Selective Inhibition of Acetylcholine-Evoked Responses of α7 Neuronal Nicotinic Acetylcholine Receptors by Novel tris- and tetrakis-Azaaromatic Quaternary Ammonium Antagonists

Abstract

A family of 20 tris-azaaromatic quaternary ammonium (AQA) compounds were tested for their inhibition of ␣7 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. The potency of inhibitory activity was related to the hydrophobic character of the tris head groups. Two tris-AQA compounds were studied in detail: the highly effective inhibitor 1,3,5-tri-[5-(1-quinolinum)-pent-1-yn-1-yl]-benzene tribromide (tPyQB) and the less potent antagonist 1,3,5,-tri-{5-[1-(2-picolinium)]-pent-1-yn-1-yl}benzene tribromide (tPy2PiB). In addition, we evaluated 1,2,4,5-tetra-{5-[1-(3-benzyl)pyridinium]pent-1-yl}benzene tetrabromide (tkP3BzPB), a tetrakis-AQA with very hydrophobic headgroups. We compared the activity of the AQA compounds to the frequently used ␣7-antagonist methyllycaconitine (MLA). Both tPyQB and tkP3BzPB were selective antagonists of ␣7. However, although inhibition by tPyQB was reversible within 5 min, the recovery time constant for tkP3BzPB inhibition was 26.6 Ϯ 0.8 min, so that the equilibrium inhibition in the prolonged presence of nanomolar concentrations of tkP3BzPB was nearly 100%. The potency, selectivity, and slow reversibility of tkP3BzPB were comparable with or greater than that of MLA. The inhibitory actions of tPyQB, tPy2PiB, and tkP3BzPB were evaluated on the acetylcholine (ACh)-evoked responses of native nAChRs in rat brain slices. The ␣7-mediated responses of hippocampal interneurons were effectively reduced by 1 M tPyQB and tkP3BzPB but not tPy2PiB. In rat medial septum, tkP3BzPB produced a greater inhibition of ACh-evoked responses of cells with fast inward currents (type I) than of cells with predominantly slow kinetics (type II), suggesting that tkP3BzPB can block ␣7 yet preserve the responsiveness of non-␣7 receptors. These agents might be helpful in elucidating complex receptor responses in brain regions with mixed populations of nAChRs.Nicotinic acetylcholine receptors (nAChRs) are distributed throughout the central and peripheral nervous systems (Role and Berg, 1996;Wonnacott, 1997). Nine neuronal ␣ subunits (␣2-␣10) and three neuronal ␤ subunits (␤2-␤4) have thus far been identified and cloned in vertebrate systems. One type of neuronal nAChR is formed by the assembly of ␣ and ␤ subunits, with functional properties depending on both ␣ and ␤ subunits within the receptor complex (Buisson and Bertrand, 2002). In Xenopus laevis oocytes, pairwise combinations of some neuronal ␣ and ␤ subunits form functional receptors. However, the existence of complex subtypes consisting of more than two different subunits has been documented in native brain regions. In addition to the heteromeric recep-

Multiple Modes of α7 nAChR Noncompetitive Antagonism of Control Agonist-Evoked and Allosterically Enhanced Currents

“…The second class of nAChR noncompetitive antagonists studied (BTMPS, TMPH, and tkP3BzPB) has mechanisms that can be distinguished based on conformational state dependence and ion channel sequence (Papke, 1993;Papke et al, 2005;López-Hernández et al, 2009). They produced blocked times far too long to be resolved on the level of singlechannel currents, even those that have been protracted by the application of a PAM.…”

Merging old and new perspectives on nicotinic acetylcholine receptors

“…The usual α7-selective antagonist of choice is methyllycaconitine (MLA). While in acute co-application experiments MLA is neither particularly potent nor selective for α7 receptors [93] due to the slow reversibility of the α7 block, pre-applications of MLA block α7 receptors effectively at concentrations of 10–30 nM with little effect on other receptors at concentrations < 100 nM. Although normally used for in vitro or ex vivo experiments, MLA has also been used in vivo with systemic delivery and apparent effects in the CNS [94].…”

Structure–Activity Relationship Studies of Sulfonylpiperazine Analogues as Novel Negative Allosteric Modulators of Human Neuronal Nicotinic Receptors

“…14,15,16 The target site of many nAChR drug discovery programs is the orthosteric (agonist binding) site of nAChRs 9,17 and few laboratories have had some success in identifying molecules that show some selectivity using this approach. 18,19 However, one difficulty with this strategy is the high degree of amino acid sequence homology among nAChR α and β subunits in the ligand binding domain for acetylcholine, making it difficult to develop drugs which specifically target nAChR subtypes. 9,17 Thus, the development of selective nAChR drugs directed at orthosteric sites progresses slowly.…”