PT  - JOURNAL ARTICLE
AU  - Paweł Kozielewicz
AU  - Carl-Fredrik Bowin
AU  - Ainoleena Turku
AU  - Gunnar Schulte
TI  - A NanoBRET-Based Binding Assay for Smoothened Allows Real-time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-cyclopamine
AID  - 10.1124/mol.119.118158
DP  - 2020 Jan 01
TA  - Molecular Pharmacology
PG  - 23--34
VI  - 97
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/97/1/23.short
4100  - http://molpharm.aspetjournals.org/content/97/1/23.full
SO  - Mol Pharmacol2020 Jan 01; 97
AB  - Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein 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 allowed 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 is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, 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 the 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.