Negative Allosteric Modulators That Target Human α4β2 Neuronal Nicotinic Receptors

Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. We have previously reported on the pharmacological activity of several compounds that act as negative allosteric modulators (NAMs) of nAChRs. In the following studies, the effects of 30 NAMs from our small chemical library on both human alpha 4 beta 2 (H alpha 4 beta 2) and human alpha 3 beta 4 (H alpha 3 beta 4) nAChRs expressed in human embryonic kidney ts201 cells were investigated. During calcium accumulation assays, these NAMs inhibited nAChR activation with IC50 values ranging from 2.4 mu M to more than 100 mu M. Several NAMs showed relative selectivity for H alpha 4 beta 2 nAChRs with IC50 values in the low micromolar range. A lead molecule, KAB-18, was identified that shows relative selectivity for H alpha 4 beta 2 nAChRs. This molecule contains three phenyl rings, one piperidine ring, and one ester bond linkage. Structure-activity relationship (SAR) analyses of our data revealed three regions of KAB-18 that contribute to its relative selectivity. Predictive three-dimensional quantitative SAR (comparative molecular field analysis and comparative molecular similarity indices analysis) models were generated from these data, and a pharmacophore model was constructed to determine the chemical features that are important for biological activity. Using docking approaches and molecular dynamics on a H alpha 4 beta 2 nAChR homology model, a binding mode for KAB-18 at the alpha/beta subunit interface that corresponds to the predicted pharmacophore is described. This binding mode was supported by mutagenesis studies. In summary, these studies highlight the importance of SAR, computational, and molecular biology approaches for the design and synthesis of potent and selective antagonists targeting specific nAChR subtypes.