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

Abstract

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 α4β2 nAChRs using a series of computational and in vitro approaches. At this site, we have identified negative allosteric modulators that selectively inhibit human α4β2 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 β2 subunit were shown to participate in the inhibitory properties of the selective antagonist KAB-18 and provided insights into its antagonism of human α4β2 nAChRs. SAR studies with KAB-18 analogues and various mutant α4β2 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 α4β2 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.

Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

“…Mutational analyses provide evidence for PAM binding sites in the ECD at noncanonical interfaces analogous to dFBr Site III in a3b2 nAChRs at the b2/a3 interface (Seo et al, 2009) and in a4b2 nAChRs at the a4/a4 interface, which is also an ACh binding site (Mazzaferro et al, 2014;Olsen et al, 2014). Mutational analyses and computational docking also provide evidence for negative allosteric modulator binding sites equivalent to dFBr Site I at the canonical interfaces in a4b2 and a3b4 nAChRs (Grishin et al, 2010;Henderson et al, 2012). Based on our results, [ 3 H]dFBr should serve as an appropriate photoreactive PAM to identify its binding sites in a4b2 nAChRs and to identify other allosteric modulators that competitively inhibit photolabeling.…”

Inside-out neuropharmacology of nicotinic drugs

“…However, inverse agonists, allosteric modulators (positive and negative), and noncompetitive channel blockers may stabilize AChRs quite effectively and likely in a manner similar to nicotine. In fact, allosteric modulators may be preferred as they do not interact with the orthosteric site and possess more potential for subtype-selective interactions (Henderson et al, 2012; Henderson et al, 2010). …”

Discovery of benzamide analogs as negative allosteric modulators of human neuronal nicotinic receptors: Pharmacophore modeling and structure–activity relationship studies

“…Other amino acids (Glu60 and Ser97) that contribute to interactions between ligands and receptors occurring through the ‘β subunit site’ also share a limited degree of sequence conservation among subunits (amino acid identity of 63% and 39%, respectively). 17 The diversity within the ‘β subunit site’ and the identification of subtype-selective NAMs acting on this site provide support for our approach of targeting allosteric binding sites to develop selective nAChR antagonists. In this study, the physicochemical properties of NAMs that selectively target hα4β2 nAChRs as well as the biochemical characteristics of their binding sites (i.e., the ‘β subunit site’) mentioned above were exploited.…”

“…Since major amino acid residues comprising this site are located at the β subunit, we named this site the ‘β subunit site’. The ‘β subunit site’ is sequentially and structurally diverse among subtypes, which provides an explanation for the relative subtype-selectivity shown with some of the NAMs 17 : among residues positioned within the ‘β subunit site’, Thr58, Serl33, Serl38, Serl42, Phell8, and Serl37 showed reduced sequence conservation (less than 26% amino acid identity across nAChR subunits). Other amino acids (Glu60 and Ser97) that contribute to interactions between ligands and receptors occurring through the ‘β subunit site’ also share a limited degree of sequence conservation among subunits (amino acid identity of 63% and 39%, respectively).…”