FZD7 inhibitors(Karolinska Institutet)

Selective inhibitors of Frizzled (FZD) GPCRs are highly sought after as potentially highly efficacious and safe treatments for cancer as well as tools in regenerative medicine and fundamental science. In recent years, there have been several reports claiming the identification of small molecule agents that are selective toward certain FZD proteins using a variety of approaches. However, the majority of these studies lacked a selective functional assay to validate their functionality. In this study, we describe the development and application of a selective assay for individual FZD proteins. Our findings indicate that the majority of reported compounds lack the capacity to inhibit the functioning of the claimed FZD proteins when stimulated by a Wnt ligand in the canonical pathway. Instead, the compounds demonstrate a broad range of off-target effects, including inhibition of downstream pathway component(s) (3235-0367, SRI35959, carbamazepine, niclosamide), lack of activity (FzM1), and surprising antagonism of firefly luciferase (F7H). The only compound that fulfills the expected selectivity profile is peptide Fz7-21. These results highlight the necessity of implementing rigorous testing of the screening-derived compounds in selective functional assays and are important for the field of drug discovery and development targeting the highly demanded Wnt-FZD pathway.

Frizzled (Fzd) proteins are seven transmembrane receptors that belong to a novel and separated family of G-protein-coupled receptors (GPCRs). The Fzd receptors can respond to Wnt proteins to activate the canonical β-catenin pathway which is important for both initiation and progression of cancers. Disruption of the Wnt/β-catenin signal thus represents an opportunity for rational cancer prevention and therapy. Of the 10 members of the Fzd family, Fzd7 is the most important member involved in cancer development and progression. In the present studies, we applied structure-based virtual screening targeting the transmembrane domain (TMD) of Fzd7 to select compounds that could potentially bind to the Fzd7-TMD and block the Wnt/Fzd7 signaling and further evaluated them in biological assays. Six small molecule compounds were confirmed as Fzd7 inhibitors. The best hit, SRI37892, significantly blocked the Wnt/Fzd7 signaling with IC₅₀ values in the sub-micromolar range and inhibited cancer cell proliferation with IC₅₀ values around 2 μM. Our results provide the first proof of concept of targeting Fzd-TMD for the development of Wnt/Fzd modulators. The identified small molecular Fzd7 inhibitors can serve as a useful tool for studying the regulation mechanism(s) of Wnt/Fzd7 signaling as well as a starting point for the development of cancer therapeutic agents.