Journal article
Molecular Pharmacology, 2019
APA
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Kozielewicz, P., Bowin, C.-F., Turku, A., & Schulte, G. (2019). A NanoBRET-Based Binding Assay for Smoothened Allows Real-time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-cyclopamine. Molecular Pharmacology.
Chicago/Turabian
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Kozielewicz, P., Carl-Fredrik Bowin, Ainoleena Turku, and G. Schulte. “A NanoBRET-Based Binding Assay for Smoothened Allows Real-Time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-Cyclopamine.” Molecular Pharmacology (2019).
MLA
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Kozielewicz, P., et al. “A NanoBRET-Based Binding Assay for Smoothened Allows Real-Time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-Cyclopamine.” Molecular Pharmacology, 2019.
BibTeX Click to copy
@article{p2019a,
title = {A NanoBRET-Based Binding Assay for Smoothened Allows Real-time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-cyclopamine},
year = {2019},
journal = {Molecular Pharmacology},
author = {Kozielewicz, P. and Bowin, Carl-Fredrik and Turku, Ainoleena and Schulte, G.}
}
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.