期刊
SCIENTIFIC REPORTS
卷 10, 期 1, 页码 -出版社
NATURE RESEARCH
DOI: 10.1038/s41598-020-70332-3
关键词
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资金
- Deutsche Forschungsgemeinschaft (DFG) [GRK 1910]
- Projekt DEAL
Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H-2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pK(d) = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pK(i) values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H-2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.
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