A Pharmacophore for Drugs Targeting the α4α4 Binding Site of the (α4)3(β2)2 Nicotinic Acetylcholine Receptor

Neuronal nicotinic acetylcholine receptors (nAChRs) have an established role in pain pathways and devastating neurodegenerative diseases; however, few drugs have been successfully developed to target them. The most abundant nAChR in the brain, the alpha 4 beta 2 nAChR, is assembled from five subunits in a 3 alpha:2 beta stoichiometry-(alpha 4)3(beta 2)2. This receptor contains a unique agonist-binding site at the alpha 4 alpha 4 interface in addition to two classical agonist-binding sites at alpha 4 beta 2 interfaces. Most known agonists target both alpha 4 alpha 4 and alpha 4 beta 2 sites, however, a few compounds with selectivity for the alpha 4 alpha 4 site have been identified. These alpha 4 alpha 4 selective compounds have a modulator-like effect akin to benzodiazepines in the gamma-aminobutyric acid type A receptor, which is desirable from a drug development perspective. The two most well characterised alpha 4 alpha 4 selective compounds are CMPI and NS9283. Both are structurally very different from classical agonists, and it is puzzling how they occupy the same binding site. In the search for a common pharmacophore, we conducted extensive molecular dynamics simulations with both classical agonists and site-selective non-classical compounds. Analyses of the simulations revealed that the alpha 4 alpha 4 binding site contains a unique pocket not found in the alpha 4 beta 2 binding site. CMPI and NS9283 were observed to bind in this pocket, thereby explaining why they are selective for the alpha 4 alpha 4 binding site. The proposed binding mode featured a closed-loop C conformation, which is strongly correlated with agonism in nAChRs and explained key site-directed mutagenesis data for both compounds. Based on this binding mode, we proposed a pharmacophore for drugs targeting the alpha 4 alpha 4 binding site. The proposed pharmacophore captures the essence of the original model, that is, nicotinic agonists act as a bridge between protein subunits. The pharmacophore model we propose is unique to the alpha 4 alpha 4 binding site and provides a template for developing new site-selective therapeutic agents.image