A Robust and High-Capacity [ 35 S]GTP γ S Binding Assay for Determining Antagonist and Inverse Agonist Pharmacological Parameters of Histamine H 3 Receptor Ligands

Abstract

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 (pK _b) values determined for nearly 200 structurally diverse H₃ antagonists were very similar to the respective negative logarithm of the equilibrium inhibition constant values from N-α-[³H]methylhistamine competition binding assays. H₃ antagonists also concentration-dependently decreased basal [³⁵S]GTPγS binding, thereby displaying inverse agonism at the constitutively active H₃ receptor. At maximally effective concentrations, non-imidazole H₃ antagonists 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 pK _b values. Both H₃ receptor 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 H₃ receptor antagonists/inverse agonists.