Discovery of a Novel Inhibitor of the Hedgehog Signaling Pathway through Cell‐based Compound Discovery and Target Prediction

Cell‐based assays enable monitoring of small‐molecule bioactivity in a target‐agnostic manner and help uncover new biological mechanisms. Subsequent identification and validation of the small‐molecule targets, typically employing proteomics techniques, is very challenging and limited, in particular if the targets are membrane proteins. Herein, we demonstrate that the combination of cell‐based bioactive‐compound discovery with cheminformatic target prediction may provide an efficient approach to accelerate the process and render target identification and validation more efficient. Using a cell‐based assay, we identified the pyrazolo‐imidazole smoothib as a new inhibitor of hedgehog (Hh) signaling and an antagonist of the protein smoothened (SMO) with a novel chemotype. Smoothib targets the heptahelical bundle of SMO, prevents its ciliary localization, reduces the expression of Hh target genes, and suppresses the growth of Ptch^+/− medulloblastoma cells.