期刊
ANALYTICAL BIOCHEMISTRY
卷 436, 期 1, 页码 1-9出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.12.013
关键词
Leptin receptor; Leptin antagonist; Binding; HTRF; High-throughput screening
资金
- Institut National de la Sante et de la Recherche Medicale (INSERM)
- Centre National de la Recherche Scientifique (CNRS)
- Agence Nationale de la Recherche [ANR-2010-BLANC-NRPLEP]
- European Union [241592]
- Region Ile de France
- Fondation pour la Recherche Medicale
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. (C) 2013 Elsevier Inc. All rights reserved.
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