A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

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.