Characterization of Binding Site Interactions and Selectivity Principles in the α3β4 Nicotinic Acetylcholine Receptor

Abstract

Nicotinic acetylcholine receptors (nAChRs) play an important role in neurotransmission and are also involved in addiction and several disease states. There is significant interest in therapeutic targeting of nAChRs; however, achieving selectivity for one subtype over others has been a longstanding challenge, given the close structural similarities across the family. Here, we characterize binding interactions in the α3β4 nAChR subtype via structure−function studies involving noncanonical amino acid mutagenesis and two-electrode voltage clamp electrophysiology. We establish comprehensive binding models for both the endogenous neurotransmitter ACh and the smoking cessation drug cytisine. We also use a panel of C(10)-substituted cytisine derivatives to probe the effects of subtle changes in the ligand structure on binding. By comparing our results to those obtained for the well-studied α4β2 subtype, we identify several features of both the receptor and agonist structure that can be utilized to enhance selectivity for either α3β4 or α4β2. Finally, we characterize binding interactions of the α3β4-selective partial agonist AT-1001 to determine factors that contribute to its selectivity. These results shed new light on the design of selective nAChR-targeted ligands and can be used to inform the design of improved therapies with minimized off-target effects.

What We Have Gained from Ibogaine: α3β4 Nicotinic Acetylcholine Receptor Inhibitors as Treatments for Substance Use Disorders

“…Single-site mutagenesis by Knox et al revealed two amino acids to be particularly important for dictating this subtype selectivity. They found mutating Leu121 in β4 to phenylalanine present in β2 led to a 3-fold loss in potency, likely due to steric clash between the bromine atom and the large phenylalanine side chain . Decreasing the steric bulk in the binding site by mutating Ile113 to the smaller valine found in β2 also led to a decrease in potency, indicating that AT-1001 forms important hydrophobic interactions with the complementary face of the binding site.…”

“…These approaches, of course, require a clearly defined binding site. And while computational studies have been used to propose binding sites for α3β4 ligands, experimental evidence from mutated receptors and the incorporation of non-natural amino acids has proven considerably more useful in defining the ligand–receptor interactions that dictate ligand binding . In addition to developing the existing classes of α3β4 nAChR inhibitors, it is also important to continue to identify and explore new chemotypes that target these receptors.…”

“…These residues are highly conserved within the orthosteric site of all nAChR subtypes, and similar interactions are observed with other agonists at α4β2 and α3β4 nAChRs . Interestingly, a recent study using fluorinated, non-canonical amino acid incorporation suggests that these hydrogen-bond and cation−π interactions are weaker in AT-1001 compared to other nAChR agonists such as cytisine and acetylcholine . To explain the high subtype selectivity of AT-1001, Hibbs and co-workers compared the binding pockets of the α3β4 and α4β2 nAChRs, found that the α3β4 site is slightly larger, and proposed that steric interactions from the bromine in AT-1001 hinder its binding to α4β2.…”

“…37 Interestingly, a recent study using fluorinated, non-canonical amino acid incorporation suggests that these hydrogen-bond and cation−π interactions are weaker in AT-1001 compared to other nAChR agonists such as cytisine and acetylcholine. 148 To explain the high subtype selectivity of AT-1001, Hibbs and co-workers compared the binding pockets of the α3β4 and α4β2 nAChRs, found that the α3β4 site is slightly larger, and proposed that steric interactions from the bromine in AT-1001 hinder its binding to α4β2. Single-site mutagenesis by Knox et al revealed two amino acids to be particularly important for dictating this subtype selectivity.…”

Cation-𝜋 Interaction as a Key Player in Healthcare: A Mini-Review

“…endogenous neurotransmitter ACh and the smoking cessation drug cytisine. They examined the impact of selective cytosine derivatives on the ligand structure, concluding that certain nicotinic acetylcholine receptor (nAChR) targeted ligands can be availed to improve the design of advanced therapies [47].…”