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Abstract:Guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assays were established and utilized as a reliable and high-capacity functional assay for determining antagonist and inverse agonist pharmacological parameters of novel histamine H3ligands, at the recombinant human H3receptor. [35S]GTPγS binding assays were performed with membranes prepared from human embryonic kidney 293 cells stably expressing the full-length (445 amino acids) human H3receptor isoform, at approximately 1 pmol/mg of protein. Utilizing robotic liquid handling, assay filtration, and scintillation counting in a 96-well format, concentration–response curves were determined for up to 40 compounds per assay. The imidazole-containing H3receptor antagonist ciproxifan and the non-imidazole antagonist ABT-239 inhibited (R)-α-methylhistamine (RAMH)-stimulated [35S]GTPγS binding in a competitive manner, and negative logarithm of the dissociation equilibrium constant (pKb) values determined for nearly 200 structurally diverse H3antagonists were very similar to the respective negative logarithm of the equilibrium inhibition constant values from N-α-[3H]methylhistamine competition binding assays. H3antagonists also concentration-dependently decreased basal [35S]GTPγS binding, thereby displaying inverse agonism at the constitutively active H3receptor. At maximally effective concentrations, non-imidazole H3antagonists inhibited basal [35S]GTPγS binding by approximately 20. For over 100 of these antagonists, negative logarithm of the 50 effective concentration values for inverse agonism were very similar to the respective pKbvalues. Both H3receptor agonist-dependent and -independent (constitutive) [35S]GTPγS binding were sensitive to changes in assay concentrations of sodium, magnesium, and the guanine nucleotide GDP; however, the potency of ABT-239 for inhibition of RAMH-stimulated [35S]GTPγS binding was not significantly affected. These robust and reliable [35S]GTPγS binding assays have become one of the important tools in our pharmacological analysis and development of novel histamine H3receptor antagonists/inverse agonists. [ABSTRACT FROM AUTHOR]