Kinetic and equilibrium characterization of vesamicol receptor-ligand complexes with picomolar dissociation constants

Previous studies from this laboratory characterized 83 analogs of vesamicol by their potencies for inhibition of acetylcholine active transport by synaptic vesicles isolated from Torpedo electric organ. Examination of the more potent of these compounds, plus five new analogs, by kinetic and equilibrium measurements on complexes with the vesamicol receptor (VR) revealed nine analogs that are significantly more potent than vesamicol. Equilibrium measurements were performed at very low protein concentrations and extended incubation times, which allowed the characterization of very high affinity analogs. Better understanding of the structural binding requirements of the VR has resulted, and a spatial map of allowed hydrophobicity has been clearly established. Three analogs were resolved, and they displayed enantioselectivity ratios as high as 260 for binding to the VR (10-times higher than that of vesamicol). The most potent analog, 4-aminobenzovesamicol (ABV), was synthesized in tritiated form and shown to dissociate from the VR with a half-life of about 14 hr at 20 degrees. The estimated dissociation constant is < or = 6.5 +/- 0.5 pM. By reciprocal kinetic experiments with vesamicol and ABV, coincidence of the two binding sites on vesicles was established. The high affinity and enantioselectivity of ABV and other similar analogs, coupled with good chemical and radiochemical stability, make these ligands attractive for the study of the VR in complex tissues. The observed difference between the equilibrium dissociation constant for the vesamicol-VR complex as estimated by titration with [3H]vesamicol (7.6 nM) and by displacement of subsaturating [3H]vesamicol by nonlabeled vesamicol (1.0 nM) suggests that high and low affinity populations of the VR exist.