Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery

Abstract

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

Fig. 1.

Chemical structures.

Fig. 2.

Effects of drugs on adrenal neurosecretion stimulated via activation of native nAChRs. Cultured bovine adrenal chromaffin cells expressing α3β4^* nAChRs were treated for 15 min with the drug before their stimulation with 10 μM nicotine in the continued presence of the drug. Concentration-response curves shown in A and B correspond to group A in Fig. 1. The concentration-response curves in C, D, and E correspond to groups B, C, and D, respectively. The concentration-response curves in F correspond to groups E and F. Values represent means ± S.E.M. (n = 3–8). Results are expressed as a percentage of control, nicotine-stimulated neurosecretion.

Fig. 3.

Comparison of effects of agonists and antagonists on HEK 293 cells expressing either rat or bovine recombinant nAChRs. KXα3β4R2 (□) and BMα3β4(▪) cells were loaded with fluo-4-AM for 60 min. For BMα3β4 cells, experiments were performed in buffer containing 20 mM calcium. Fluorescence was measured after stimulation with increasing concentrations of nicotine (A) or epibatidine (B). Effects of d-tubocurarine (C), mecamylamine (D), tetracaine (E), and COB-2 (F) on nicotine (100 μM)-stimulated increases in calcium accumulation were also determined. Results are expressed as either the percentage of the effect of 100 μM nicotine (A), the percentage of the effect of 1 μM epibatidine (B), or the percentage of control, nicotine-stimulated peak fluorescence levels (C–F). Values represent means ± S.E.M. (n = 3–4).

Fig. 4.

Effects of drugs on intracellular calcium levels stimulated via activation of recombinant nAChRs. Cultured KXα3β4R2 cells were loaded with fluo-4-AM for 60 min and treated with the drug 40 s before their stimulation with 100 μM nicotine in the continued presence of the drug. Concentration-response curves shown in A and B correspond to group A in Fig. 1. The concentration-response curves in C to E correspond to groups B, C, and D, respectively. The concentration-response curves in F correspond to groups E and F. Values represent means ± S.E.M. (n = 3–4). Results are expressed as percentage of control, nicotine-stimulated peak fluorescence levels.

Fig. 5.

Comparison of effects of drugs on recombinant and native nAChRs. Linear regression analysis of IC₅₀ values of all our drugs on nicotine-stimulated calcium accumulation (recombinant nAChRs) and adrenal neurosecretion (native nAChRs) was performed. Actual IC₅₀ values for recombinant nAChRs are found in Table 1. Values for native nAChRs are from McKay et al. (2007) and Bergmeier et al. (2004).

Fig. 6.

Effects of nAChR Antagonists on KCl-stimulated Neurosecretion. KCl-stimulated neurosecretion studies were performed, as described under Materials and Methods, using a single concentration (10 μM) of each compound. Results are expressed as percentage of control, KCl-stimulated neurosecretion. Values represent means ± S.E.M. (n = 4–9).

Fig. 7.

Regression analyses comparing the effects of antagonists on recombinant and native nAChRs. Linear regression analysis of IC₅₀ values obtained using nAChR antagonists with little or no non-nAChR effects (A: COB-3, APB-10, APB-6, APB-12, COB-1, PPB-9, APB-7, JHB-7, PPB-6, and COB-2) or nAChR antagonists with large non-nAChR actions (B: KAB-28, KAB-14, DDR-18, JHB-2, IB-10, DDR-1, DDR-17, KAB-38, JHB-1, and KAB-18) on nicotine-stimulated increase in intracellular calcium (recombinant nAChRs) and adrenal neurosecretion (native nAChRs). Actual IC₅₀ values are found in Table 1. Dotted lines represent linear regression analyses when the data are forced through x = 0, y = 0.

Fig. 8.

Effects of nAChR antagonists on [³H]epibatidine binding to native nAChRs. Competition binding experiments were performed on bovine adrenal medulla membrane homogenates using indicated concentrations of the molecules. Data are expressed as a percentage of control specific binding. Values represent means ± S.E.M. (n = 4).

Fig. 9.

Effects of APB-8 on [³H]epibatidine binding. [³H]epibatidine homologous competition binding experiments were performed on adrenal medulla membranes in the absence (•) and presence of 10 μM APB-8 (○). Data are expressed as percentage specific [³H]epibatidine binding or as specific [³H]epibatidine binding in femtomoles per milligram of protein (inset). Triangles represent [³H]epibatidine binding in the absence of epibatidine (inset). Values represent means ± S.E.M. (n = 4).

Fig. 10.

Docking of COB-3 to a computational model of the α3β4 nAChR. A, position of the most frequent COB-3 (pink) docking cluster on the rat α3β4 nAChR LBD model. The docking position is at the α/β interface on the pore-side of the channel. Three of the five subunits are not shown for clarity. B, detailed interactions of COB-3 at the α/β interface as viewed from inside the pore. The α3 subunit is shown in cyan and the β4 subunit is in blue. Epibatidine is shown in gray at the agonist binding site.