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The range of assay technologies for the binding and signaling has been developed in HTS laboratories for the identification of hit or lead compounds acting on GPCRs. The [35S] GTPγS binding assay still remains to be a useful and simple technique to demonstrate receptor activation and is one of the few functional, cell-free assays. However, its radioactive nature imposes clear limitations to its use in regular laboratory practice and in high-throughput experimentations. Herein, we have developed a new non-radioactive version of the assay using europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence. In continuation with our efforts, this assay was adapted for Histamine 3 receptors. The assay format was specifically evaluated by testing known histamine 3 agonists (Imetit, Immepip, Methylhistamine, Proxifan and Histamine) and antagonists (GSK189254, Clobenpropit and Thioperamide) drugs. Under optimized assay conditions, the potencies (pEC50 & PKB) in the binding assay are in good agreement with those obtained previously in the isotopic functional activity assay. The Eu-GTP binding assay was observed to be highly robust (Z’ factor 0.84) with high percentage over basal counts. This assay can be utilized as a component of screening cascade for the screening of Histamine 3 receptor antagonists.