title: Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.
abstract: The free fatty acid receptor 1 (FFA1/GPR40) is a potential target for treatment of type 2 diabetes. Although several potent agonists have been described, there remains a strong need for suitable tracers to interrogate ligand binding to this receptor. We address this by exploring fluorophore-tethering to known potent FFA1 agonists. This led to the development of 4, a high affinity FFA1 tracer with favorable and polarity-dependent fluorescent properties. A close to ideal overlap between the emission spectrum of the NanoLuciferase receptor tag and the excitation spectrum of 4 enabled the establishment of a homogeneous BRET-based binding assay suitable for both detailed kinetic studies and high throughput competition binding studies. Using 4 as a tracer demonstrated that the compound acts fully competitively with selected synthetic agonists but not with lauric acid and allowed for the characterization of binding affinities of a diverse selection of known FFA1 agonists, indicating that 4 will be a valuable tool for future studies at FFA1.
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark , Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Christiansen
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow , Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hudson
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark , Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Anders Højgaard
authors.initials: AH
authors.lastname: Hansen
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow , Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark , Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Trond
authors.initials: T
authors.lastname: Ulven
mesh_terms: D001596:Benzylamines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D010069:Oxadiazoles / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D001596:Benzylamines; C473034:FFAR1 protein, human; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D043562:Receptors, G-Protein-Coupled; C011514:4-nitrobenzofurazan; C030796:benzylamine