Alan P. Robertson

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions

Graphical abstract

The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.

Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect ∼1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5∶1 (Asu-unc-38∶Asu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1∶5 Asu-unc-38∶Asu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite-specific screens for future anthelmintics.

Pharmacology of N-, L-, and B-subtypes of nematode nAChR resolved at the single-channel level in Ascaris suum

Pharmacological experiments on Ascaris suum have demonstrated the presence of three (N‐, L‐, and B‐) subtypes of cholinergic receptor mediating contraction of body wall muscle in parasitic nematodes (1). In the present study, these ionotropic acetylcholine (ACh) receptors (nAChRs) were activated by levamisole and bephenium under patch‐clamp conditions and competitively antagonized by paraherquamide and 2‐desoxoparaherquamide. A number of recordings exhibited three separate current amplitude levels, indicating the presence of small, intermediate, and large conductance subtypes of receptor. The mean conductance of the small conductance subtype, G25, was 22 ± 1 pS; the intermediate conductance channel, G35, was 33 ± 1 pS; and the large conductance channel, G45, was 45 ± 1 pS. The small channel was not antagonized significantly by paraherquamide and was identified as the N‐subtype. The intermediate channel was preferentially activated by levamisole rather than bephenium and antagonized by paraherquamide: the intermediate channel was identified as the L‐subtype. The large conductance channel was preferentially activated by bephenium, antagonized more by 2‐desoxoparaherquamde than by paraherquamide and was identified as the B‐subtype. These observations reveal that the three channel subtypes have different selectivity for cholinergic anthelmintics. The different selectivity of these compounds should be considered when dealing with drug resistant infections.—Qian, H., Martin, R. J., and Robertson, A. P. Pharmacology of N‐, L‐, and B‐subtypes of nematode nAChR resolved at the single‐chain level in Ascaris Suum. FASEB J. 20, E2108–E2116 (2006)

Mode of action of levamisole and pyrantel, anthelmintic resistance, E153 and Q57

Here we review molecular information related to resistance to the cholinergic anthelmintics in nematodes. The amount of molecular information available varies between the nematode species, with the best understood so far being C. elegans. More information is becoming available for some other parasitic species. The cholinergic anthelmintics act on nematode nicotinic acetylcholine receptors located on somatic muscle cells. Recent findings demonstrate the presence of multiple types of the nicotinic receptors in several nematodes and the numerous genes required to form these multimeric proteins. Not only are the receptors the product of several genes but they are subject to modulation by several other proteins. Mutations altering these modulatory proteins could alter sensitivity to the cholinergic anthelmitics and thus lead to resistance. We also discuss the possibility that resistance to the cholinergic anthelmintics is not necessarily the result of a single mutation but may well be polygenic in nature. Additionally, the mutations resulting in resistance may vary between different species or between resistant isolates of the same species. A list of candidate genes to examine for SNPs is presented.

Levamisole receptors: a second awakening

Levamisole and pyrantel are old (1965) but useful anthelmintics that selectively activate nematode acetylcholine ion channel receptors; they are used to treat roundworm infections in humans and animals. Interest in their actions has surged, giving rise to new knowledge and technical advances, including an ability to reconstitute receptors that reveal more details of modes of action/resistance. We now know that the receptors are plastic and may form diverse species-dependent subtypes of receptor with different sensitivities to individual cholinergic anthelmintics. Understanding the biology of the levamisole receptors is expected to inform other studies on anthelmintics (ivermectin and emodepside) that act on ion channels.

Levamisole resistance resolved at the single-channel level in Caenorhabditis elegans

Sydney Brenner promoted Caenorhabditis elegans as a model organism, and subsequent investigations pursued resistance to the nicotinic anthelmintic drug levamisole in C. elegans at a genetic level. These studies have advanced our understanding of genes associated with neuromuscular transmission and resistance to the antinematodal drug. In lev‐8 and lev‐1 mutant C. elegans, levamisole resistance is associated with reductions in levamisole‐activated whole muscle cell currents. Although lev‐8 and lev‐1 are known to code for nicotinic acetylcholine receptor (nAChR) subunits, an explanation for why these currents get smaller is not available. In wild‐type adults, nAChRs aggregate at neuromuscular junctions and are not accessible for single‐channel recording. Here we describe a use of LEV‐10 knockouts, in which aggregation is lost, to make in situ recordings of nAChR channel currents. Our observations provide an explanation for levamisole resistance produced by LEV‐8 and LEV‐1 mutants at the single‐channel level.—Qian, H., Robertson, A. P., Powell‐Coffman, J. A., and Martin, R. J. Levamisole resistance resolved at the single‐channel level in Caenorhabditis elegans. FASEB J. 22, 3247–3254 (2008)

Drug resistance and neurotransmitter receptors of nematodes : recent studies on the mode of action of levamisole

Here we review recent studies on the mode of action of the cholinergic anthelmintics (levamisole, pyrantel etc.). We also include material from studies on the free living nematode Caenorhabditis elegans. The initial notion that these drugs act on a single receptor population, while attractive, has proven to be an oversimplification. In both free living and parasitic nematodes there are multiple types of nicotinic acetylcholine receptor (nAChR) on the somatic musculature. Each type has different (sometimes subtly so) pharmacological properties. The implications of these findings are: (1) combinations of anthelmintic that preferentially activate a broad range of nAChR types would be predicted to be more effective; (2) in resistant isolates of parasite where a subtype has been lost, other cholinergic anthelmintics may remain effective. Not only are there multiple types of nAChR, but relatively recent research has shown these receptors can be modulated; it is possible to increase the response of a parasite to a fixed concentration of drug by altering the receptor properties (e.g. phosphorylation state). These findings offer a potential means of increasing efficacy of existing compounds as an alternative to the costly and time consuming development of new anthelmintic agents.

Methyridine (2-[2-methoxyethyl]-pyridine]) and levamisole activate different ACh receptor subtypes in nematode parasites: a new lead for levamisole-resistance

The development of resistance to all chemotherapeutic agents increases and needs to be addressed. We are interested in resistance in parasitic nematodes to the anthelmintic levamisole. During studies on methyridine, we found that it gave us a new insight into pharmacological changes associated with levamisole resistance. Initially, electrophysiological investigation using a two‐micropipette current‐clamp recording technique revealed that methyridine acts as a cholinergic agonist on nematode muscle receptors (Ascaris suum). Methyridine (>30 μM) produced reversible concentration‐dependent depolarizations and increases in input conductance. Mecamylamine (30 μM) and paraherquamide (0.3 μM) produced reversible antagonism of the depolarization and conductance responses to methyridine. These observations suggest that methyridine, like acetylcholine and levamisole, gates ion channels on the muscle of parasitic nematodes. The antagonistic effects of dihydro‐β‐erythroidine and paraherquamide on methyridine‐induced contractions of A. suum muscle flaps were then examined to determine if methyridine showed subtype selectivity for N‐subtype (nicotine‐sensitive) or L‐subtype (levamisole‐sensitive) acetylcholine receptors. Dihydro‐β‐erythroidine weakly antagonized the effects of methyridine (but had no effect on levamisole responses). The antagonism of methyridine (pA2, 5.9) and nicotine (pA2, 6.1) by paraherquamide was similar, but was less than the antagonism of levamisole (pA2, 7.0). The antagonist profiles suggested that methyridine has a selective action on the N‐subtype rather than on the L‐subtype. A novel use for a larval inhibition migration assay was made using L3 larvae of Oesophagostomum dentatum. Inhibitory effects of nicotine, levamisole, pyrantel and methyridine on the migration of larvae of levamisole‐sensitive (SENS) and levamisole‐resistant (LEV‐R) isolates were tested at different concentrations. Levamisole and pyrantel (putative L‐subtype‐selective agonists) concentration–response plots were displaced to the right in LEV‐R isolates. Nicotine (an N‐subtype‐selective agonist) and methyridine produced little shift in concentration–response plots in the LEV‐R isolates. Resistance dose ratios were used to calculate the relative selectivity, ρL, for the L‐type receptor (levamisole ρL=1.0; pyrantel ρL=0.93; methyridine ρL=0.17; nicotine ρL=0.06). These observations reveal an N‐subtype‐selective action of methyridine and suggest that levamisole resistance may be associated with a loss of the L‐subtype, but not the N‐subtype receptors. The pharmacology of methyridine suggests an approach for the treatment of levamisole‐resistant parasites.

Investigation of acetylcholine receptor diversity in a nematode parasite leads to characterization of tribendimidine- and derquantel-sensitive nAChRs.

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important “classical” anthelmintics like levamisole and pyrantel, as well as “novel” anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.

Ion-channels on parasite muscle: pharmacology and physiology.

Ion-channels are essential components of excitable cells. This fact has been exploited in the development of anthelmintic agents; the majority of which act on nematode ion channels. The purpose of this review is to describe the site of action of some frequently used anthelmintic compounds: nAChRs and levamisole/pyrantel; Glu-Cls and avermectins/mylbemycins; GABA receptors and piperazine. Also described is some of the physiological and pharmacological data on other nematode muscle ion-channels which may prove attractive targets for future anthelmintic development: Ca2+ activated Cl− channels; peptide gated chloride Cl− channels; Ca2+ channels and potassium channels. Emphasis is placed on the pharmacological and physiological data from parasite tissue. Information on the genes involved in ion-channel formation and modulation are reviewed in detail elsewhere in this issue.