Analgesic Effect of Nicotine and Exploration of Binding Sites for α4β2 Nicotinic Acetylcholine Receptor Positive Allosteric Modulators

Abstract

Pain is a leading cause of the global burden of disease and disability. Nicotinic acetylcholine receptors (nAChRs), especially the α4β2 subtype, are widely expressed throughout the central nervous system (CNS) and peripheral nervous system (PNS), making them critical analgesic targets for the development of novel drugs. In the early part of this thesis, the analgesic effects of nicotine were quantified through meta-analysis. Sixteen eligible articles, including five studies on pain tolerance (n = 210), five studies on pain threshold (n = 210), and 11 studies on pain scores (n = 1,148) were included. Prolonged laboratory-induced pain threshold and tolerance time and mild postoperative pain relief were observed in patients who received acute nicotine therapy. In addition, a network meta-analysis was conducted to compare the effectiveness of nicotine transdermal patch and nasal spray for postoperative pain. Eight eligible studies (n = 555) were included. The results showed that the nicotine transdermal patch had a stronger analgesic effect than the nicotine nasal spray did. In addition, the analgesic effect of the nicotine transdermal patch peaked at 1-1.5 hours. The results of our analysis revealed that nicotine produces a rapid analgesic effect in humans, but the long-term use of nicotine is likely to cause hyperalgesia. The side effects of nicotine have been reported in several studies, likely due to the absence of nAChR subtype selectivity of nicotine. This limits the clinical applications of nicotine. Consequently, there is a need to study nAChR-targeted compounds with high receptor selectivity in order to replace nicotine.

Pharmacological studies of subtype-selective positive allosteric modulators (PAMs) with regard to the α4β2 nAChR (the most abundant subtype of nAChR in humans) are described in the following part of this thesis. The α4β2 nAChR is the most highly expressed subtype of nAChRs in the human brain, where it forms a high-affinity binding site for nicotine. NS206 was identified as a selective PAM of α4β2 nAChRs. LY2087101 and dFBr have pharmacological profiles similar to those of NS206, suggesting that they may bind to the same binding site(s) in wild-type (α4)3(β2)2 nAChRs. Notably, the α4 and β2 subunits assembled into a mixture of (α4)2(β2)3 (high-affinity) and (α4)3(β2)2 (low-affinity) nAChRs in Xenopus laevis oocytes, and the two stoichiometries differ significantly in their functional and pharmacological properties, for instance, the (α4)3(β2)2 nAChR exhibits a biphasic ACh concentration-response relationship, whereas the (α4)2(β2)3 nAChR exhibits a monophasic ACh concentration-response relationship. Therefore, the pharmacological studies on NS206 have been conducted on both stoichiometries. A uniform population of either (α4)3(β2)2 or (α4)2(β2)3 nAChRs in oocytes was obtained by injecting with α4 and β2 cRNA at ratios of 10:1 and 1:4, respectively. The addition of NS206 increased the height of peak responses at (α4)3(β2)2 and (α4)2(β2)3 nAChRs (2.04 ± 0.64 vs. 0.88 ± 0.13, and 2.61 ± 0.42 vs. 0.96 ± 0.03, respectively), which is a statistically significant difference. However, a change in EC50s of the ACh concentration-response curves was not observed. In addition, no significant additive effect or change in potency was observed when comparing the NS206 + LY2087101 and NS206 + dFBr groups to the NS206, LY2087101 or dFBr groups.

To determine the binding sites of NS206, amino acid substitutions of phenylalanine-substituted leucine at position 256 (α4L256F) and leucine-substituted phenylalanine at position 316 (α4F316L) in the transmembrane domain (TMD) of the α4-subunit were performed using site-directed mutagenesis technique. Compared with the wild-type (α4)3(β2)2 and (α4)2(β2)3 nAChRs, the mutated receptors with amino acid substitutions α4L256F and α4F316L significantly reduced NS206 potentiated maximum responses of ACh-evoked currents [(α4)3(β2)2: 6.91 ± 0.57 vs. 6.23 ± 4.35 and 0.84 ± 1.18, respectively; (α4)2(β2)3: 4.14 ± 0.38 vs. 2.37 ± 0.1 and 0.94 ± 0.14, respectively]. In addition, the NS206 potency showed a right-shifted trend for the mutated nAChRs. These results indicate that α4L256 and α4F316 are potential binding sites for NS206 in α4β2 nAChRs.

In summary, this thesis first discussed the involvement of nAChRs in pain by determining the analgesic effect of nicotine on laboratory-induced pain and acute postoperative pain. Furthermore, we compared the analgesic effects of two common clinical nicotine dosage forms, the nicotine transdermal patch and nasal spray, and determined the time of the peak analgesic effect. We found that the analgesic effect of the nicotine transdermal patch was better than that of nicotine nasal spray, peaking at approximately 1-1.5 hours. This thesis studied the pharmacological profiles of NS206 and investigated the binding interactions of NS206, LY2087101, and dFBr. We found that adding NS206 hardly changed the EC50s of ACh concentration-response curves, but significantly increased the height of peak responses at (α4)3(β2)2 and (α4)2(β2)3 nAChRs. Furthermore, no significant additive effect or change in potency was observed when comparing the NS206 + LY2087101 and NS206 + dFBr groups to the NS206, LY2087101 or dFBr groups. Finally, we identified two potential binding sites of NS206. Compared to the wild-type (α4)3(β2)2 and (α4)2(β2)3 nAChRs, the mutated receptors with amino acid substitutions α4L256F and α4F316L significantly reduced NS206 potentiated maximum responses of ACh-evoked currents. Furthermore, the NS206 potency showed a right-shifted trend for mutated nAChRs. These results indicate that α4L256 and α4F316 are the potential binding sites of NS206 on the α4β2 nAChRs. Combined with molecular modelling these results may facilitate delineation of the binding mode of the studied compounds and may thereby pave the way for the future rational design and discovery of novel modulators.