Sulfonium Ligands of the α7 nAChR

Abstract

The α7 nicotinic acetylcholine receptor (nAChR) is an important target given its role in cognitive function as well as in the cholinergic anti-inflammatory pathway, where ligands that are effective at stabilizing desensitized states of the receptor are of particular interest. The typical structural element associated with a good desensitizer is the ammonium pharmacophore, but recent work has identified that a trivalent sulfur, in the positively charged sulfonium form, can substitute for the nitrogen in the ammonium pharmacophore. However, the breadth and scope of employing the sulfonium group is largely unexplored. In this work, we have surveyed a disparate group of sulfonium compounds for their functional activity with α7 as well as other nAChR subtypes. Amongst them, we found that there is a wide range of ability to induce α7 desensitization, with 4-hydroxyphenyldimethylsulfonium and suplatast sulfonium salts being the most desensitizing. The smallest sulfonium compound, trimethylsulfonium, was a partial agonist for α7 and other neuronal nAChR. Molecular docking into the α7 receptor extracellular domain revealed preferred poses in the orthosteric binding site for all but one compound, with typical cation-pi interactions as seen with traditional ammonium compounds. A number of the compounds tested may serve as useful platforms for further development of α7 desensitizing ability and for receptor subtype selectivity.

Keywords: desensitize; isostere; nicotinic acetylcholine receptor; silent agonist; sulfonium.

Figure 1

The structures of sulfonium compounds described in this study. Full compound names are defined in Materials and Methods.

Figure 2

Responses of α7 nAChR to sulfonium compounds. (A) Net-charge responses to the applications of the test compounds applied at 100 µM. Net charge was calculated as the integrated area of inward current over a 120 s interval following the 30 s-pre-application period used to establish an initial baseline. Note that points less than zero do not represent outward current responses, per se, but rather reductions in the holding current during the post application period. (B) Averaged net-charge responses (±SEM, n = 8) to applications of S1 across a range of concentrations. Responses were measured relative to preceding 60 µM ACh control responses and then adjusted for the difference between ACh controls and ACh maximum responses. See text for curve-fit values.

Figure 3

Responses of α7 nAChR to sulfonium compounds coapplied with PAMs. (A) Potentiation of α7 responses with 30 µM racemic TQS. Shown are the average net-charge responses of seven or eight oocytes ± SEM. (B) Concentration-response studies of peak currents generated by compounds that gave positive responses with TQS, coapplied with 10 µM PNU-120596. Each point represents the average (±SEM) of at least four oocytes. See Table 1 for curve-fit values.

Figure 4

Effects sulfonium compounds on α7 ACh-evoked net-charge responses. (A) Effects of the sulfonium compounds at 100 µM co-applied with 60 µM relative to responses to 60 µM ACh applied alone. Data are the averages (±SEM) from seven or eight cells under each condition. See Table S2 for ANOVA. (B) Effects of select sulfonium compounds at 1 mM co-applied with 60 µM relative to responses to 60 µM ACh applied alone. Data are the averages (±SEM) from seven or eight cells under each condition. Statistical significance was determined by pairwise t-test with p values corrected for multiple comparisons; * p < 0.05, ** p < 0.001.

Figure 5

Inhibition of α7 ACh-evoked responses by S8. (A) Concentration-response studies of α7 net-charge responses to 60 µM ACh co-applied with increasing concentrations of S8. To confirm the stability of the ACh responses, applications of ACh plus S8 were alternated with applications of ACh alone. Data are the averages of eight cells (±SEM). See text for curve-fit values. (B) Effects of 5 µM S8 on responses to either 60 µM or 1 mM ACh. The traces shown across the top are the averaged raw data from seven cells, each normalized to the peak current of the first ACh control response shown. In order to assure that the S8 was present at 5 µM throughout the ACh-evoked responses, a 30 s pre-application was made of 5 µM S8 alone. The lower traces show averaged responses to 60 µM or 1 mM ACh alone or with the 5 µM S8 pre- and co-application. The details of the data are described in Methods.

Figure 6

Activation of heteromeric neuronal nAChR by S1. Concatamers were used in order to obtain receptors with known subunit composition. The β2-6-α4 concatamer was co-expressed with monomeric α4, β2, or α5 to obtain receptors with the composition α4(3)β2(2), α4(2)β2(3), or α4(2)β2(2)α5, respectively. A concatamer of five linked subunits was used to obtain the β3α4β2 α6β2 receptors. The α3β4 receptors were formed from the co-expression of α3 and β4 subunit monomers at equal ratios. Data are the average peak currents (n = 6–8 oocytes ± SEM), measured relative to initial ACh control responses for each oocyte (concentrations listed in Methods). Responses were subsequently adjusted for the relative values of the ACh controls and the ACh maximum responses for each of the various subtypes, determined in previous experiments. Curve-fit values are provided in Table 2.

Figure 7

Inhibition of heteromeric nAChR by sulfonium compounds. (A) Inhibition of α3β4 100 µM ACh-evoked peak currents by the co-application of the sulfonium compounds. Data are the averaged of six to eight oocytes (±SEM). To confirm the stability of the ACh responses, applications of ACh plus the sulfonium compounds were alternated with applications of ACh alone. Curve-fit values are provided in Table 3. (B) Inhibition of ACh control peak-current responses of α4-containing nAChR formed with concatamers by 100 µM of the sulfonium compounds indicated. Data are the average of six to eight oocytes (±SEM).

Figure 8

Silent agonism of an α4β2 mutant receptor that is sensitive to the α7 PAM TQS. (A) The averaged raw data of responses of eight oocytes expressing the β2-6-α4 concatamer and the β2L15’M mutant to 100 µM of the sulfonium agonist S1, applied alone or co-applied with 30 µM racemic TQS. The insert shows a schematic of the subunit configuration predicted for these receptors, with a single mutant β2 subunit outside of the two pairs of α4 and β2 subunits that would form the ACh binding sites. (B) Evaluation of the effects of the sulfonium compounds, applied alone at 100 µM or co-applied with 30 µM TQS to α4(2)β2(2)β2L15’M receptors. The dashed line represents our reliable limit of detection for a peak current response relative to application artifacts, which, for these experiments, was 20 nA. Data are the averages of seven to eight oocytes (±SEM), normalized to 10 µM ACh control responses from the same cells.

Figure 9

Molecular electrostatic potential (MEP) of compounds S5, S9, and S10. Positive regions are colored blue; negative regions are colored red. The arrow points to the sulfonium sulfur atom. The left panel compares the MEP of S5 and S10; the right panel compares the MEP of S5 and S9. Note that S5 is presented from two different perspectives in the two figures to best highlight differences between it and either S9 or S10.