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PLoS One
2016 Jan 01;111:e0146854. doi: 10.1371/journal.pone.0146854.
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Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms.
Abongwa M
,
Buxton SK
,
Robertson AP
,
Martin RJ
.
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Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex effects of macrocyclic lactones that may be exploited in combinations with other anthelmintics.
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Fig 1. Structures of derquantel and abamectin.A: Structure of derquantel. B: Structure of abamectin. Abamectin is a mixture of avermectin B1a and avermectin B1b. Avermectin B1a differs from avermectin B1b by a functional group at the ‘R’ position, and makes up more than 80% of abamectin, while avermectin B1b makes up less than 20% of abamectin.
Fig 2. Acetylcholine and pyrantel concentration-response relationships for the Ode-UNC-29:Ode-UNC-63:Ode-UNC-38 receptor.A: Representative trace (inward currents, holding potential -60mV, from oocytes expressing Ode-UNC-29:Ode-UNC-63:Ode-UNC-38) following 10 seconds applications of different acetylcholine concentrations, from 0.1 μM to 100 μM. B: Representative trace (inward currents, holding potential -60mV, from oocytes expressing Ode-UNC-29:Ode-UNC-63:Ode-UNC-38) of the 10 seconds application of different pyrantel concentrations, from 0.03 μM to 10 μM. An initial 10 seconds application of 100 μM acetylcholine served as the control. C: Concentration-response plots for acetylcholine (n = 4, black) and pyrantel (n = 6, red). Results were normalized to 100 μM acetylcholine current responses and expressed as mean ± S.E.M.
Fig 3. Pyrantel concentration-response relationships in the presence of 0.03 μM and 0.1 μM abamectin.A: Representative trace (inward currents, holding potential -60mV, from oocytes expressing Ode-UNC-29:Ode-UNC-63:Ode-UNC-38) of the 10 seconds application of the control 100 μM acetylcholine, followed by a 10 minutes application of abamectin (0.03 μM) and finally, a 10 seconds application of different pyrantel concentrations in the continued presence of abamectin. B: Concentration-response plots for pyrantel in the absence (n = 6, red) and presence of 0.03 μM abamectin (n = 5, orange) and 0.1 μM abamectin (n = 6, dark blue). Results were normalized to 100 μM acetylcholine current responses and expressed as mean ± S.E.M. Notice 0.03 μM abamectin caused an inhibition of current responses to pyrantel, and this inhibition was greater with 0.1 μM abamectin.
Fig 4. Effects of increasing concentrations of abamectin (above 0.1 μM) on the pyrantel concentration-response plots.A: Concentration-response plots for pyrantel in the absence (n = 6, red) and presence of 0.1 μM abamectin (n = 6, dark blue); 0.3 μM abamectin (n = 5, dark green); 1 μM abamectin (n = 6, pink); 10 μM abamectin (n = 5, light blue). Results were normalized to 100 μM acetylcholine current responses and expressed as mean ± S.E.M. Increasing the concentration of abamectin from 0.1 μM to 0.3, 1 and 10 μM rather caused a reduction in the inhibition instead of a potentiation. B: Bar chart showing the mean ± S.E.M of the maximum current responses (Rmax) for pyrantel and the different abamectin concentrations. Rmax for 0.1 μM abamectin (n = 6, dark blue), 0.3 μM abamectin (n = 5, dark green), 1 μM abamectin (n = 6, pink) and 10 μM abamectin (n = 5, light blue) were significantly lower than Rmax for pyrantel alone (n = 6, red). * p < 0.05, ** p < 0.01 and *** p < 0.001, unpaired two-tailed student t-test. C: Model of ligand sites of action. Pyrantel binds to the orthosteric sites opening the channel. Low concentrations of abamectin (0.03 and 0.1 μM) bind to a negative allosteric site (NAM) in the lipid phase of the channel, inhibiting opening. Higher concentrations of abamectin (0.3, 1 and 10 μM) bind to a positive allosteric site (PAM) increasing opening. D: Abamectin Rmax inhibition (blue curve) and inhibition reduction (red curve) dose response plots. The data points for inhibition used data from 0.03 μM and 0.1 μM abamectin from Fig 3. The data points for inhibition reduction used data from Fig 4B.
Fig 5. Effects of 0.1 μM derquantel alone, 0.3 μM abamectin alone, and derquantel and abamectin combination (0.1 μM derquantel + 0.3 μM abamectin) on the pyrantel concentration-response plots.A: Representative trace (inward currents from oocytes expressing Ode-UNC-29:Ode-UNC-63:Ode-UNC-38) of the 10 seconds application of the control 100 μM acetylcholine, followed by a 10 minutes application of 0.3 μM abamectin and finally, a 10 seconds application of different pyrantel concentrations in the continued presence of abamectin. B: Representative trace (inward currents from oocytes expressing Ode-UNC-29:Ode-UNC-63:Ode-UNC-38) of the 10 seconds application of the control 100 μM acetylcholine, followed by a 10 minutes application of derquantel and abamectin combination and finally, a 10 seconds application of different pyrantel concentrations in the continued presence of the derquantel and abamectin combination. C: Concentration-response plots for pyrantel in the absence (n = 6, red) and presence of 0.1 μM derquantel (n = 4, light purple); 0.3 μM abamectin (n = 5, dark green); 0.1 μM derquantel + 0.3 μM abamectin combination (n = 6, olive green). Results were normalized to 100 μM acetylcholine current responses and expressed as mean ± S.E.M. Inhibition with 0.1 μM derquantel + 0.3 μM abamectin combination was greater than that with 0.1 μM derquantel alone and 0.3 μM abamectin alone. The calculated additive effect for the combination of derquantel and abamectin (broken black) was not statistically different (p > 0.05, paired two-tailed student t-test) from the observed additive effect for the combination of derquantel and abamectin (olive green). D: Bar chart showing the mean ± S.E.M of the maximum current responses (Rmax) for 0.1 μM derquantel (n = 4, light purple); 0.3 μM abamectin (n = 5, dark green); 0.1 μM derquantel + 0.3 μM abamectin combination (n = 6, olive green). Rmax for the combination of 0.1 μM derquantel + 0.3 μM abamectin was significantly smaller than Rmax for 0.1 μM derquantel alone and for 0.3 μM abamectin alone. * p < 0.05 and *** p < 0.001, unpaired two-tailed student t-test.
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