Effect of Novel Negative Allosteric Modulators of Neuronal Nicotinic Receptors on Cells Expressing Native and Recombinant Nicotinic Receptors: Implications for Drug Discovery

Abstract

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of ␣3␤4* nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant ␣3␤4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native ␣3␤4* nAChR, with IC 50 values ranging from 0.4 to 13.0 M. Using cells expressing recombinant ␣3␤4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC 50 values ranging from 0.7 to 38.2 M. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 M. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/ 51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

Negative Allosteric Modulators That Target Human α4β2 Neuronal Nicotinic Receptors

“…A previously reported procedure that has been used for rat ␣3␤4 nAChRs stably expressed in HEK ts201 cells has been adapted for this work (McKay et al, 2007;Gonzá lez-Cestari et al, 2009). For the calcium accumulation assays, HEK ts201 cells stably expressing either H␣4␤2 or H␣3␤4 nAChRs (obtained from Professor Jon Lindstrom, University of Pennsylvania, Philadelphia, PA) were used.…”

“…We have discovered a class of nAChR NAMs that target a novel negative allosteric site on nAChRs (McKay et al, 2007;Gonzá lez-Cestari et al, 2009). Using our small chemical library of structurally similar NAMs, the present study describes structure-activity relationship (SAR) analyses on recombinant human ␣4␤2 (H␣4␤2) and recombinant human ␣3␤4 (H␣3␤4) nAChRs.…”

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

“…24 Previous SAR established that our COB analogues are the most potent NAMs on Hα4β2 and Hα3β4 nAChRs. 25 Although they are not selective, we decided to gain insight concerning their interactions in this allosteric site.…”

“…We have also shown previously that KAB-18 does not compete with the agonist for the orthosteric site using binding studies in native bovine and recombinant nAChRs. 24 With these results, three amino acids have been identified to be important for the inhibitory effects of KAB-18 at the allosteric site (Phe118, Thr58, and Glu60).…”

“…This site was identified through a combination of homology modeling, blind docking, and molecular dynamics studies and was confirmed through the use of site-directed mutagenesis and structure-based virtual screening. 24,25,28,29 At this site, NAMs have been shown to selectively antagonize Hα4β2 nAChRs over Hα3β4 nAChRs. 25,26,29,30 To develop more potent and selective NAMs for nAChRs, the putative binding site and binding mode of KAB-18 must be refined.…”