4-4-difluoro-4-bora-3a-4a-diaza-s-indacene and cyclopamine

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD

Topics: Binding Sites; Bioluminescence Resonance Energy Transfer Techniques; Boron Compounds; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Frizzled Receptors; Humans; Molecular Dynamics Simulation; Mutation; Phosphoproteins; Protein Conformation; Smoothened Receptor; Veratrum Alkaloids

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.

Topics: Binding Sites; Biological Assay; Bioluminescence Resonance Energy Transfer Techniques; Boron Compounds; Cinnamates; Drug Discovery; Gene Knockout Techniques; Hedgehog Proteins; HEK293 Cells; Humans; Ligands; Luciferases; Morpholines; Nanostructures; Purines; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids

Topics: Animals; Boron Compounds; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Discovery; Drug Evaluation, Preclinical; Fluorescent Dyes; Humans; Mice; Molecular Docking Simulation; Molecular Structure; NIH 3T3 Cells; Receptors, G-Protein-Coupled; Structure-Activity Relationship; Veratrum Alkaloids