Challenging Reported\nFrizzled-Targeting Compounds\nin Selective Assays Reveals Lack of Functional Inhibition and Claimed\nProfiles

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