Molecular Pharmacology of Gelsemium Alkaloids on Inhibitory Receptors

Abstract

Indole alkaloids are the main bioactive molecules of the Gelsemium genus plants. Diverse reports have shown the beneficial actions of Gelsemium alkaloids on the pathological states of the central nervous system (CNS). Nevertheless, Gelsemium alkaloids are toxic for mammals. To date, the molecular targets underlying the biological actions of Gelsemium alkaloids at the CNS remain poorly defined. Functional studies have determined that gelsemine is a modulator of glycine receptors (GlyRs) and GABA_A receptors (GABA_ARs), which are ligand-gated ion channels of the CNS. The molecular and physicochemical determinants involved in the interactions between Gelsemium alkaloids and these channels are still undefined. We used electrophysiological recordings and bioinformatic approaches to determine the pharmacological profile and the molecular interactions between koumine, gelsemine, gelsevirine, and humantenmine and these ion channels. GlyRs composed of α1 subunits were inhibited by koumine and gelsevirine (IC₅₀ of 31.5 ± 1.7 and 40.6 ± 8.2 μM, respectively), while humantenmine did not display any detectable activity. The examination of GlyRs composed of α2 and α3 subunits showed similar results. Likewise, GABA_ARs were inhibited by koumine and were insensitive to humantenmine. Further assays with chimeric and mutated GlyRs showed that the extracellular domain and residues within the orthosteric site were critical for the alkaloid effects, while the pharmacophore modeling revealed the physicochemical features of the alkaloids for the functional modulation. Our study provides novel information about the molecular determinants and functional actions of four major Gelsemium indole alkaloids on inhibitory receptors, expanding our knowledge regarding the interaction of these types of compounds with protein targets of the CNS.

Keywords: GABAA receptor; Gelsemium alkaloids; bioinformatics; electrophysiology; glycine receptor.

Figure 1

Modulation of recombinant GlyRs and GABA_ARs by Gelsemium alkaloids. (A) Current traces before and during the application of koumine, gelsevirine, or humantenmine to cells expressing α1GlyRs. (B) Concentration response curves (0.01–300 μM) of alkaloids on homomeric α1, α2, and α3 GlyRs. Currents were evoked using 35 μM (α1), 60 μM (α2), or 65 μM (α3) of the agonist glycine. The dashed lines describe the gelsemine sensitivity [14]. (C) Current traces before and during the application of koumine, gelsevirine, or humantenmine to cells expressing α1β GlyRs. (D) Concentration response curves (0.01–300 μM) of alkaloids on α1β, α2β, and α3β GlyRs. The currents were evoked using 30 μM (α1β), 60 μM (α2β), or 70 μM (α3β) of glycine. The dashed lines describe the gelsemine sensitivity [14]. (E) Current traces before and during the application of koumine or humantenmine (50 μM) to cells expressing α1β2γ2 GABA_ARs. (F) The graph summarizes the sensitivity of GABA-evoked currents to 50 μM of koumine or humantenmine. Currents were evoked using 1 μM of GABA. *, p < 0.05, koumine-induced inhibition of α1β2γ2 GABA_ARs versus α1GlyRs; *** p < 0.001; koumine-induced inhibition of α1β2γ2 GABA_ARs versus α2 and α3GlyRs. ANOVA followed by Tukey post hoc test, F(3, 17) = 0.2916. Data are presented as means ± SEM.

Figure 2

Putative binding sites of Gelsemium alkaloids within the orthosteric sites of GlyRs and GABA_ARs. (A) The left panel shows gelsemine binding to homopentameric α1GlyRs. Panels on the right show an enhanced view of the predicted binding of gelsemine, koumine, gelsevirine, andhumantenmine to the orthosteric sites of α1, α2, and α3GlyRs. Glycine binding is shown in Figure S3. (B) The boxed graphs summarize the docking scores for the gelsemine (GEL), koumine (KOU), gelsevirine (GEV), humantenmine (HUM), and strychnine (STN) interaction to the orthosteric sites. (C) The left panel shows the binding of gelsemine to α1β2γ2 GABA_ARs. Panels on the right show an augmented vision of the putative binding of gelsemine, koumine, humantenmine, and bicuculline to the α1β2γ2 GABA_AR orthosteric site. (D) The graph shows the docking score values for the interaction of gelsemine (GEL), koumine (KOU), humantenmine (HUM), and bicuculline (BIC) to the orthosteric site. The boxed graphs show medians (middle line) and quartile ranges (25–75, box borders). Whiskers indicate the maximal and the minimal docking score values. The parameters of strychnine and bicuculline are also shown as reference compounds. The number of binding conformations for each alkaloid were as follows: gelsemine (13), koumine (30), gelseverine (19), humantenmine (62), strychnine (37), and bicuculline (16).

Figure 3

Relevance of the ECD for the subunit-specific actions of gelsemine on α1GlyRs. (A) Structural outlook of wild-type and chimeric receptors studied. (B) Current traces show the effects of gelsemine (10 μM or 50 μM) on wild-type and chimeric α1α2 or α2α1 GlyRs. Currents were evoked using 35 μM (α1), 60 μM (α2), 35 μM (α1α2), and 65 μM (α2α1) of glycine. (C) Summary of gelsemine effects on wild-type and chimeric GlyRs. Differences were not significant. α1 (n = 9), α2 (n = 6), α1α2 (n = 7), α2α1 (n = 10). Unpaired Student’s t-test: 10 μM, p = 0.91; 50 μM, p = 0.93. Data are presented as means ± SEM.

Figure 4

Molecular analysis of Gelsemium alkaloid’s interactions with amino acids within the orthosteric site of α1GlyRs. (A) Two-dimensional structures of gelsemine, koumine, gelsevirine, and humantenmine (pH 7.0) and interaction diagrams of the alkaloids with residues of the orthosteric site of α1GlyRs. Interactions between α1GlyR and each alkaloid are described (4 Å cutoff). The purple arrows indicate hydrogen bonds, while the red lines represent pi–cation interactions. The green line symbolizes a pi–pi interaction. Numbered residues are depicted by colored drops. The color code describes the amino acid properties (green, hydrophobic residues; red, negatively charged residues; blue, positively charged residues; cyan, polar residues; light yellow, glycine). (B) Sample current traces showing the sensitivity loss to gelsemine (200 μM) of α1GlyR F63A and G160E mutants. The currents were evoked using 2 mM (F63A) or 1 mM (G160E) of glycine. (C,D) The bar plots describe the potentiation percentage induced by gelsemine (C) or the inhibition percentage induced by gelsemine, koumine, or gelsevirine (D) on wild-type or mutated receptors. For graph (C), WT (n = 9), F63A (n = 4), G160E (n = 9). For graph (D), gelsemine, WT (n = 6), F63A (n = 4), G160E (n = 6); koumine, WT (n = 5), F63A (n = 4), G160E (n = 5); gelsevirine, WT (n = 6), F63A (n = 4), G160E (n = 6). ANOVA followed by Tukey post hoc test. Differences were significant. **, p < 0.01, F(2, 18) = 3.41, gelsemine-induced potentiation of wild-type α1GlyRs versus F63A and G160E (C). For gelsemine inhibition: ***, p < 0.001, F(2, 11) = 1.47; for koumine inhibition: **, p < 0.01, ***, p < 0.001, F(2, 10) = 2.50; for gelsevirine inhibition: ***, p < 0.001, F(2, 12) = 0.32 (D). Data are presented as means ± SEM. (E) Pharmacophore modeling of Gelsemium alkaloids. Strychnine is also shown as a reference competitive alkaloid.