Abstract
The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4β2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4β2 receptors. Crystal structures of five agonists with efficacies at α4β2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.
Highlights
Molecular features governing ␣42 nicotinic acetylcholine receptor (nAChR) efficacy have remained elusive
Evaluation of Lymnaea stagnalis (Ls)-acetylcholine-binding proteins (AChBPs) as Model System for nAChR ␣42 Agonists—We selected Ls-AChBP as the model protein for our structural studies because Ls-AChBP, unlike Aplysia californica (Ac)-AChBP, contains a, with respect to nAChRs, conserved tryptophan, Trp-53 (Ls-AChBP numbering), which is known to be functionally important in nAChRs (25, 49 –51) [52] and to influence ligand binding affinities [15, 53] and even ligand binding modes as evident from comparing Ls- and Ac-AChBP crystal structures with the same ligand [16, 17]
Nicotine, epibatidine, and cytisine were initially tested as reference compounds resulting in inhibition constants of 83, 0.097, and 8.2 nM, respectively (Table 1), which is in agreement with previously reported data [14, 15, 54]
Summary
Molecular features governing ␣42 nAChRs efficacy have remained elusive. Results: Binding studies, electrophysiology, and structural data from co-crystallization with Ls-AChBP are reported for a series of ␣42 agonists. Success of future drug discovery projects may depend on the ability to selectively target subpopulations or individual receptor subtypes in a custom-tailored manner to produce drugs with less side effects To reach this goal, a better understanding of the molecular determinants responsible for binding and not the least functional profiles of drugs at individual receptor subtypes is required, and comprehensive studies of high resolution co-crystal structures may significantly aid this process. All five selected compounds bind with subnanomolar affinity to ␣42 receptors (see Table 1), and they have previously been shown to possess significantly different functional profiles using fluorescent-based assays [31]. We confirmed the use of Ls-AChBP as the model system for nAChR ␣42 agonists based on a comparison of binding affinities to the two proteins and co-crystallized compounds 1-5 with
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