Choudhary S et al. (2019), Menthol acts as a positive allosteric modulator...

XB-ART-55651

Int J Parasitol Drugs Drug Resist 2019 Apr 01;9:44-53. doi: 10.1016/j.ijpddr.2018.12.005.

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Menthol acts as a positive allosteric modulator on nematode levamisole sensitive nicotinic acetylcholine receptors.

Choudhary S , Marjianović DS , Wong CR , Zhang X , Abongwa M , Coats JR , Trailović SM , Martin RJ , Robertson AP .

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The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100 μM the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1 μM and 10 μM menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1 μM menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics.

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Species referenced: Xenopus laevis
Genes referenced: pam

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	Fig. 1. Chemical structures of monoterpenoid compounds used in the present study.
	Fig. 2. Agonist effects of monoterpenoid compounds on levamisole sensitive O. dentatum nAChR. A. Sample traces for the agonist experiment. B. Bar chart (expressed as meanâ¯Â±â¯SEM%) showing agonistic effects of the monoterpenoid compounds: menthol (6.5â¯Â±â¯2.4%, nâ¯=â¯6), geraniol (0.9â¯Â±â¯0.2%, nâ¯=â¯6), cinnamaldehyde (0.6â¯Â±â¯0.2%, nâ¯=â¯6), citronellol (0.4â¯Â±â¯0.2%, nâ¯=â¯6), menthyl acetate (0.3â¯Â±â¯0.1%, nâ¯=â¯6), eugenol (0.3â¯Â±â¯0.2%, nâ¯=â¯6), phenethyl propionate (0.3â¯Â±â¯0.1%, nâ¯=â¯6), limonene oxide (0.3â¯Â±â¯0.2%, nâ¯=â¯6), carvacrol (0.2â¯Â±â¯0.2%, nâ¯=â¯6), menthone (0.2â¯Â±â¯0.1%, nâ¯=â¯6), pulegone (0.1â¯Â±â¯0.1%, nâ¯=â¯6) and carvone (0.0â¯Â±â¯0.0%, nâ¯=â¯6).
	Fig. 3. Effect of monoterpenoid compounds as antagonists on levamisole sensitive O. dentatum receptor acetylcholine mediated responses. A. Bar graph showing the rank order potency of monoterpenoid compounds as antagonists. Results were expressed as % mean inhibitionâ¯Â±â¯SEM of currents elicited by 100â¯Î¼M ACh: limonene oxide (36.0â¯Â±â¯3.2%, nâ¯=â¯6)â¯>â¯citronellol (18.0â¯Â±â¯1.6%, nâ¯=â¯6)â¯>â¯carvone (14.0â¯Â±â¯0.6%, nâ¯=â¯6)â¯>â¯carvacrol (11.0â¯Â±â¯1.6%, nâ¯=â¯6)â¯=â¯pulegone (11.0â¯Â±â¯2.6%, nâ¯=â¯6)â¯=â¯eugenol (11.0â¯Â±â¯1.0%, nâ¯=â¯6). Inset: image showing predicted acetylcholine response in the absence of limonene oxide (dotted line) and inhibition of acetylcholine mediated response in the presence of limonene oxide (highlighted in green). B. Acetylcholine current responses, recorded from the Xenopus oocyte expressing the levamisole sensitive channel, alone and in the presence of 100â¯Î¼M carvacrol (nâ¯=â¯6) & 100â¯Î¼M limonene oxide (nâ¯=â¯5). C. Concentration-response plots for acetylcholine alone (nâ¯=â¯6, black) and acetylcholine in the presence of 100â¯Î¼M carvacrol (nâ¯=â¯6, maroon) & 100â¯Î¼M limonene oxide (nâ¯=â¯5, green). pEC50 (meanâ¯Â±â¯SEM), EC50 (mean, Î¼M), Hill slope (nH) (meanâ¯Â±â¯SEM) and Imax (meanâ¯Â±â¯SEM%) values were respectively: 5.3â¯Â±â¯0.0, 5.3â¯Î¼M, 2.0â¯Â±â¯0.2 and 104.6â¯Â±â¯1.6 for acetylcholine alone; 5.4â¯Â±â¯0.2, 4.0â¯Î¼M, 2.2â¯Â±â¯1.7 and 62.8â¯Â±â¯7.0 in the presence of limonene oxide; 5.7â¯Â±â¯0.1, 2.0â¯Î¼M, 1.7â¯Â±â¯1.1 and 74.1â¯Â±â¯3.2 in the presence of carvacrol. Bottom was constrained to zero for curve fitting. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 4. Effects of menthol as a PAM on levamisole sensitive O. dentatum nAChR acetylcholine responses. A. Sample traces for two-electrode voltage-clamp recording showing inward currents in response to ascending concentrations of acetylcholine alone, acetylcholine in the presence of 0.1â¯Î¼M and 10â¯Î¼M menthol. B. Concentration-response relationships for acetylcholine alone (nâ¯=â¯7, black), acetylcholine in the presence of 0.1â¯Î¼M (nâ¯=â¯6, blue) and 10â¯Î¼M menthol (nâ¯=â¯7, purple). pEC50 (meanâ¯Â±â¯SEM), EC50 (mean, Î¼M), Hill slope (nH) (meanâ¯Â±â¯SEM) and Imax (meanâ¯Â±â¯SEM%) values were respectively: 5.3â¯Â±â¯0.0, 5.0â¯Î¼M, 1.4â¯Â±â¯0.1 and 107.5â¯Â±â¯2.6 for acetylcholine alone; 6.4â¯Â±â¯0.1, 0.4â¯Î¼M, 1.0â¯Â±â¯0.1 and 109.0â¯Â±â¯3.6 in the presence of 0.1â¯Î¼M menthol; 6.5â¯Â±â¯0.1, 0.3â¯Î¼M, 1.0â¯Â±â¯0.1 and 110.5â¯Â±â¯3.7 in the presence of 10â¯Î¼M menthol. Bottom was constrained to zero for curve fitting. C. Bar chart showing comparison of pEC50 (expressed as meanÂ Â±Â SEM) for acetylcholine in the presence and absence of menthol. ****Pâ¯<â¯0.0001; significantly different as indicated; extra sum of squares F-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 5. Effects of menthol on levamisole sensitive nAChR levamisole responses. A. Representative current traces for two-electrode voltage-clamp recording showing inward currents in response to ascending application of levamisole alone, levamisole in the presence of 0.1â¯Î¼M and 10â¯Î¼M menthol. B. Concentration-response relationships for levamisole alone (nâ¯=â¯7, steel gray) and in the presence of 0.1â¯Î¼M (nâ¯=â¯6, light green) and 10â¯Î¼M menthol (nâ¯=â¯7, dark green). pEC50 (meanâ¯Â±â¯SEM), EC50 (mean, Î¼M), Hill slope (nH) (meanâ¯Â±â¯SEM) and Imax (meanâ¯Â±â¯SEM%) values were respectively: 6.3â¯Â±â¯0.1, 0.5â¯Î¼M, 1.0â¯Â±â¯0.2 and 96.3â¯Â±â¯5.2 for levamisole alone; 6.5â¯Â±â¯0.1, 0.3â¯Î¼M, 1.1â¯Â±â¯0.2 and 94.6â¯Â±â¯5.0 in the presence of 0.1â¯Î¼M menthol; 6.8â¯Â±â¯0.2, 0.2â¯Î¼M, 1.0â¯Â±â¯0.2 and 89.2â¯Â±â¯6.0 in the presence of 10â¯Î¼M menthol. Bottom was constrained to zero for curve fitting. C. Bar chart summarizing the results showing comparison of pEC50 (expressed as meanÂ Â±Â SEM) for levamisole in the presence and absence of menthol. *Pâ¯<â¯0.05; significantly different as indicated; extra sum of squares F-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 6. Effects of menthol and carvacrol on nicotine sensitive A. suum ACR-16 nAChR acetylcholine responses. A. Representative current traces for two-electrode voltage-clamp recording showing inward currents in response to increasing acetylcholine concentrations in the presence of menthol (10â¯Î¼M) and carvacrol (10â¯Î¼M and 100â¯Î¼M) B. Concentration-response plot for acetylcholine alone (nâ¯=â¯7, black) and acetylcholine in the presence of menthol (nâ¯=â¯6, red). pEC50 (meanâ¯Â±â¯SEM) and EC50 (mean, Î¼M) values were respectively: 5.0â¯Â±â¯0.1 and 11.1â¯Î¼M for acetylcholine alone, 5.0â¯Â±â¯0.1 and 9.5â¯Î¼M in the presence of 10â¯Î¼M menthol. Hill slope (nH) (meanâ¯Â±â¯SEM) values were: 1.6â¯Â±â¯0.3 for acetylcholine alone; 2.5â¯Â±â¯1.2 in the presence of 10â¯Î¼M menthol. Imax (meanâ¯Â±â¯SEM%) values were: 109.1â¯Â±â¯6.6 for acetylcholine alone; 101.6â¯Â±â¯7.5 in the presence of 10â¯Î¼M menthol. Bottom was constrained to zero for curve fitting. C. Concentration-response plots for acetylcholine alone (nâ¯=â¯7, black) and in the presence of carvacrol (nâ¯=â¯6, blue). pEC50 (meanâ¯Â±â¯SEM), EC50 (mean, Î¼M), Hill slope (nH) (meanâ¯Â±â¯SEM) and Imax (meanâ¯Â±â¯SEM%) values were respectively: 5.1â¯Â±â¯0.1, 8.1â¯Î¼M, 2.5â¯Â±â¯0.8 and 94.6â¯Â±â¯5.9 in the presence of 10â¯Î¼M carvacrol; 5.1â¯Â±â¯0.1, 8.2â¯Î¼M, 2.0â¯Â±â¯0.7 and 55.8â¯Â±â¯4.4 in the presence of 100â¯Î¼M carvacrol. Bottom was constrained to zero for curve fitting. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 7. A. Sample trace for the A. suum muscle flap contraction experiments. Inset: concentration-response plots for acetylcholine alone (nâ¯=â¯6, black) and in the presence of 0.1â¯Î¼M menthol (nâ¯=â¯6, red). pEC50 (meanâ¯Â±â¯SEM) and EC50 (mean, Î¼M) values were respectively: 5.2â¯Â±â¯0.2 and 7.0â¯Î¼M for acetylcholine alone; 5.2â¯Â±â¯0.2 and 6.5â¯Î¼M in the presence of 0.1â¯Î¼M menthol. Rmax (meanâ¯Â±â¯SEM, g) values were: 1.7â¯Â±â¯0.2â¯g for acetylcholine alone and 2.0â¯Â±â¯0.2â¯g in the presence of 0.1â¯Î¼M menthol. Hill slope (nH) (meanÂ Â±Â SEM) values were: 0.8Â Â±Â 0.2 for acetylcholine alone and 0.8Â Â±Â 0.1 in the presence of 0.1Â Î¼M menthol. Bottom was constrained to zero for curve fitting. B. Bar chart summarizing the results showing significant potentiation in amplitude of acetylcholine mediated contractions in the presence of 0.1Â Î¼M menthol. PÂ <Â 0.05, **Pâ¯<â¯0.001; significantly different as indicated; paired t-tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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