CHARACTERIZATION OF THE BINDING OF A POTENT, SELECTIVE, RADIOIODINATED ANTAGONIST TO THE HUMAN NEUROKININ-1 RECEPTOR

We have synthesized a potent, selective, radioiodinated antagonist of the human neurokinin-1 (NK1) receptor and have characterized its binding to the cloned receptor expressed in Chinese hamster ovary cells. (cis)-2-(Diphenylmethyl)-N-[(2-iodophenyl)-methyl]-1-azabicyclo[2.2.2]octan-3-amine (L-703606) inhibits binding of I-125-Tyr8-substance P to the human NK1 receptor with an IC50 of 2 nM. This compound is a competitive antagonist of substance P-induced inositol phosphate generation, with a K(b) of 29 nm. [I-125]L-703606 binds to a single class of high affinity binding sites in human NK1/Chinese hamster ovary cell membranes (K(d) = 0.3 nM). Substance P inhibits the binding of [I-125]L-703606 to 65% of the NK1 receptor sites with a K(d) of 0.04 +/-0.03 nM and to the remaining 35% of the sites with a K(d) of 1.5 +/- 0.7 nM. Addition of the nonhydrolyzable GTP analog guanylyl-5'-(beta,gamma-imido)diphosPhate [Gpp(NH)P] shifts >90% of the binding sites to the lower affinity state. In addition, Gpp(NH)p markedly alters the dissociation of substance P from the NK1 receptor by increasing the number of sites in the low affinity, rapidly dissociating state. However, Gpp(NH)p does not affect the rate of dissociation of [I-125]L-703606. These data suggest that the pharmacological properties of [I-125]L-703606 binding to the human NK1 receptor are similar to those of antagonists of nonpeptide guanine nucleotide-binding protein-coupled receptors and that this ligand will be useful for the biochemical and pharmacological characterization of the human NK1 receptor.