Abstract
Background and Purpose Smoothened (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.
Experimental Approach In 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).
Key Results The 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.
Conclusions and Implications The 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.
What is already known Cyclopamine 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.
What this study adds We 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.
What is the clinical significance The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.
Footnotes
Abbreviations: SMO, Smoothened; GPCR, G protein-coupled receptor; NanoBRET, Nanoluciferase-based bioluminescence resonance energy transfer; Nluc, Nanoluciferase; Hh, Hedgehog; SMO, Smoothened; Gli, Glioma-associated oncogene