Arecoline concentration-response studies of α4- and α6-containing receptors.

<p>Data are the averages of at least 5 cells for each subtype: the α6-containing receptor produced with an α6β2β3α4β2 concatamer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.ref033" target="_blank">33</a>] and the high sensitivity (HS, α4(2) β2(3)) and low sensitivity (LS, α4(3) β2(2)) α4β2 nAChR produced with the β2-α4 concatamer and monomers. Responses were calculated relative to ACh control responses measured four minutes prior to the arecoline applications and then adjusted for the ratio between the ACh controls and ACh maximum responses determined in previous experiments. EC₅₀s were 14 ± 3, 21 ± 4, and 75 ± 7 μM for HS α4β2, α6-containing, and LS α4β2 receptors, respectively.</p

Heteromeric AChR sensitivity to areca nut infusion.

<p>Areca nut infusion was prepared as described above. The traces were scaled to ACh controls in each cell prior to calculating averages and S.E.M. The α4β2 receptors were formed from the co-expression of monomers. The infusion was applied either alone or was co-applied with ACh. The inserts below some of the traces display the same data scaled up by a factor of 10, and the traces shown represent the average of the normalized responses (black line) ± the S.E.M. (shaded band) calculated for each of the 10,500 points in the 210 s traces (acquired at 50 Hz). Consistent with the arecoline data, the infusion produced small activation of the nAChR and inhibited the responses to ACh at the control concentration. Consistent with effects of the nut infusion of α7 receptors in the absence of PNU-120596 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.g003" target="_blank">Fig 3</a>), there was a profound inhibition of subsequent ACh-evoked responses.</p

Arecoline activation of other nAChR subtypes.

<p>100 μM arecoline was applied to cells expressing the nAChR subunits indicated. Responses of human α3β4, α7, and mouse muscle (α1β1εδ) subunits were barely at the threshold of detection, less than 1% the ACh maximum, extrapolated from comparisons to ACh controls and ACh concentration-response studies conducted previously. The responses of cells expressing α4β2 or a concatamer containing α6 and β3 in addition to α4 and β2 were substantially larger and well above the threshold of detection.</p

Modulation of HS α4β2 receptors with a low concentration of arecoline.

<p>(A) Partial agonists for α4β2 nAChR such as varenicline and cytisine [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.ref035" target="_blank">35</a>] modulate the sensitivity of the receptors to the endogenous activator ACh through pre-desensitization for prolonged periods, even when present at very low concentrations. They can also stimulate low levels of tonic activation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.ref035" target="_blank">35</a>]. After obtaining initial control responses to ACh, a steady flow of 3 μM arecoline was applied to the bath. After 8 minutes the responses to a control application of ACh was reduced approximately 50%. (B) The perfusion of 3 μM arecoline was continued, and 100 μM mecamylamine was applied along with the arecoline to reveal the mecamylamine-sensitive steady-state current. The decrease in inward current shown is the averaged response of seven cells. Normalized relative to initial ACh controls and adjusted for ACh maximum, these currents indicated steady-state activation of approximately 1% ACh maximum.</p

The effects of areca nut infusion (see Methods) on oocytes expressing α7 nAChR.

<p>Cells were initially tested for their responses to control applications of 60 μM ACh prior to the application of the filtered nut infused solution. After a 4-minute wash, the infusion solution ± 10 μM PNU-120596 was applied (0.4 ml over 12 seconds) followed by another application of 60 μM ACh. The cells were voltage clamped at -60 mV, and the traces shown represent the average response (black line) ± the S.E.M. (shaded band) calculated for each of the 10,500 points in the 210 s traces (acquired at 50 Hz). For application of the infusion solution alone n = 8, and for the data obtained in the presence of PNU-120596 (n = 5 cells).</p

Effects 100 μM arecoline (structure illustrated) on oocytes expressing α7 nAChR.

<p>Cells were tested for their responses to control applications of 60 μM ACh prior to the application of the test solution. The second of two such control applications is shown. After a 4 minute wash period, 100 μM arecoline ± 10 μM PNU-120596 was applied (0.4 ml over 12 seconds) followed by another application of 60 μM ACh, as shown. Prior to the calculation of the multi-cell averages, each single cell response was normalized to the average of the two initial controls obtained from that cell. The cells were voltage clamped at -60 mV, and the traces shown represent the average of the normalized responses (black line) ± the S.E.M. (shaded band) calculated for each of the 10,500 points in the 210 s traces (acquired at 50 Hz). For arecoline alone (n = 8), and for arecoline plus PNU-120596 (n = 7). In order to allow for comparison between experiments, the data for the responses to the nut infusion plus PNU-120596 shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.g002" target="_blank">Fig 2</a> were also normalized to their respective controls and are displayed along with the arecoline plus PNU-120596 data in the insert.</p

Arecoline concentration-response studies.

<p>(A) Oocytes expressing α7 were tested with co-applications of 30 μM PNU-120596 plus varying concentrations of arecoline. Both peak currents and net charge responses were calculated and normalized to the average of two initial 60 μM ACh control responses in the same cells. The EC₅₀ values were 60 ± 7 and 93 ± 2 μM for peak currents and net charge, respectively. Relative to ACh controls, the I_max values were 15 ± 1 and 49 ± 1 for peak currents and net charge, respectively. (B) Since in the absence of a PAM, silent agonists can function as antagonists of typical agonists, the potency of arecoline for antagonizing 60 μM ACh-evoked responses was tested. Arecoline was surprisingly ineffective at inhibiting ACh responses, with an IC₅₀ > 1000 μM.</p

Agonist and silent agonist activity of muscarinic cholinergic agonists.

<p>The pharmacophore for silent agonism of α7 is distinct from that for activation in the absence of a PAM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.ref025" target="_blank">25</a>]. Since arecoline is known to be a muscarinic agonist, we tested additional compounds with muscarinic activity for their ability to activate α7 in the absence and presence of 10 μM PNU-120596. The structures of the test compounds are shown, as well as, that of nicotine for comparison.</p

Sulfonium as a Surrogate for Ammonium: A New α7 Nicotinic Acetylcholine Receptor Partial Agonist with Desensitizing Activity

Weak partial agonists that promote a desensitized state of the α7 nicotinic acetylcholine receptor (nAChR) have been associated with anti-inflammatory effects. Exemplar compounds feature a tertiary or quaternary ammonium group. We report the synthesis, structure, and electrophysiological evaluation of 1-ethyl-4-phenylthiomorpholin-1-ium triflate, a weak partial agonist with a sulfonium isostere of the ammonium pharmacophore. These results offer new insights in understanding nAChR–ligand interactions and provide a new chemical space to target the α7 nAChR

New Alpha9 nAChR Ligands Based on a 5‑(Quinuclidin-3-ylmethyl)-1,2,4-oxadiazole Scaffold

Several lines of evidence have indicated that nicotinic acetylcholine receptors (nAChR) that contain α9 subunits, probably in combination with α10 subunits, may be valuable targets for the management of pain associated with inflammatory diseases through a cholinergic anti-inflammatory system (CAS), which has also been associated with α7 nAChR. Both α7- and α9-containing neuronal nAChR can be pharmacologically distinguished from the high-affinity nicotinic receptors of the brain by their sensitivity to α-bungarotoxin, but in other ways, they have quite distinct pharmacological profiles. The early association of α7 with CAS led to the development of numerous new ligands, variously characterized as α7 agonists, partial agonists, or silent agonists that desensitized α7 receptors without activation. Subsequent reinvestigation of one such family of α7 ligands based on an N,N-diethyl-N′-phenylpiperazine scaffold led to the identification of potent agonists and antagonists for α9. In this paper, we characterize the α9/α10 activity of a series of compounds based on a 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole (QMO) scaffold and identify two new potent ligands of α9, QMO-28, an agonist, and QMO-17, an antagonist. We separated the stereoisomers of these compounds to identify the most potent agonist and discovered that only the 3R isomer of QMO-17 was an α9 antagonist, permitting an in silico model of α9 antagonism to be developed. The α9 activity of these compounds was confirmed to be potentially useful for CAS management of inflammatory pain in cell-based assays of cytokine release