Mobasher M et al. (2026), Putative α7-selective ligands interact with α9-...

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Front Immunol 2026 Mar 11;17:1773637. doi: 10.3389/fimmu.2026.1773637.

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Putative α7-selective ligands interact with α9-containing nicotinic acetylcholine receptors and modulate immune functions of human mononuclear phagocytes.

Mobasher M, Hone AJ, Pilzecker M, Bozic D, Hecker A, McIntosh JM, Kirschner KN, Grau V, Papke RL, Andleeb H, Richter K.

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INTRODUCTION: Nicotinic acetylcholine receptors (nAChRs) on immune cells are promising therapeutic targets for the treatment of inflammatory diseases and pain. Both α7 and α9* nAChRs (*denotes the potential presence of other nAChR subunits) have been implicated as mediators of the cholinergic anti-inflammatory system (CAS). This study investigated the binding sites of α7-selective ligands on these receptors and their effects on ATP-dependent release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 by human mononuclear phagocytes. MATERIALS AND METHODS: The effects of classical ligands (e.g. ACh, nicotine), unconventional (phosphocholine), putative α7-specific ligands (S24795, PNU-282987 and methyllycaconitine), on the ATP-induced IL-1β release were studied in lipopolysaccharide-primed human monocytic THP-1 cells and THP-1-derived macrophages. Electrophysiological two-electrode voltage-clamp measurements were conducted on Xenopus laevis oocytes expressing human α7, α9 or α9α10 nAChRs. Molecular docking was performed using the crystal structure of the homomeric human α7 receptor (PDB ID: 7EKI) and a modeled pentameric assembly of the homomeric α9 extracellular domain (PDB ID: 6HY7). In addition, the homomeric α10 extracellular domain was generated by homology modeling using 6HY7 as the template. RESULTS: In cytokine-release experiments, the nAChR agonists efficiently inhibited ATP-mediated IL-1β release. This inhibitory effect was reversed by specific antagonistic conopeptides [V11L;V16D]ArIB (α7 antagonist) and RgIA4 (α9 and α9α10 antagonist), indicating the involvement of nAChRs containing subunits α7, α9 and/or α10. Electrophysiological measurements suggested an interaction of putative α7-specific ligands with human α9* nAChRs. In molecular docking simulations, all tested ligands showed reasonable binding affinity to homomeric α7, α9 and α10 nAChR models near the C-loop region of the binding pocket. CONCLUSION: Our findings provide a more nuanced framework for interpreting the roles of nAChR subtypes in non-neuronal immune modulation, highlighting the complexity and potential importance of α9* nAChRs in the context of inflammation and innate immunity. The results underscore the importance of considering the nAChR subunit α9 when developing α7-selective ligands for immunomodulation and provides novel insights into the role of α9* nAChRs as potential therapeutic targets for inflammatory diseases and pain.

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	Figure 1. Chemical structures of the molecules investigated to modulate the ATP-induced release of the pro-inflammatory cytokine interleukin (IL)-1β by mononuclear phagocytes.
	Figure 2. The putative α7-selective nicotinic acetylcholine receptor (nAChR) agonists S24795 (S24) and PNU-282987 (PNU) inhibit the BzATP-mediated interleukin (IL)-1β release. Monocytic THP-1 cells and THP-1 cell-derived M1-like macrophages were primed for 5 h with lipopolysaccharide (LPS; 1 µg/ml). The P2X7 receptor agonist BzATP was added for another 40 min to trigger IL-1β release, which was quantified by ELISA (A,C) and by measuring IL-1α/β-equivalent bioactive concentrations using HEK-Blue™ IL-1R cells in a QUANTI-Blue™ assay (B, D). The BzATP- (100 µM) induced release of IL-1β was investigated in the presence and absence of the nAChR agonists S24 (50 µM) and PNU (10 µM), acetylcholine (ACh, 10 µM) or phosphocholine (PC, 200 µM). The amount of IL-1β released in response to BzATP was calculated by subtracting the IL-1β concentrations of cells treated with LPS alone. In each experiment, the IL-1β concentrations obtained after stimulation with BzATP were set to 100% and all other values were calculated accordingly. Data are presented as individual data points, bars represent median, whiskers percentiles 25 and 75. *p ≤ 0.05, different from LPS-primed cells stimulated with BzATP alone. Friedman test followed by the Wilcoxon signed-rank test.
	Figure 3. The effect of α7 nicotinic acetylcholine receptor (nAChR) agonists S24795 (S24) and PNU-282987 (PNU) on the BzATP-mediated release of interleukin (IL)-1β is sensitive to methyllycaconitine (MLA). Monocytic THP-1 cells (A) and THP-1 cell-derived M1-like macrophages (B) were primed for 5 h with LPS (LPS; 1 µg/ml). The P2X7 receptor agonist BzATP was added for another 40 min to trigger IL-1β release, which was measured by ELISA. The BzATP- (100 µM) induced release of IL-1β was investigated in the presence and absence of the nAChR agonists S24 (50 µM) and PNU (10 µM), or the ligands acetylcholine (ACh, 10 µM) and phosphocholine (PC, 200 µM). In addition, MLA was co-applied. The amount of IL-1β released in response to BzATP was calculated by subtracting the IL-1β concentrations measured in supernatants of cells treated with LPS alone. In each experiment, the IL-1β concentrations obtained after stimulation with BzATP were set to 100% and all other values were calculated accordingly. Data are presented as individual data points, bars represent median, whiskers percentiles 25 and 75. *p ≤ 0.05, different from LPS-primed cells stimulated with BzATP alone; #p ≤ 0.05, different from LPS-primed cells stimulated with BzATP plus an agonist. Friedman test followed by the Wilcoxon signed-rank test.
	Figure 4. The effect of the putative α7-selective nicotinic acetylcholine receptor (nAChR) agonists S24795 (S24) and PNU-282987 (PNU) on the BzATP-mediated cytokine release is sensitive to the α-conopeptides [V11L,V16D]ArIB and RgIA4. Monocytic THP-1 cells and THP-1 cell-derived M1-like macrophages were primed for 5 h with LPS (LPS; 1 µg/ml). The P2X7 receptor agonist BzATP was added for another 40 min to trigger the release of IL-1β (A, C) and IL-18 (B, D), which was measured by ELISA. The BzATP- (100 µM) induced release of IL-1β and IL-18 was investigated in the presence and absence of the α7 nAChR agonists S24 (50 µM) and PNU (10 µM). To test for the involvement of nAChR subunits the conopeptides Rg1A4 (RgIA; 200 nM) or [V11L,V16D]ArIB (500 nM) were co-applied. The amount of IL-1β and IL-18 released in response to BzATP was calculated by subtracting the IL-1β concentrations measured in supernatants of cells treated with LPS alone. In each experiment, the IL-1β concentrations obtained after stimulation with BzATP were set to 100% and all other values were calculated accordingly. Data are presented as individual data points, bars represent median, whiskers percentiles 25 and 75. *p ≤ 0.05, different from LPS-primed cells stimulated with BzATP alone; #p ≤ 0.05, different from LPS-primed cells stimulated with BzATP plus an agonist. Friedman test followed by the Wilcoxon signed-rank test.
	Figure 5. The putative α7-selective agonists S24795 and PNU-282987 interact with α9α10 nAChRs. Xenopus laevis oocytes heterologously expressing human α9α10 nicotinic acetylcholine receptor (nAChR) were subjected to two-electrode voltage-clamp experiments and exposed to the α7-selective agonists S24795 or PNU-282987. (A, B) Oocytes expressing α9α10 nAChRs were stimulated with 1 s pulses of acetylcholine (ACh; 60 µM) until steady baseline responses were observed, then stimulated with S24795 (A) or PNU-282987 (B) and the responses compared to those evoked by ACh. The asterisks indicate 1 s pulses of S24795 (red) or PNU-282987 (green). (C, D) S24795 and PNU-282987 were also tested for antagonist activity by perfusion of the compounds during stimulation with ACh. Oocytes were stimulated with 1 s pulses of ACh until steady baseline responses were observed, then the control solution was switched to one containing S24795 (C); 10 min perfusion indicated by the horizontal red bar above the current traces) or PNU-282987 (D); 5 min perfusion, horizontal green bar). Representative current traces are shown (A-D) from 5–8 oocytes each.
	Figure 6. Cartoon highlighting the extracellular domain with two chains of α7, α9, and α10 of homomeric nicotinic acetylcholine receptor (nAChR). Docked ligands in the α7 [shown in grey; (A, B)], α9 [shown in cyan; (C, D)], and α10 [shown in pink; (E, F)] nAChR are shown. Docked poses of representative compounds S24795 (green), PNU-282987 (red), methyllycaconitine (MLA) (grey), acetylcholine (yellow), and nicotine (Pink), demonstrate binding within the C-loop region and its vicinity. Electrostatic surface potentials for complementary chain of a7, a9 and a10 are displayed in (B, D, F) respectively.
	Figure 7. Binding mode and ligand interaction diagram for putative α7-selective nicotinic acetylcholine receptor (nAChR) agonists in the extracellular domain of homomeric α9 and α10 nAChRs. Ligand interaction diagrams for S24795 (A, D), PNU-282987 (B, E), and methyllycaconitine (MLA; (C, F) in the α9 (A–C) and the α10 (D-F) nAChR subunit. Ligand and residues are shown in sticks and distances and interactions are shown in lines: blue lines indicate hydrogen bonds, gray dotted lines indicate hydrophobic interactions, yellow dotted lines indicate salt-bridges interactions, green dotted lines indicate (π-stacking or π-cation), and green lines represent halogen-bonds interaction.