Abstract
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. 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, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine) and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay, which is superior to commonly used SMO ligand binding assays, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs.
SIGNIFICANCE STATEMENT 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 transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.
- Allosterism
- G protein-coupled receptors (GPCRs)
- High throughput screening
- Ligand docking
- Receptor structure
- Smoothened (SMO)
- The American Society for Pharmacology and Experimental Therapeutics