A Robust and High-Capacity [35S]GTPγS Binding Assay for Determining Antagonist and Inverse Agonist Pharmacological Parameters of Histamine H3 Receptor Ligands

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 H3 ligands, at the recombinant human H3 receptor. [35S]GTPγS binding assays were performed with membranes prepared from human embryonic kidney 293 cells stably expressing the full-length (445 amino acids) human H3 receptor 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 H3 receptor 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...