Development of Flashplate™ Technology to Measure [35S]GTPyS Binding to Chinese Hamster Ovary Cell Membranes Expressing the Cloned Human 5-HTIB Receptor

Abstract

With the exponential rate at which proposed drugs are being produced for disease therapy, it is now essential to automate assays used to screen these compounds and increase throughput. This has been rapidly adopted for simple radioligand binding assays but is less amenable for certain functional screens. [35S]GTPγS binding represents a convenient method for screening ligands that bind to G protein-coupled receptors and, ultimately, stimulate G-protein activation. In this study we have investigated the use of 96-well FlashPlates™ (NEN DuPont, Stevenage, England) to measure [35S]GTPγS binding to human 5-HT1B receptors expressed in Chinese hamster ovary cells. The cells were added to the individual wells of the FlashPlate and incubated with [35S]GTPγS in the presence or absence of test drug and bound radioactivity measured in a 96-well spectrometer. 5-HT produced a stimulation of basal [35S]GTPγS binding, which was robust within and between experiments, with pEC50 = 8.1. The 5-HT1B partial agonist GR127935 (2′n-methyl-4′-5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) caused a partial stimulation (pEC5o = 8.3, intrinsic activity = 0.7), and the selective 5-HTIB receptor antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2′-methyl-4′-[(5-methyl-1,2,4-oxadiazole-3-yl)biphenyl-4-yl]carbonyl}furo[2,3-flindole-3-spiro-4′-piperidine oxalate) displayed inverse agonism with pEC50 = 7.6. These results are similar to those obtained using the conventional filtration method and indicate that FlashPlate technology can provide a rapid method for measuring [35S]GTPγS binding.