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Richard J Martin - One of the best experts on this subject based on the ideXlab platform.
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Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Saurabh Verma, Alan P. Robertson, Sudhanva S. Kashyap, Richard J MartinAbstract:Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to Caenorhabditis elegans because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite Brugia malayi, which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (M-, L-, P-, and N-). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the unc-38 and/or unc-29 genes. Derquantel behaved as a competitive antagonist and distinguished M-nAChRs activated by morantel (Kb 13.9 nM), P-nAChRs activated by pyrantel (Kb 126 nM), and L-nAChRs activated by levamisole (Kb 0.96 µM) and Bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing N-type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the C. elegans model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of unc-38+unc-29 (L- and/or P-receptors) inhibited motility but knockdown of acr-16+acr-26 (M- and/or N-receptors) did not.
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Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues
British journal of pharmacology, 2016Co-Authors: Melanie Abongwa, Alan P. Robertson, Samuel K Buxton, Elise Courtot, Claude L. Charvet, Cédric Neveu, Ciaran J Mccoy, Saurabh Verma, Richard J MartinAbstract:SummaryBackground and Purpose Control of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR-16, from a nematode parasite. Experimental Approach Using RT-PCR, molecular cloning and two-electrode voltage clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested. Key Results Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, Bephenium, tribendimidine and pyrantel) did not activate Asu-ACR-16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3-bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had lower calcium permeability than α7 receptors. Conclusions and Implications We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
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Pharmacological profile of [i]Ascaris suum[/i] ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in [i]Ascaris[/i] tissues
British Journal of Pharmacology, 2016Co-Authors: Melanie Abongwa, Alan P. Robertson, Samuel K Buxton, Elise Courtot, Claude L. Charvet, Cédric Neveu, Ciaran J Mccoy, Saurabh Verma, Richard J MartinAbstract:Control of nematode parasite infections relies largely on anthelminthic drugs, several of which act on nicotinic acetylcholine receptors (nAChRs) and there are concerns about the development of resistance. There is an urgent need for development of new resistance-busting drugs and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of the homomeric nAChR, ACR-16, from a nematode parasite.[br/] Using RT-PCR, molecular cloning, and two-electrode voltage-clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum, then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of the receptor to cholinergic anthelmintics and a range of nicotinic agonists was then tested.[br/] Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from A. suum somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, we observed that existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, Bephenium, tribendimidine & pyrantel) did not activate Asu-ACR-16 (except for small currents to oxantel). Other nAChR agonists: nicotine, acetylcholine, cytisine, 3-bromocytisine and epibatidine produced robust current responses which desensitized at a rate that varied with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin, and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had a lower calcium permeability than α7 receptors.[br/] We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
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Tribendimidine: mode of action and nAChR subtype selectivity in Ascaris and Oesophagostomum.
PLoS neglected tropical diseases, 2015Co-Authors: Alan P. Robertson, Samuel K Buxton, Sreekanth Puttachary, Richard J MartinAbstract:The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like Bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.
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Voltage-clamp of oocytes injected with O. dentatum Ode-unc-29 and Ode-unc-63 nAChR subunits.
2014Co-Authors: Samuel K Buxton, Alan P. Robertson, Jacques Cabaret, Melanie Abongwa, Elise Courtot, Cédric Neveu, Claude Charvet, Jacques Cortet, Nicolas Peineau, Richard J MartinAbstract:(A) Diagram of possible subunit arrangements of Ode-unc-29 and Ode-unc-63. X represents either UNC-63 or UNC-29 subunit. PyR, pyrantel; Tbd, tribendimidine, ACh, acetylcholine; Nic, nicotine; Bep, Bephenium; The, thenium. (B) Representative traces showing the inward currents in oocytes injected with 1∶1 Ode-unc-29 and Ode-unc-63. (C) Bar chart (mean ± se) of agonists-elicited currents in the Ode-(29 - 63) Pyr-nAChR, (paired t-test, **p
Alan P. Robertson - One of the best experts on this subject based on the ideXlab platform.
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Carvacrol acts as a potent selective antagonist of different types of nicotinic acetylcholine receptors and enhances the effect of monepantel in the parasitic nematode Ascaris suum.
Veterinary parasitology, 2020Co-Authors: Djordje S. Marjanović, Alan P. Robertson, Nemanja Zdravkovic, Mirjana Milovanović, Jelena Nedeljković Trailović, Zoran Todorovic, Sasa M. TrailovicAbstract:The neuromuscular system of parasitic nematodes has proven to be an efficient pharmacological target for antihelmintics. Some of the most frequently used antiparasitic drugs are agonists or antagonists of nicotinic acetylcholine receptors (nAChRs). The antinematodal mechanism of action of carvacrol involves the inhibition of parasite muscle contraction. We have examined the interaction of carvacrol with antinematodal drugs that are agonists of different subtypes of nAChRs and monepantel, which is a non-competitive antagonist of this receptor in A. suum. Additionally, we investigated the effect of carvacrol on the muscle type of nAChRs in the mammalian host. As orthosteric agonists of nAChR, pyrantel, morantel and befinijum lead to dose-dependent contractions of the neuromuscular preparation of Ascaris suum. Carvacrol 100 μM decreased the Emax of pyrantel, morantel and Bephenium by 29%, 39% and 12 %, 39 % and 12 % respectively. The EC50 ratio was 3.43, 2.95 and 2.47 for pyrantel, morantel and bephinium, respectively. Carvacrol 300 u μM reduces the Emax of pyrantel, morantel and Bephenium by 71%, 80% and 75 %, 80 % and 75 % respectively. The EC50 ratio for pyrantel, morantel and Bephenium was 3.88, 3.19 and 4.83 respectively. Furthermore, carvacrol enhances the inhibitory effect of monepantel on A. suum contractions, which may have an effective clinical application. On the other hand, tested concentrations of carvacrol did not significantly affect the EFS-induced contractions of the rat diaphragm, indicating a lack of interaction with the postsynaptic nAChR at the muscle end plate in mammals, but the highest concentration (300 μM) caused a clear tetanic fade. Carvacrol exhibited a time and dose-dependent effect on the Rota-rod performances of rats with a high value of the ED50 (421.6 mg/kg). In our research, carvacrol dominantly exhibited characteristics of a non-competitive antagonist of nAChR in A. suum, and enhances the inhibitory effect of monepantel. The combination of monepantel and carvacrol may be clinically very effective, and the carvacrol molecule itself can be used as a promising platform for the development of new anthelmintic drugs.
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Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Saurabh Verma, Alan P. Robertson, Sudhanva S. Kashyap, Richard J MartinAbstract:Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to Caenorhabditis elegans because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite Brugia malayi, which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (M-, L-, P-, and N-). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the unc-38 and/or unc-29 genes. Derquantel behaved as a competitive antagonist and distinguished M-nAChRs activated by morantel (Kb 13.9 nM), P-nAChRs activated by pyrantel (Kb 126 nM), and L-nAChRs activated by levamisole (Kb 0.96 µM) and Bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing N-type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the C. elegans model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of unc-38+unc-29 (L- and/or P-receptors) inhibited motility but knockdown of acr-16+acr-26 (M- and/or N-receptors) did not.
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Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues
British journal of pharmacology, 2016Co-Authors: Melanie Abongwa, Alan P. Robertson, Samuel K Buxton, Elise Courtot, Claude L. Charvet, Cédric Neveu, Ciaran J Mccoy, Saurabh Verma, Richard J MartinAbstract:SummaryBackground and Purpose Control of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR-16, from a nematode parasite. Experimental Approach Using RT-PCR, molecular cloning and two-electrode voltage clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested. Key Results Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, Bephenium, tribendimidine and pyrantel) did not activate Asu-ACR-16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3-bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had lower calcium permeability than α7 receptors. Conclusions and Implications We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
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Pharmacological profile of [i]Ascaris suum[/i] ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in [i]Ascaris[/i] tissues
British Journal of Pharmacology, 2016Co-Authors: Melanie Abongwa, Alan P. Robertson, Samuel K Buxton, Elise Courtot, Claude L. Charvet, Cédric Neveu, Ciaran J Mccoy, Saurabh Verma, Richard J MartinAbstract:Control of nematode parasite infections relies largely on anthelminthic drugs, several of which act on nicotinic acetylcholine receptors (nAChRs) and there are concerns about the development of resistance. There is an urgent need for development of new resistance-busting drugs and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of the homomeric nAChR, ACR-16, from a nematode parasite.[br/] Using RT-PCR, molecular cloning, and two-electrode voltage-clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum, then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of the receptor to cholinergic anthelmintics and a range of nicotinic agonists was then tested.[br/] Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from A. suum somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, we observed that existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, Bephenium, tribendimidine & pyrantel) did not activate Asu-ACR-16 (except for small currents to oxantel). Other nAChR agonists: nicotine, acetylcholine, cytisine, 3-bromocytisine and epibatidine produced robust current responses which desensitized at a rate that varied with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin, and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had a lower calcium permeability than α7 receptors.[br/] We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
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Tribendimidine: mode of action and nAChR subtype selectivity in Ascaris and Oesophagostomum.
PLoS neglected tropical diseases, 2015Co-Authors: Alan P. Robertson, Samuel K Buxton, Sreekanth Puttachary, Richard J MartinAbstract:The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like Bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.
Samuel K Buxton - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues
British journal of pharmacology, 2016Co-Authors: Melanie Abongwa, Alan P. Robertson, Samuel K Buxton, Elise Courtot, Claude L. Charvet, Cédric Neveu, Ciaran J Mccoy, Saurabh Verma, Richard J MartinAbstract:SummaryBackground and Purpose Control of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR-16, from a nematode parasite. Experimental Approach Using RT-PCR, molecular cloning and two-electrode voltage clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested. Key Results Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, Bephenium, tribendimidine and pyrantel) did not activate Asu-ACR-16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3-bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had lower calcium permeability than α7 receptors. Conclusions and Implications We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
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Pharmacological profile of [i]Ascaris suum[/i] ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in [i]Ascaris[/i] tissues
British Journal of Pharmacology, 2016Co-Authors: Melanie Abongwa, Alan P. Robertson, Samuel K Buxton, Elise Courtot, Claude L. Charvet, Cédric Neveu, Ciaran J Mccoy, Saurabh Verma, Richard J MartinAbstract:Control of nematode parasite infections relies largely on anthelminthic drugs, several of which act on nicotinic acetylcholine receptors (nAChRs) and there are concerns about the development of resistance. There is an urgent need for development of new resistance-busting drugs and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of the homomeric nAChR, ACR-16, from a nematode parasite.[br/] Using RT-PCR, molecular cloning, and two-electrode voltage-clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum, then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of the receptor to cholinergic anthelmintics and a range of nicotinic agonists was then tested.[br/] Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from A. suum somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, we observed that existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, Bephenium, tribendimidine & pyrantel) did not activate Asu-ACR-16 (except for small currents to oxantel). Other nAChR agonists: nicotine, acetylcholine, cytisine, 3-bromocytisine and epibatidine produced robust current responses which desensitized at a rate that varied with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin, and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had a lower calcium permeability than α7 receptors.[br/] We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
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Tribendimidine: mode of action and nAChR subtype selectivity in Ascaris and Oesophagostomum.
PLoS neglected tropical diseases, 2015Co-Authors: Alan P. Robertson, Samuel K Buxton, Sreekanth Puttachary, Richard J MartinAbstract:The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like Bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.
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Voltage-clamp of oocytes injected with O. dentatum Ode-unc-29 and Ode-unc-63 nAChR subunits.
2014Co-Authors: Samuel K Buxton, Alan P. Robertson, Jacques Cabaret, Melanie Abongwa, Elise Courtot, Cédric Neveu, Claude Charvet, Jacques Cortet, Nicolas Peineau, Richard J MartinAbstract:(A) Diagram of possible subunit arrangements of Ode-unc-29 and Ode-unc-63. X represents either UNC-63 or UNC-29 subunit. PyR, pyrantel; Tbd, tribendimidine, ACh, acetylcholine; Nic, nicotine; Bep, Bephenium; The, thenium. (B) Representative traces showing the inward currents in oocytes injected with 1∶1 Ode-unc-29 and Ode-unc-63. (C) Bar chart (mean ± se) of agonists-elicited currents in the Ode-(29 - 63) Pyr-nAChR, (paired t-test, **p
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Voltage-clamp of oocytes injected with different combinations of the four O. dentatum nAChR subunits.
2014Co-Authors: Samuel K Buxton, Alan P. Robertson, Jacques Cabaret, Melanie Abongwa, Elise Courtot, Claude L. Charvet, Cédric Neveu, Jacques Cortet, Nicolas Peineau, Richard J MartinAbstract:(A) Depiction of a possible arrangement of O. dentatum UNC-29, UNC-63 & UNC-38. ‘X’ represents any of the subunits. PyR, pyrantel; Tbd, tribendimidine, ACh, acetylcholine; Nic, nicotine; Bep, Bephenium; The, thenium. (B) Representative traces of inward currents elicited by the various agonists in oocytes injected with 1∶1∶1 Ode-unc-29∶Ode-unc-63∶Ode-unc-38. Pyr & Tbd were the most potent agonists on this receptor subtype. (C) Bar chart (mean ± se) of currents elicited by the different agonists in the Ode-(29 - 38 - 63) Pyr/Tbd-nAChR (paired t-test, **p
Cédric Neveu - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues
British journal of pharmacology, 2016Co-Authors: Melanie Abongwa, Alan P. Robertson, Samuel K Buxton, Elise Courtot, Claude L. Charvet, Cédric Neveu, Ciaran J Mccoy, Saurabh Verma, Richard J MartinAbstract:SummaryBackground and Purpose Control of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR-16, from a nematode parasite. Experimental Approach Using RT-PCR, molecular cloning and two-electrode voltage clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested. Key Results Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, Bephenium, tribendimidine and pyrantel) did not activate Asu-ACR-16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3-bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had lower calcium permeability than α7 receptors. Conclusions and Implications We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
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Response of H. contortus reconstituted receptors in Xenopus oocytes.
2016Co-Authors: Thomas B. Duguet, Claude L. Charvet, Cédric Neveu, Sean G. Forrester, Claudia M. Wever, Joseph A. Dent, Robin N. BeechAbstract:Two electrode voltage clamp experiments were performed on oocytes injected with Hco-unc-63, Hco-unc-38, Hco-acr-8, and Hco-ric-3.1, Hco-unc-74, Hco-unc-50 cRNAs. Hco-unc-29.1, unc-29.3 and unc-29.4 were combined independently with the cRNA mixture. A), B) and C) Representative recording traces from single oocytes perfused with 100 μM of the following cholinergic agonists: acetylcholine (ACh), Dimethylpiperazinium (DMPP), Pyrantel (PYR), Nicotine (NIC), Bephenium (BEPH) and Levamisole (LEV). D), E) and F) Representative recording traces from single oocytes continuously perfused with 100 μM ACh. Oocytes were perfused with the following cholinergic antagonists: D-tubocurarine (dTC, 100 μM), Dihydro-β-erythroidine (DHβE, 10 μM) and Mecamylamine (MECA, 30 μM). Black horizontal bars show the time period of agonist and or antagonist application. G), H) and I) Concentration-response curves for the L-AChR1.1, L-AChR1.3 and L-AChR1.4 for ACh (black circles) and LEV (white squares). All responses are normalized to 100 μM ACh, which corresponds to the saturating dose. The ACh and LEV 50% effective concentration (EC50) values as well as Hill coefficients are indicated in Table 2. Error bars represent SD.
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Pharmacological profile of [i]Ascaris suum[/i] ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in [i]Ascaris[/i] tissues
British Journal of Pharmacology, 2016Co-Authors: Melanie Abongwa, Alan P. Robertson, Samuel K Buxton, Elise Courtot, Claude L. Charvet, Cédric Neveu, Ciaran J Mccoy, Saurabh Verma, Richard J MartinAbstract:Control of nematode parasite infections relies largely on anthelminthic drugs, several of which act on nicotinic acetylcholine receptors (nAChRs) and there are concerns about the development of resistance. There is an urgent need for development of new resistance-busting drugs and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of the homomeric nAChR, ACR-16, from a nematode parasite.[br/] Using RT-PCR, molecular cloning, and two-electrode voltage-clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum, then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of the receptor to cholinergic anthelmintics and a range of nicotinic agonists was then tested.[br/] Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from A. suum somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, we observed that existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, Bephenium, tribendimidine & pyrantel) did not activate Asu-ACR-16 (except for small currents to oxantel). Other nAChR agonists: nicotine, acetylcholine, cytisine, 3-bromocytisine and epibatidine produced robust current responses which desensitized at a rate that varied with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin, and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had a lower calcium permeability than α7 receptors.[br/] We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
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Pharmacological profiles of Hco-26/27 and Peq-26/27.
2015Co-Authors: Elise Courtot, Claude L. Charvet, Nicolas Peineau, Robin N. Beech, Abdallah Harmache, Adrian J. Wolstenholme, Lindy Holden-dye, Vincent O’connor, Debra J. Woods, Cédric NeveuAbstract:(A and B) Representative recording traces from a single oocyte expressing Hco-26/27 (A) or Peq-26/27 (B) challenged with 100 μM ACh and 100 μM of different anthelmintic compounds (morantel (Mor), pyrantel (Pyr), oxantel (Oxa), levamisole (Lev), Bephenium (Beph) and nicotine (Nic)). The bars indicate the time period of the agonist application. (C and D) Scatter plot (mean ± SEM) of normalized currents elicited by 100 μM of anthelmintic compounds on Hco-26/27 (C) or Peq-26/27 (D). Currents have been normalized to and compared with 100 μM ACh currents. Paired Student’s t-test, ***p
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Voltage-clamp of oocytes injected with O. dentatum Ode-unc-29 and Ode-unc-63 nAChR subunits.
2014Co-Authors: Samuel K Buxton, Alan P. Robertson, Jacques Cabaret, Melanie Abongwa, Elise Courtot, Cédric Neveu, Claude Charvet, Jacques Cortet, Nicolas Peineau, Richard J MartinAbstract:(A) Diagram of possible subunit arrangements of Ode-unc-29 and Ode-unc-63. X represents either UNC-63 or UNC-29 subunit. PyR, pyrantel; Tbd, tribendimidine, ACh, acetylcholine; Nic, nicotine; Bep, Bephenium; The, thenium. (B) Representative traces showing the inward currents in oocytes injected with 1∶1 Ode-unc-29 and Ode-unc-63. (C) Bar chart (mean ± se) of agonists-elicited currents in the Ode-(29 - 63) Pyr-nAChR, (paired t-test, **p
Andrew C Kotze - One of the best experts on this subject based on the ideXlab platform.
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In vitro levamisole selection pressure on larval stages of Haemonchus contortus over nine generations gives rise to drug resistance and target site gene expression changes specific to the early larval stages only.
Veterinary parasitology, 2015Co-Authors: Ranbir S Sarai, Steven R Kopp, Malcolm R Knox, Glen T Coleman, Andrew C KotzeAbstract:There is some evidence that resistance to levamisole and pyrantel in trichostrongylid nematodes is due to changes in the composition of nicotinic acetylcholine receptors (nAChRs) which represent the drug target site. Altered expression patterns of genes coding for nAChR subunits, as well as the presence of truncated versions of several subunits, have been implicated in observed resistances. The studies have mostly compared target sites in worm isolates of very different genetic background, and hence the ability to associate the molecular changes with drug sensitivity alone have been clouded to some extent. The present study aimed to circumvent this issue by following target site gene expression pattern changes as resistance developed in Haemonchus contortus worms under laboratory selection pressure with levamisole. We applied drug selection pressure to early stage larvae in vitro over nine generations, and monitored changes in larval and adult drug sensitivities and target site gene expression patterns. High level resistance developed in larvae, with resistance factors of 94-fold and 1350-fold at the IC50 and IC95, respectively, in larval development assays after nine generations of selection. There was some cross-resistance to Bephenium (70-fold increase in IC95). The expression of all the putative subunit components of levamisole-sensitive nAChRs, as well as a number of ancillary protein genes, particularly Hco-unc-29.1 and -ric-3, were significantly decreased (up to 5.5-fold) in the resistant larvae at generation nine compared to the starting population. However, adult worms did not show any resistance to levamisole, and showed an inverse pattern of gene expression changes, with many target site genes showing increased expression compared to the starting population. A comparison of the larval/adult drug sensitivity data with the known relationships for field-derived isolates indicated that the adults of our selected population should have been highly resistant to the drug if the larval/adult sensitivity relationships were in accordance with previous field isolates. Hence, our selected worms showed a life-stage drug sensitivity pattern quite different to that seen in the field. The present study has highlighted an association between drug target site changes and resistance to levamisole in H. contortus larvae. However, it has also highlighted the artificial nature of the larval selection method with levamisole, as the resistance phenotype and the associated molecular changes were only observed in the drug-pressured life stage. The study therefore reinforces the need for caution in extrapolating larval-based laboratory selection outcomes to field resistances.
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phenotypic characterization of two ancylostoma caninum isolates with different susceptibilities to the anthelmintic pyrantel
Antimicrobial Agents and Chemotherapy, 2008Co-Authors: Steven Kopp, G T Coleman, James S Mccarthy, Andrew C KotzeAbstract:The anthelmintic pyrantel plays an important role in the control of gastrointestinal helminths of humans and domestic animals. Despite the demonstration of pyrantel resistance in several helminth species over the last 20 years, the resistance mechanism remains unclear. It has been hypothesized that resistance may arise as a consequence of changes to the relative proportions of subpopulations of nicotinic acetylcholine receptors (nAchRs). To test this hypothesis, we examined the responses of two isolates of the canine hookworm Ancylostoma caninum with low-level resistance (isolate NT) and high-level resistance (isolate PR) to pyrantel to nicotinic agonist drugs reported to be selective for three nAchR subtypes. We used larval motility and conformation assays and force transduction experiments with adult worms. Pyrantel and levamisole were less potent against larvae of isolate PR than larvae of isolate NT (up to an 18-fold increase in the 50% inhibitory concentration); on the other hand, Bephenium was more potent against larvae of isolate PR than larvae of isolate NT (twofold) and nicotine had the same potency against larvae of both isolates. In adults, pyrantel, levamisole, and nicotine were less potent against isolate PR than isolate NT (two- to threefold), but the potency of Bephenium against the two isolates was equivalent. Our data indicate a complex pattern of nAchRs in this species and suggest that the two isolates differ in their relative sensitivities to agonists targeting different nAchRs.