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

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