A Homogeneous G Protein-Coupled Receptor Ligand Binding Assay Based on Time-Resolved Fluorescence Resonance Energy Transfer

2008 ◽  
Vol 6 (4) ◽  
pp. 543-550 ◽  
Author(s):  
Liaoyuan A. Hu ◽  
Tian Zhou ◽  
Brian D. Hamman ◽  
Qingyun Liu
Keyword(s):  
Energy Transfer ◽  
Ligand Binding ◽  
Binding Assay ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ligand Binding Assay ◽  
Time Resolved Fluorescence ◽  
Receptor Ligand ◽  
Time Resolved ◽  
G Protein Coupled

Cell-based β2-adrenergic receptor–ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence

2009 ◽  
Vol 392 (2) ◽  
pp. 103-109 ◽  
Author(s):  
Eija Martikkala ◽  
Mirva Lehmusto ◽  
Minna Lilja ◽  
Anita Rozwandowicz-Jansen ◽  
Jenni Lunden ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Adrenergic Receptor ◽  
Binding Assay ◽  
Ligand Binding Assay ◽  
Time Resolved Fluorescence ◽  
Receptor Ligand ◽  
Time Resolved ◽  
Β2 Adrenergic Receptor

scholarly journals 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

2019 ◽  
Author(s):  
Paweł Kozielewicz ◽  
Carl-Fredrik Bowin ◽  
Gunnar Schulte
Keyword(s):  
Energy Transfer ◽  
Ligand Binding ◽  
Real Time ◽  
Binding Sites ◽  
Binding Assay ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ligand Binding Assay ◽  
Binding Assays ◽  
Class F

AbstractBackground and PurposeSmoothened (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 ApproachIn 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 ResultsThe 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 ImplicationsThe 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.What is already knownCyclopamine targets SMO as antagonist and fluorescently-labelled cyclopamine has been used for fluorescence-based binding assays for SMO. Structural analysis has suggested two binding sites on SMO, one in the receptor core and one the CRD.What this study addsWe 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 in live cells in real time.What is the clinical significanceThe assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.

Time-Resolved Fluorescence Resonance Energy Transfer Shows that the Bacterial Multidrug ABC Half-Transporter BmrA Functions as a Homodimer†

Biochemistry ◽  
2005 ◽  
Vol 44 (11) ◽  
pp. 4312-4321 ◽  
Author(s):  
Olivier Dalmas ◽  
Marie-Ange Do Cao ◽  
Miguel R. Lugo ◽  
Frances J. Sharom ◽  
Attilio Di Pietro ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance
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