title: Design and validation of a homogeneous time-resolved fluorescence-based leptin receptor binding assay.
abstract: The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.
authors: Institut National de la Santé et de la Recherche Médicale (INSERM), U1016, Institut Cochin, 75014 Paris, France.
authors: Virginie
authors: V
authors: Vauthier
authors: Carine
authors: C
authors: Derviaux
authors: Najim
authors: N
authors: Douayry
authors: Thomas
authors: T
authors: Roux
authors: Eric
authors: E
authors: Trinquet
authors: Ralf
authors: R
authors: Jockers
authors: Julie
authors: J
authors: Dam
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D002478:Cells, Cultured; D005453:Fluorescence; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006727:Hormone Antagonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D020738:Leptin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D054411:Receptors, Leptin / Q000032:analysis / Q000378:metabolism*; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D006727:Hormone Antagonists; C515448:LEPR protein, human; D020738:Leptin; D008024:Ligands; D054411:Receptors, Leptin; D000469:Alkaline Phosphatase