Defining the Putative Inhibitory Site for a Selective Negative Allosteric Modulator of Human α4β2 Neuronal Nicotinic Receptors

Neuronal nicotinic receptors (nAChRs) have been implicated in several diseases and disorders such as autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, and nicotine addiction. To understand the role of nAChRs in these conditions, it would be beneficial to have selective molecules that target specific nAChRs in vitro and in vivo. Our laboratory has previously identified a novel allosteric site on human alpha 4 beta 2 nAChRs using a series of computational and in vitro approaches. At this site, we have identified negative allosteric modulators that selectively inhibit human alpha 4 beta 2 nAChRs, a subtype implicated in nicotine addiction. This study characterizes the allosteric site via site-directed mutagenesis. Three amino acids (Phe118, Glu60, and Thr58) on the beta 2 subunit were shown to participate in the inhibitory properties of the selective antagonist KAB-18 and provided insights into its antagonism of human alpha 4 beta 2 nAChRs. SAR studies with KAB-18 analogues and various mutant alpha 4 beta 2 nAChRs also provided information concerning how different physiochemical features influence the inhibition of nAChRs through this allosteric site. Together, these studies identify the amino acids that contribute to the selective antagonism of human alpha 4 beta 2 nAChRs at this allosteric site. Finally, these studies define the physiochemical features of ligands that influence interaction with specific amino acids in this allosteric site.