Received for publication August 14, 2008.
Revised October 7, 2008.
Accepted for publication October 7, 2008.

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 previously reported on the pharmacological activity of several compounds that appear to act non-competitively to inhibit the activation of 34* nAChRs. In the following studies the effects of 51 structurally-similar molecules on native and recombinant 34 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native 34* nAChR with IC₅₀ values ranging from 0.4 to 13.0 µM. Using cells expressing recombinant 34 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity) with IC₅₀ values ranging from 0.7 to 38.2 µM. 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 µM. 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, these studies identify several molecules for potential development as negative allosteric modulators and document the importance of multiple screening assays for nAChR drug discovery.

Key words: allosteric, antagonists, drug discovery, nicotinic, receptor, structure-activity