Functional expression of a glutamate-gated chloride channel (GLC-3) from adult Brugia malayi.

Parasitic worms are causative agents for six highly prevalent neglected tropical diseases of humans which include ascariasis, lymphatic filariasis, schistosomiasis, trichuriasis, hookworm infection, and onchocerciasis that affect a high percentage of the world’s population. Resistance to available anthelminthic drugs especially for the benzimidazole anthelmintic agents (e.g., albendazole and mebendazole) and ivermectin is a serious concern (this is a real problem for veterinary medicine and a growing concern in human medicine). Previous studies involving ivermectin have shown that it has a limited action against adult filarial worms. Here, we describe the functional expression of a glutamate-gated chloride channel (GLC-3) from adult Brugia malayi, a filarial parasite. We expressed GLC-3 in Xenopus laevis oocytes and used two-electrode voltage-clamp electrophysiology to study the resulting ion channel. Application of various receptor agonists (1 mM): L-aspartate, glycine, γ-aminobutyric acid (GABA), α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) failed to activate the GLC3 receptor. Application of 1mM L-glutamate and ibotenate produced robust inward currents. Further experiments revealed that GLC-3 is a glutamate-gated channel with an EC50 of 64.8 4.01 µM and a Hill coefficient of 2.56 0.46 µM. We recorded the responses to 300 µM L-glutamate over a range of holding potentials (-80 to +20 mV) and constructed a current-voltage plot. The current-voltage relationship revealed a reversal potential (Erev) of -35.3 3.2 mV indicating the GLC-3 channel is selective for chloride ions. The GluCl channel antagonists picrotoxin and fipronil had negligible inhibitory effects on the L-glutamate responses. We investigated the ivermectin effects on the GLC-3 receptor which reveals ivermectin to be an agonist with an EC50 of 3.6 nM (pEC50 = -2.4 ± 0.4). Interestingly, we also observed that Ivermectin has an inhibitory effect on the GLC-3 glutamate response at very low concentrations, IC50 = 73 pM (pIC50 = -4.1 ± 11.4). This is in stark contrast to the previously reported positive allosteric (PAM) effects of ivermectin on many invertebrate GluCls

Selective activity of extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi

<p>Abstract</p> <p>Background</p> <p>The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of <it>Margaritaria discoidea </it>and <it>Homalium africanum </it>on <it>Onchocerca ochengi</it>, a model parasite for <it>O. volvulus</it>. These plants are used to treat the disease in North West Cameroon.</p> <p>Methods</p> <p>Sixteen crude extracts were prepared from various parts of <it>M. discoidea </it>and <it>H. africanum </it>using different organic solvents. The filaricidal activities were determined <it>in vitro</it>. Cytotoxicity of the active extracts was assessed on monkey kidney epithelial cells <it>in vitro </it>and the selectivity indices (SI) of the extracts determined. Acute toxicity of the promising extracts was investigated in mice.</p> <p>Results</p> <p>Four out of the 16 extracts showed microfilaricidal activity based on motility reduction, whereas, none showed macrofilaricidal activity based on the MTT/formazan assay. The methylene chloride extract of <it>H. africanum </it>leaves (HLC) recorded the lowest IC₅₀of 31.25 μg/mL and an IC₁₀₀of 62.5 μg/mL. The SI for the active extracts ranged from 0.5 - 2.63. No form of acute toxicity was observed in mice. Phytochemical analysis revealed the presence of anthraquinones, sterols and terpenoids in the promising extracts.</p> <p>Conclusions</p> <p>The non-polar extracts of <it>M. discoidea </it>and <it>H. africanum </it>are potential sources of new microfilaricidal lead compounds, and the results support their use in traditional medicine.</p

Monepantel is a non-competitive antagonist of nicotinic acetylcholine receptors from Ascaris suum and Oesophagostomum dentatum

Zolvix (R) is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltagec-lamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype from A. suum comprising of ACR-16; a pyrantel/tribendimidine preferring heteromeric subtype from O. dentatum comprising UNC-29, UNC-38 and UNC-63 subunits; and a levamisole preferring subtype (O. dentatum) comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents thereby ruling out an agonist action. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6 +/- 3.1 nM and 0.2 +/- 2.3 mu M). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the inhibition was a mixture of competitive and non-competitive antagonism. Our findings suggest that monepantel is active on multiple nAChR subtypes

Potential new drug targets and therapeutic approaches for parasitic nematode infections

Parasitic nematode infections remain a serious global public health threat to humans and animals. These infections cause debilitating conditions in humans and significant economic losses through infection of livestock and crop damage. Control of parasitic nematode diseases has continued to rely on use of anthelmintic drugs as there is presently no effective vaccine for the majority of these infections. Over the years, anthelmintic drug discovery has been very slow, and the majority of anthelmintic drugs used in human medicine today were initially developed for animal use as those affected live in the world’s poorest nations that lack the financial means to afford conventional drugs. However, resistance to all the major anthelmintic drug classes has been reported in numerous veterinary parasite species and there are increasing concerns of resistance development in human parasite species. The emergence of widespread resistance therefore underscores the urgent need for the search of new targets, leads and strategies for anthelmintic drug discovery and development. We have identified the nicotinic acetylcholine receptor (nAChR) subunit ACR-16 from Ascaris suum and showed that ACR-16 forms a functional homopentameric nAChR when expressed in Xenopus oocytes. A. suum is a gastrointestinal (GI) tract parasitic nematode of pigs that is very closely related to the human equivalent A. lumbricoides. ACR-16 is not activated by cholinergic anthelmintics and, although ACR-16 is most closely related to vertebrate 7 receptors based on amino acid sequence alignment, it has some marked pharmacological differences from vertebrate 7 receptors. As opposed to vertebrate 7 receptors, ACR-16 was insensitive to -bungarotoxin and the effects of ivermectin, genistein and PNU120596 on ACR-16 were inhibitory rather than potentiating. The relative calcium permeability ratio for ACR-16 was about 50x lower than that for vertebrate 7 receptors. We have showed using reverse transcription polymerase chain reaction (RT-PCR) that mRNA for ACR-16 is widely distributed throughout Ascaris tissues; suggesting ACR-16 may have functions other than neurotransmission. Our results showed, for the first time, the characterization of the pharmacology of ACR-16 from a parasitic nematode. Based on the pharmacology and expression pattern of ACR-16 in different tissues of the parasite, we suggest ACR-16 is an attractive new anthelmintic drug target with ‘resistance-busting’ properties that should be further exploited for therapeutic drug development. Secondly, we have showed that the plants Daniellia oliveri and Psorospermum febrifugum have potential as sources of lead compounds for the development of the much-needed filaricidal drugs to treat onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis). We prepared extracts of different polarities from D. oliveri and P. febrifugum and showed these extracts to be active against Onchocerca ochengi microfilariae and adults, and against adult Brugia pahangi based on visual motility scoring, MTT/formazan assay and the Worminator motility measurement system. Importantly, some extracts with O. ochengi microfilariae activity were also active on the adult worm. These extracts also showed activity against adult B. pahangi. We further fractionated the active extracts using Sep-Pak cartridges and High Performance Liquid Chromatography (HPLC) and showed some of these fractions retained activity against adult B. pahangi. We recommend that the active HPLC fractions be further purified to allow for the isolation of the bioactive compounds. This could significantly contribute to chemotherapeutic control of filarial infections currently hampered by the lack of macrofilaricides (adulticides). Lastly, we have demonstrated that use of combination therapy over single drug therapy is a potentially useful tool for increasing efficacy, spectrum of action and reducing the likelihood of resistance development. We showed the combination of derquantel and abamectin to produce a greater inhibition of acetylcholine and pyrantel responses of expressed pyrantel/tribendimidine nAChRs from Oesophagostomum dentatum than derquantel or abamectin used alone. These nAChRs comprise of UNC-29, UNC-63 and UNC-38 subunits. Our results also showed abamectin acts on nAChRs, in addition to its known effects on glutamate-gated chloride channels (GluCls), implying abamectin has multiple targets. We further showed the action of abamectin on the expressed O. dentatum pyrantel/tribendimidine nAChRs to be bi-phasic, suggesting two allosteric sites of action: a high affinity negative allosteric modulation (NAM) site causing antagonism at lower concentrations (≤0.1 à  à µM) and a lower affinity positive allosteric modulation (PAM) site causing a reduction in the antagonism at higher concentrations (0.3 à  à µM).</p

Functional expression of a glutamate-gated chloride channel (GLC-3) from adult Brugia malayi.

Parasitic worms are causative agents for six highly prevalent neglected tropical diseases of humans which include ascariasis, lymphatic filariasis, schistosomiasis, trichuriasis, hookworm infection, and onchocerciasis that affect a high percentage of the world’s population. Resistance to available anthelminthic drugs especially for the benzimidazole anthelmintic agents (e.g., albendazole and mebendazole) and ivermectin is a serious concern (this is a real problem for veterinary medicine and a growing concern in human medicine). Previous studies involving ivermectin have shown that it has a limited action against adult filarial worms. Here, we describe the functional expression of a glutamate-gated chloride channel (GLC-3) from adult Brugia malayi, a filarial parasite. We expressed GLC-3 in Xenopus laevis oocytes and used two-electrode voltage-clamp electrophysiology to study the resulting ion channel. Application of various receptor agonists (1 mM): L-aspartate, glycine, γ-aminobutyric acid (GABA), α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) failed to activate the GLC3 receptor. Application of 1mM L-glutamate and ibotenate produced robust inward currents. Further experiments revealed that GLC-3 is a glutamate-gated channel with an EC50 of 64.8 4.01 µM and a Hill coefficient of 2.56 0.46 µM. We recorded the responses to 300 µM L-glutamate over a range of holding potentials (-80 to +20 mV) and constructed a current-voltage plot. The current-voltage relationship revealed a reversal potential (Erev) of -35.3 3.2 mV indicating the GLC-3 channel is selective for chloride ions. The GluCl channel antagonists picrotoxin and fipronil had negligible inhibitory effects on the L-glutamate responses. We investigated the ivermectin effects on the GLC-3 receptor which reveals ivermectin to be an agonist with an EC50 of 3.6 nM (pEC50 = -2.4 ± 0.4). Interestingly, we also observed that Ivermectin has an inhibitory effect on the GLC-3 glutamate response at very low concentrations, IC50 = 73 pM (pIC50 = -4.1 ± 11.4). This is in stark contrast to the previously reported positive allosteric (PAM) effects of ivermectin on many invertebrate GluCls.</p

Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms.

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

Characterization of the Ascaris suum homomeric nicotinic acetylcholine receptor, ACR-16, in Xenopus laevis oocytes

International audienc

The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening

Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 μM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 μM; fmoc-1, IC50, 10.00 ± 1.38 μM; fmoc-2, IC50, 16.67 ± 1.95 μM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization. Keywords: Asu-ACR-16, Structure-based drug discovery, Homology modeling, Orthosteric site, Allosteric modulator, Xenopus expressio

Asu-ACR-16: A dispersed nAChR drug target in muscle, intestine and other tissues of Ascaris

International audienc