Elsevier

Neuropharmacology

Volume 91, April 2015, Pages 34-42
Neuropharmacology

The analgesic-like properties of the alpha7 nAChR silent agonist NS6740 is associated with non-conducting conformations of the receptor

https://doi.org/10.1016/j.neuropharm.2014.12.002Get rights and content

Highlights

  • NS6740 is an α7 nicotinic acetylcholine receptor (nAChR) silent agonist.

  • NS6740 induces non-conducting conformational states of the receptor.

  • NS6740 is effective in the treatment of inflammatory and neuropathic pain in mouse.

  • NS6740 reduces the aversive component of pain.

  • The α7 nAChR silent agonists may represent viable targets for novel analgesics.

Abstract

The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of neurological disorders including chronic pain and inflammatory diseases. Since α7 can function as a ligand-gated ion channel, drug development initially focused on ligands that were selective activators of the α7 ion channel. However, the best α7 drugs for chronic pain and inflammation indications may not be ion channel activators but rather “silent agonists”, which bind to the receptor but preferentially induce non-conducting states that modulate signal transduction in non-neuronal cells. One such compound is NS6740. We show that NS6740 selectively induces prolonged desensitization of α7 nAChRs. There are two forms of α7 desensitization that can be distinguished by their sensitivity to the positive allosteric modulators (PAMs). At high concentrations, NS6740 preferentially induces PAM-insensitive desensitization, which over the course of several minutes reverts to the sensitive form. NS6740 was tested in several pain models after in vivo administration in the mouse. Although it had no effects in acute thermal pain, NS6740 induced significant dose- and time-dependent antinociceptive activity in formalin- and acetic acid-induced nociceptive behaviors as well as in the chronic constrictive nerve injury (CCI) model for neuropathic pain. The antinociceptive activity of NS6740 in these models was α7-dependent. In addition, NS6740 administration reversed pain-induced aversion, an important affective component of pain. The time and concentration dependence of the effects were consistent with NS6740 induction of PAM-insensitive non-conducting states, suggesting that signal transduction required for analgesia is accomplished by α7 receptors in that conformation.

Introduction

The α7 nicotinic acetylcholine receptor (nAChR) silent agonist (Chojnacka et al., 2013) NS6740 first appeared in the literature as a straw man in an analysis of α7 ligands for activity in a test for cognitive enhancement (Briggs et al., 2009). NS6740, which has very little efficacy for ion channel activation of α7 under control conditions, was tested along with the more efficacious analog NS6784 in a mouse inhibitory avoidance model, and while NS6784 improved performance, NS6740 did not. NS6740 was revealed to be a silent agonist, a ligand that binds to the site for orthosteric activation but more effectively promotes the conformational changes associated with desensitization than activation, when it was used in combination with the type II positive allosteric modulator (PAM) PNU-120596 and shown to generate currents. PNU-120596 destabilizes a form of desensitization that is unique to α7, thereby promoting protracted bursts of channel opening (Williams et al., 2011). The conclusion of Briggs et al. (2009) was that α7 channel activation was required for α7-mediated cognitive effects. However, even under the best of conditions the open probability of α7 nAChR is very low (Williams et al., 2011), and recent studies of α7-mediated signal transduction in non-neuronal cells has promoted the alternative hypothesis that, at least for some indications or stimuli, α7 signaling can be ion channel independent. This hypothesis was strongly supported by a subsequent study of NS6740 as a modulator of the inflammatory function of microglia (Thomsen and Mikkelsen, 2012) which showed that NS6740 and GTS-21, another α7 selective partial agonist with low ion channel efficacy, were more effective at suppressing LPS-stimulated secretion of TNF-α in rat cultured microglia than were the more efficacious agonists, SSR180711, A-582941, and choline.

The basic pharmacological data on NS6740 are limited, even in regard to its putative selectivity for α7. We have tested NS6740 on α3β4 and α4β2 as well as α7 nAChR to confirm its selectivity for α7 compared to these heteromeric nAChR subtypes. We have also investigated the time and concentration dependence of the effects of NS6740 on human α7 nAChR with and without the modulatory effects of PNU-120596. We found that NS6740 shows a remarkable concentration dependence in its ability to synergize with PNU-120596 and produce ion channel activation, and that above a certain concentration NS6740 induces non-conducting states of the receptor that are insensitive to the activation-promoting effects of the PAM.

Just as NS6740 was shown to be effective at modulating the inflammatory responses of microglia cells in vitro, we report that it is effective in well-established mouse models of chronic pain, including peripheral neuropathy (chronic constriction nerve injury, CCI) and tonic inflammatory pain (the formalin test). The behavioral and pharmacological profile of NS6740 in these models is consistent with the induction of non-conducting conformational states of the receptor. Results obtained from these studies will further understanding of the α7 nAChR subtype in pain regulation and be helpful for the targeting of novel α7 silent agonists as therapeutic agents for the treatment of chronic pain.

Section snippets

Heterologous expression of nAChRs in Xenopus laevis oocytes

Human nAChR clones were obtained from Dr. J. Lindstrom (University of Pennsylvania, Philadelphia, PA). The human resistance-to-cholinesterase 3 (RIC-3) clone, obtained from Dr. M. Treinin (Hebrew University, Jerusalem, Israel), was co-injected with α7 to improve the level and speed of α7 receptor expression without affecting the pharmacological properties of the receptors (Halevi et al., 2003). Subsequent to linearization and purification of the plasmid cDNAs, cRNAs were prepared using the

In vitro results

We tested the effects of NS6740 at several concentrations on heteromeric α3β4 and α4β2 and homomeric α7 nAChR expressed in Xenopus oocytes. Responses to applications of NS6740 along with control ACh-evoked responses obtained from the same cells obtained before and after NS6740 applications are shown in Fig. 1. Our limit of detection for receptor-mediated activity is approximately 0.05% of the ACh controls, since the application of Ringer's solution alone can cause a small stimulus artifact (not

Discussion

The identification of α7 nAChRs as a potential therapeutic target for several diseases, including pain and inflammation (de Jonge and Ulloa, 2007, Medhurst et al., 2008, Munro et al., 2012), has stimulated the development of many α7 nAChR-selective drugs. The present study investigated the in vitro and in vivo pharmacological properties of NS6740, an α7 nAChR silent agonist. Our results show for the first time that NS6740 was effective in mouse models of tonic and chronic pain via α7 nAChR

Disclosures

This work was supported by a grant from a Pilot Project from VCU Massey Cancer Center (A-35337) and National Institute on Drug Abuse [grant DA032246] to MID, National Institute of Health grants [GM57481] (RLP) and [DA027113] (GAT). The authors report no conflicts of interest. Deniz Bagdas would like to thank The Scientific and Technical Research Council of Turkey (TUBITAK) for her postdoctoral research scholarship (2219-2013).

Acknowledgments

The authors wish to thank Tie Han for his technical assistance and maintenance of the breeding colony. OpusXpress experiments were conducted by Clare Stokes, Shehd Abdullah Abbas Al Rubaiy, Lu Wenchi Corrie, and Christopher W. Kinter.

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