A Robust and High-Capacity [³⁵S]GTPγS Binding Assay for Determining Antagonist and Inverse Agonist Pharmacological Parameters of Histamine H₃Receptor Ligands

Guanosine 5'-O-(3-[S-35]thio) triphosphate ([S-35]GTP gamma S) 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 H-3 ligands, at the recombinant human H-3 receptor. [S-35]GTP gamma S binding assays were performed with membranes prepared from human embryonic kidney 293 cells stably expressing the full-length (445 amino acids) human H-3 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 H-3 receptor antagonist ciproxifan and the nonimidazole antagonist ABT-239 inhibited (R)-alpha-methylhistamine (RAMH)-stimulated [S-35] GTP gamma S binding in a competitive manner, and negative logarithm of the dissociation equilibrium constant (pK(b)) values determined for nearly 200 structurally diverse H-3 antagonists were very similar to the respective negative logarithm of the equilibrium inhibition constant values from N-alpha-[H-3] methylhistamine competition binding assays. H-3 antagonists also concentration- dependently decreased basal [S-35]GTP gamma S binding, thereby displaying inverse agonism at the constitutively active H-3 receptor. At maximally effective concentrations, non-imidazole H-3 antagonists inhibited basal [S-35]GTP gamma S 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 pKb values. Both H-3 receptor agonist-dependent and -independent (constitutive) [S-35]GTP gamma S 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 [S-35]GTP gamma S binding was not significantly affected. These robust and reliable [S-35]GTP gamma S binding assays have become one of the important tools in our pharmacological analysis and development of novel histamine H-3 receptor antagonists/inverse agonists.