Background and PurposeSmoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here, we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists.\n\nExperimental ApproachIn the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allows kinetic analysis of ligand-receptor binding in living HEK293 cells and competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine).\n\nKey ResultsThe NanoBRET binding assay for SMO is sensitive and superior to purely fluorescence-based binding assays. BODIPY-cyclopamine showed complex binding parameters suggesting separate binding sites.\n\nConclusions and ImplicationsThe NanoBRET ligand binding assay for SMO provides a fast, sensitive and reliable alternative to assess SMO ligand binding. Furthermore, this assay is sufficiently sensitive to dissect a SANT-1-sensitive and a SANT-1-insensitive cyclopamine binding site in the 7TM core, and will be important to further dissect and understand the molecular pharmacology of Class F receptors.\n\nWhat is already knownCyclopamine targets SMO as antagonist and fluorescently-labelled cyclopamine has been used for fluorescence-based binding assays for SMO. Structural analysis has suggested two binding sites on SMO, one in the receptor core and one the CRD.\n\nWhat this study addsWe established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on SMO in live cells in real time.\n\nWhat is the clinical significanceThe assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.
