compound BMD4503-2, which recovered the downregulated activity of the Wnt/β-catenin signaling pathway by competitive binding to the LRP5/6-sclerostin complex. Overall, this study showed that the optimized structure-based drug design was a promising approach for the development of small-molecule inhibitors of the LRP5/6-sclerostin interaction. A novel scaffold offered considerable insights into the structural basis for binding to LRP5/6 and disruption of the sclerostin-mediated inhibition of Wnt signaling.     

Mesd is a general inhibitor of different Wnt ligands in Wnt/LRP signaling and inhibits PC-3 tumor growth in vivo

Mesd is a specialized chaperone for Wnt co-receptor low-density lipoprotein receptor-related protein-5 (LRP5) and LRP6, which contain four β-propeller/epidermal growth factor modules, named E1 to E4 from N- to C-terminal, in their extracellular domains. Herein, we demonstrated that recombinant Mesd protein is a general Wnt inhibitor that blocks Wnt/β-catenin signaling induced not only by LRP6 E1-E2-binding Wnts but also by LRP6 E3-E4-binding Wnts. We also found that Mesd suppressed Wnt/β-catenin signaling induced by Wnt1 in prostate cancer PC-3 cells, and inhibited tumor growth in PC-3 xenograft model. Our results indicate that Mesd is a universal inhibitor of Wnt/LRP signaling on the cell surface.     

Syndecan-4 inhibits Wnt/β-catenin signaling through regulation of low-density-lipoprotein receptor-related protein (LRP6) and R-spondin 3

Regulation of Wnt signaling is crucial for embryonic development and adult homeostasis. Here we study the role of Syndecan-4 (SDC4), a cell-surface heparan sulphate proteoglycan, and Fibronectin (FN), in Wnt/β-catenin signaling. Gain- and loss-of-function experiments in mammalian cell lines and Xenopus embryos demonstrate that SDC4 and FN inhibit Wnt/β-catenin signaling. Epistatic and biochemical experiments show that this inhibition occurs at the cell membrane level through regulation of LRP6. R-spondin 3, a ligand that promotes canonical and non-canonical Wnt signaling, is more prone to potentiate Wnt/β-catenin signaling when SDC4 levels are reduced, suggesting a model whereby SDC4 tunes the ability of R-spondin to modulate the different Wnt signaling pathways. Since SDC4 has been previously related to non-canonical Wnt signaling, our results also suggest that this proteoglycan can be a key component in the regulation of Wnt signaling.     

Dishevelled-DEP domain interacting protein (DDIP) inhibits Wnt signaling by promoting TCF4 degradation and disrupting the TCF4/β-catenin complex

The TCF4/β-catenin complex, the executor of canonical Wnt/β-catenin signaling, is regulated by a variety of factors. Among these, Dishevelled (Dvl) is a critical regulator that releases β-catenin from degradation and stabilizes TCF4/β-catenin complex. Here, we report that DDIP (Dishevelled-DEP domain Interacting Protein, also named as Spats1, spermatogenesis associated, serine-rich 1), a novel protein that interacts with Dvl, regulates Wnt signaling. We provide evidence that DDIP suppresses Lef-1 luciferase reporter activity stimulated by Wnt1, Dvl2 or β-catenin, interacts with the TCF4/β-catenin complex, and disrupts the interaction of TCF4 and β-catenin by promoting TCF4 degradation through the proteasome pathway. Our results indicate that DDIP is a negative regulator of the canonical Wnt signaling.     

Reconstitution of a Frizzled8��Wnt3a��LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/β-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/β-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD·Wnt3a·LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth β-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/β-catenin signaling by Dkk1.     

Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway

The Wnt/β-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/β-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced β-catenin accumulation, and inhibit Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC(50) values less than 1 μM for prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/β-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.     

Waif1/5T4 inhibits Wnt/β-catenin signaling and activates noncanonical Wnt pathways by modifying LRP6 subcellular localization

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.