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 α4β2 nAChR, is assembled from five subunits in a 3α:2β stoichiometry-(α4)₃(β2)₂. This receptor contains a unique agonist-binding site at the α4α4 interface in addition to two classical agonist-binding sites at α4β2 interfaces. Most known agonists target both α4α4 and α4β2 sites, however, a few compounds with selectivity for the α4α4 site have been identified. These α4α4 selective compounds have a modulator-like effect akin to benzodiazepines in the γ-aminobutyric acid type A receptor, which is desirable from a drug development perspective. The two most well characterised α4α4 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 α4α4 binding site contains a unique pocket not found in the α4β2 binding site. CMPI and NS9283 were observed to bind in this pocket, thereby explaining why they are selective for the α4α4 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 α4α4 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 α4α4 binding site and provides a template for developing new site-selective therapeutic agents.