Parasitic nematodes express a large number of distinct nicotinic acetylcholine receptors and these in turn are the targets of many classes of anthelmintic drug. This complexity poses many challenges to the field, including sorting the exact subunit composition of each of the receptor subtypes and how much they vary between species. It is clear that the model organism Caenorhabditis elegans does not recapitulate the complexity of nicotinic pharmacology of many parasite species and data using this system may be misleading when applied to them. The number of different receptors may allow nematodes some plasticity which they can exploit to evolve resistance to a specific cholinergic drug; however, this may mean that combinations of cholinergic agents may be effective at sustainably controlling them. Resistance may involve the expression of truncated receptor subunits that affect the expression levels of the receptors via mechanisms that remain to be deciphered.

Introduction

The majority of anthelmintic drugs in current use act on the parasite nervous system, especially ion channels [1], which make excellent drug targets for these organisms as inhibiting, or more commonly, activating them has rapid and often fatal effects. They also usually result in paralysis of the worm, a phenotype which can be readily monitored and lends itself to at least some degree of automation for screening purposes in drug discovery and development [26]. Even if the paralysis itself is not directly fatal, in the case of gastrointestinal nematodes it is likely to result in the parasites being quickly removed from their predilection site via normal peristalsis. Several different ion channels have been exploited in the development of anthelmintic drugs, including the ligand-gated chloride channels gated by glutamate and GABA, targets of the avermectin/milbemycin class [7] as well as older compounds such as piperazine [8,9], and voltage-gated potassium channels, the target of octadepsipeptides, such as emodepside [1012]. However, the greatest number of compounds and drug classes act at nicotinic acetylcholine receptors (nAChRs), especially those found at the neuromuscular junction (Table 1) [1315], though it is important to remember that these receptors are also expressed at extra-synaptic sites and in tissues other than muscle, and the receptors expressed there may make an important contribution to the overall anthelmintic activity of the cholinergic drugs. Targeting the neuromuscular receptor makes a lot of sense in terms of the rapid and easily observable effects mentioned above as uncontrolled activation or inhibition of muscle contraction will obviously have a rapidly deleterious and easily observed effect on the target organism, and the rich and varied pharmacology of the nAChR offers a variety of effective targets, both orthosteric and allosteric. These targets also differ between parasite nAChRs and those of their vertebrate hosts, allowing specific and safe anthelmintic effects. Table 1 summarizes some of the quantitative pharmacological data available in the literature to illustrate this diversity, though the differences in EC50 values do not always mirror the relative potency of the compounds in vitro or in vivo.

Table 1
Anthelmintic drugs acting at nicotinic receptors
Drug Class Action EC50/IC50 
Levamisole Imidazothiazole Agonist C. elegans Lev-R (oocyte) 10.1 ± 1.8 [23
H. contortus Lev-R1 (oocyte) 6.08 ± 0.3 [27
H. contortus Lev-R2 (oocyte) 48.4 ± 0.9 [27]1 
O. dentatum 29/63/8 (oocyte) 2.2 ± 0.2 [32
O. dentatum 29/38/63/8 (oocyte) 3.1 ± 2.2 [32
A. suum 29/38 (5:1, oocyte) 3.39 [36
A. suum 38/29 (5:1 oocyte) 4.17 [36]1 
A. suum muscle strip 6.76 [56
B. malayi muscle 3.4 ± 0.6 [35
P. equorum ACR-26/27 (oocyte) 16.7 ± 1.3 [42]1 
Pyrantel Tetrahydropyrimidine Agonist O. dentatum (29/63) (oocyte) 0.09 ± 0.005 [32
O. dentatum 29/38/63 (oocyte) 0.4 ± 0.1 [32
A. suum 29/38 (5:1 oocyte) 0.25 [32
A. suum muscle strip 0.02 [56
B. malayi muscle 0.064 ± 0.002 [35
H. contortus ACR-26/27 (oocyte) 6.8 ± 1.3 [42]1 
P. equorum ACR-26/27 (oocyte) 0.98 ± 0.26 [42]1 
Oxantel Tetrahydropyrimidine Agonist A. suum 38/29 (5:1, oocyte) 2.45 [32
A. suum muscle strip 0.015 [56
Morantel Tetrahydropyrimidine Agonist H. contortus ACR-26/27 (oocyte) 29.0 ± 1.3 [42
P. equorum ACR-26/27 (oocyte) 0.32 ± 0.26 [42
B. malayi muscle 0.10 [35
A. suum ACR-16 IC50 = 5.6 ± 1.8 [40
Monepantel (Zolvix) Amino-acetonitrile derivative Agonist C. elegans 0.19 ± 0.05 [47
Tribendimidine Aminophenylamidine Agonist O. dentatum (29/63) (oocyte) 3.9 ± 0.8 [32
O. dentatum 29/38/63 (oocyte) 2.2 ± 0.5 [32
O. dentatum 29/63/8 (oocyte) 0.8 ± 0.1 [32
O. dentatum 29/38/63/8 (oocyte) 0.3 ± 0.6 [32
A. suum (muscle) 0.83 [33
Abamectin Avermectin Allosteric modulator/antagonist A. suum (pharynx) 0.42, CI 0.13–1.36 [53
2-desoxyparaherquamide (Derquantel) Spiroindole Antagonist A. suum (somatic muscle) IC50 0.22, CI 0.18–0.28 [53
Drug Class Action EC50/IC50 
Levamisole Imidazothiazole Agonist C. elegans Lev-R (oocyte) 10.1 ± 1.8 [23
H. contortus Lev-R1 (oocyte) 6.08 ± 0.3 [27
H. contortus Lev-R2 (oocyte) 48.4 ± 0.9 [27]1 
O. dentatum 29/63/8 (oocyte) 2.2 ± 0.2 [32
O. dentatum 29/38/63/8 (oocyte) 3.1 ± 2.2 [32
A. suum 29/38 (5:1, oocyte) 3.39 [36
A. suum 38/29 (5:1 oocyte) 4.17 [36]1 
A. suum muscle strip 6.76 [56
B. malayi muscle 3.4 ± 0.6 [35
P. equorum ACR-26/27 (oocyte) 16.7 ± 1.3 [42]1 
Pyrantel Tetrahydropyrimidine Agonist O. dentatum (29/63) (oocyte) 0.09 ± 0.005 [32
O. dentatum 29/38/63 (oocyte) 0.4 ± 0.1 [32
A. suum 29/38 (5:1 oocyte) 0.25 [32
A. suum muscle strip 0.02 [56
B. malayi muscle 0.064 ± 0.002 [35
H. contortus ACR-26/27 (oocyte) 6.8 ± 1.3 [42]1 
P. equorum ACR-26/27 (oocyte) 0.98 ± 0.26 [42]1 
Oxantel Tetrahydropyrimidine Agonist A. suum 38/29 (5:1, oocyte) 2.45 [32
A. suum muscle strip 0.015 [56
Morantel Tetrahydropyrimidine Agonist H. contortus ACR-26/27 (oocyte) 29.0 ± 1.3 [42
P. equorum ACR-26/27 (oocyte) 0.32 ± 0.26 [42
B. malayi muscle 0.10 [35
A. suum ACR-16 IC50 = 5.6 ± 1.8 [40
Monepantel (Zolvix) Amino-acetonitrile derivative Agonist C. elegans 0.19 ± 0.05 [47
Tribendimidine Aminophenylamidine Agonist O. dentatum (29/63) (oocyte) 3.9 ± 0.8 [32
O. dentatum 29/38/63 (oocyte) 2.2 ± 0.5 [32
O. dentatum 29/63/8 (oocyte) 0.8 ± 0.1 [32
O. dentatum 29/38/63/8 (oocyte) 0.3 ± 0.6 [32
A. suum (muscle) 0.83 [33
Abamectin Avermectin Allosteric modulator/antagonist A. suum (pharynx) 0.42, CI 0.13–1.36 [53
2-desoxyparaherquamide (Derquantel) Spiroindole Antagonist A. suum (somatic muscle) IC50 0.22, CI 0.18–0.28 [53

All values are in µM. ‘oocyte’ indicates that combinations of cRNAs encoding the subunits indicated were expressed in the Xenopus oocyte system. ‘8’ = ACR-8, ‘29’ = UNC-29, ‘38’ = UNC-38 and ‘63’ = UNC-63.

*

Partial agonist.

In nematodes, the pharmacological complexity of the nAChR is mirrored at the genetic level. nAChRs are genetically complex in most animals, as they are pentameric proteins which may be either homomeric or heteromeric and they function in the peripheral and central nervous systems and play other physiological roles, for example in the mammalian immune system [1618]. It has long been known that Caenorhabditis elegans, the ‘model’ nematode, encodes a very large number, 24–25, of nAChR subunits [19]. It has become clear that C. elegans' parasitic cousins in the phylum Nematoda also encode a large number of such subunits, many of which are conserved between the species, but several of which are not. A major current challenge for the field, and the one which this short article attempts to address, is identifying the subunit composition of the multiple receptors that are present in parasitic nematodes, and the drugs that might preferentially interact with those different receptors. This complexity also allows the parasites several ways in which to alter their receptor makeup in response to drug treatments and become resistant to them.

Levamisole receptors

Not surprisingly given the powerful genetic tractability of C. elegans, some of the first studies were carried out on the free-living species, and levamisole, possibly the most studied and widely used of the cholinergic anthelmintics, was used to isolate strains with a high degree of drug resistance following chemical mutagenesis [20,21]. These strains included those with mutations in nAChR-encoding genes and led to the eventual identification of a levamisole-sensitive receptor (Lev-R, Figure 1) expressed at the neuromuscular junction [22]. Importantly, the robust reconstitution of a receptor with very similar properties could be achieved in Xenopus oocytes by expression of five subunits, UNC-38, UNC-29, UNC-63, LEV-1 and LEV-8, plus three chaperones, RIC-3, UNC-50 and UNC-74 [23]. This receptor is also partially activated by pyrantel, but not by nicotine. As genomic sequence information became available for parasitic nematodes, it rapidly became apparent that some of these subunit genes, such as unc-38 and unc-63 are widely conserved, whereas lev-8 is not [24]. In one group of parasitic nematodes, the trichostrongylus, there are multiple copies of unc-29 and the sequence of lev-1, while conserved, does not appear to encode a signal peptide [25]. Reconstitution of a Lev-R from a parasitic nematode, Haemonchus contortus, can be accomplished via expression of Hco-UNC-38, Hco-UNC-29 (three out of the four subtypes are functional), Hco-UNC-63 and Hco-ACR-8 (nomenclature as in Beech et al. [26]) plus the H. contortus orthologs of the three chaperones, RIC-3, UNC-50 and UNC-74 [25,27]. In C. elegans, ACR-8 is not thought to normally form part of the Lev-R, though single-channel and macroscopic current recordings on mutant strains suggest a functional redundancy between this subunit and LEV-8 [28]. For H. contortus, omission of Hco-ACR-8 from the recombinant Lev-R produced a second receptor subtype with reduced activation by levamisole, but increased sensitivity to pyrantel and nicotine [27]. This result, and the association of a truncated form of Hco-ACR-8 with levamisole resistance in some isolates of H. contortus [24,29,30], might indicate that a combination of two nicotinic agonists might be more effective at not selecting for resistance than a single compound. The data from ACR-8 illustrate that differences in the subunit composition and pharmacology of nAChR between C. elegans and parasites make it difficult to infer the specific targets of anthelmintics based on studies on the model nematode [14] (Figure 1). This is exemplified by tribendimidine, another nicotinic agonist, which seems, based on genetic studies, to activate the Lev-R of C. elegans [31], but in electrophysiological experiments to be more effective at activating other receptor subtypes in Oesophagostomum dentatum and Ascaris suum [32,33].

Lev-R, Nic-R, Aad-R and Mor-R acetylcholine receptor subtypes identified in nematodes.

Figure 1.
Lev-R, Nic-R, Aad-R and Mor-R acetylcholine receptor subtypes identified in nematodes.

Representation of the putative subunit arrangement of the distinct subtypes of nematode acetylcholine receptors expressed in the Xenopus oocyte. The most potent agonist (highlighted by a blue arrow) or antagonist (highlighted by a red arrow) are indicated below each receptor subtype. When different agonists can activate a single receptor subtype, they are indicated from the most potent to the less potent. Beph, bephenium; Der, derquantel; Lev, levamisole; Mon, monepantel; Mor, morantel; Nic, nicotine; Oxa, oxantel; Pyr, pyrantel; Tbd, tribendimidine.

Figure 1.
Lev-R, Nic-R, Aad-R and Mor-R acetylcholine receptor subtypes identified in nematodes.

Representation of the putative subunit arrangement of the distinct subtypes of nematode acetylcholine receptors expressed in the Xenopus oocyte. The most potent agonist (highlighted by a blue arrow) or antagonist (highlighted by a red arrow) are indicated below each receptor subtype. When different agonists can activate a single receptor subtype, they are indicated from the most potent to the less potent. Beph, bephenium; Der, derquantel; Lev, levamisole; Mon, monepantel; Mor, morantel; Nic, nicotine; Oxa, oxantel; Pyr, pyrantel; Tbd, tribendimidine.

Electrophysiological recordings from muscle cells of parasitic nematodes have revealed a diversity of nAChR, including multiple channel subtypes sensitive to levamisole, though here the species studied have been some of the larger parasites. Patch-clamp recordings from A. suum muscle cells revealed three different types of Lev-R, designated N (nicotine), L (levamisole) and B (bephenium), based on the most efficient agonist at inducing the relevant channel conductance [34], and subsequent experiments on muscles from O. dentatum and Brugia malayi have confirmed the existence of four nAChR subtypes on muscle cells of these parasites [32,35]. To date, there have been fewer molecular studies on these species though pharmacologically distinct nAChR result from expression of differing ratios of Asu-UNC-29 and Asu-UNC-38, implying that these two subunits were able to form receptors with differing stoichiometry [36]. Expression of four O. dentatum subunits, Ode-ACR-8, Ode-UNC-29, Ode-UNC-38 and Ode-UNC-63 allowed the formation of four pharmacologically distinct receptors, all sensitive to levamisole, the conductance of one of which matched that of one of the channels identified from muscle recordings [32].

Nicotine, morantel and monepantel receptors

Although nicotine is not an agonist at the C. elegans Lev-R, there is a second nAChR expressed at the C. elegans neuromuscular junction that is nicotine sensitive — Nic-R (Figure 1) [22]. This receptor is believed to be homomeric, made up solely of ACR-16 subunits, and is widely conserved in parasitic species [3739]. The A. suum Nic-R receptor has also been studied and is blocked by morantel [40] in addition to its activation by nicotine [38]. Though this receptor is not the target of any current anthelmintics, historically nicotine was used to control parasitic species, indicating that it may be possible to develop agents that act at it. A third neuromuscular nAChR is found in a variety of animal parasites, but is not present in C. elegans, and is made up of ACR-26 and ACR-27 subunits [41,42]. This receptor, Mor-R (Figure 1), is specifically activated by morantel and to a lesser extent by pyrantel but not by oxantel, another tetrahydropyrimidine (Table 1). The H. contortus Mor-R is insensitive to levamisole though the drug is a partial agonist at the Parascaris equorum Mor-R [42]. The existence of this additional neuromuscular nAChR may help to explain how parasites can acquire levamisole resistance by reducing expression of the target receptors yet maintain sufficient motility to complete their life cycles without a dramatic loss of fitness. Such reductions in expression may be mediated by truncated forms of the subunits; in addition to the truncated Hco-ACR-8 mentioned above, truncations in Hco-UNC-63 have also been implicated in mediating levamisole resistance in H. contortus [24]. Such truncated subunits may or may not be incorporated into the mature receptor, perhaps depending on the exact nature of the truncation, but in either case it is likely that the pharmacology of the resultant nAChR will be altered, with the truncated subunits potentially conferring a dominant-negative phenotype on the Lev-R [24,27].

The diversity of nematode nAChR was further exploited in the development of monepantel, an amino-acetonitrile derivative (Table 1) which activates members of the nematode-specific DEG-3 family of homomeric nAChR, including ACR-20 and the betaine receptor ACR-23 in C. elegans [43,44]. In C. elegans, ACR-23 is expressed in four types of mechanosensory neurons (ALM, PLM, AVM and PVM), multiple interneurons and body muscles. The H. contortus monepantel-sensitive receptor (Aad-R) is encoded by Hco-mptl-1, also a member of the DEG-3 family of receptors, and resistance to the drug is associated with mutations in this gene [43,45] and in acr-23 in C. elegans [46]. The molecular target of monepantel, if any, in other species has not been defined and orthologs of Hco-mptl-1 are not obvious in the genomes of other parasitic nematodes [47].

Derquantel and abamectin

Most of the cholinergic anthelmintics are agonists, but one antagonist, 2-desoxyparaherquamide (derquantel), is commercialized in combination with abamectin (an avermectin). Derquantel is a fairly specific antagonist of the bephenium-sensitive Lev-R subtype of Ascaris [48] and acts as both a competitive and non-competitive antagonist of different recombinant Lev-R from O. dentatum [32]. It is a potent inhibitor of the Mor-R from B. malayi [35]. The role of abamectin in the combination is fascinating because avermectins are normally considered to act via glutamate-gated chloride channels [7], some of which, such as those of H. contortus, activate at nanomolar concentrations [49] though other species may be less sensitive [50]. However, ivermectin is also a positive allosteric modulator of some mammalian nAChR [51] and abamectin is a negative allosteric modulator or antagonist at nematode receptors [52,53], confirming the promiscuity of this drug class [54]. Abamectin (0.03 µM) and derquantel both reduce the effect of acetylcholine on A. suum muscle strips; if abamectin largely affects a pyrantel-sensitive receptor and Derquantel a morantel-sensitive one [53], this might explain the greater than additive effects of the combination and provides strong evidence that combining cholinergic anthelmintics is a viable approach to maintaining worm control. However, that concentration of abamectin might be expected to have significant effects on neuronal GluCls, which would not be apparent in muscle preparations.

Current challenges

Considerable progress has been made in unraveling the complexities of cholinergic signaling at the nematode neuromuscular junction, yet many questions remain. One important question is the role of the other nAChR subunits, not found at the neuromuscular junctions from present in neurons and other cell types. This has been barely addressed in parasites, though some data are available from C. elegans [55]. Even for the receptors discussed in the present study, some important questions have yet to be answered.

  • What is the subunit composition of the native receptors found at the parasitic nematode neuromuscular junction?

  • How much can parasites vary the expression of their various receptors in response to drug pressure? Are such variations only genetically caused, or are epigenetic or cellular mechanisms also involved?

  • Does the multiplicity of distinct nAChRs present at the neuromuscular junction imply that combinations of cholinergic drugs make an effective and sustainable worm control strategy?

  • Does the cholinergic activity of the avermectins contribute to their anthelmintic activity in the field, and do all members of this class have similar effects? Are there species-specific differences in the importance of nicotinic effects to their anthelmintic activity?

  • What is the role of truncated receptor subunits in mediating resistance?

Abbreviations

     
  • Lev-R

    levamisole-sensitive receptor

  •  
  • nAChRs

    nicotinic acetylcholine receptors

Funding

Research on ion channels in the Wolstenholme laboratory is supported by an award from the National Institutes of Health [R21AI125899].

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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