Anthelmintic resistance in human and animal pathogenic helminths has been spreading in prevalence and severity to a point where multidrug resistance against the three major classes of anthelmintics--the benzimidazoles, imidazothiazoles and macrocyclic lactones--has become a global phenomenon in gastrointestinal nematodes of farm animals. Hence, there is an urgent need for an anthelmintic with a new mode of action. Here we report the discovery of the amino-acetonitrile derivatives (AADs) as a new chemical class of synthetic anthelmintics and describe the development of drug candidates that are efficacious against various species of livestock-pathogenic nematodes. These drug candidates seem to have a novel mode of action involving a unique, nematode-specific clade of acetylcholine receptor subunits. The AADs are well tolerated and of low toxicity to mammals, and overcome existing resistances to the currently available anthelmintics.

Graphical abstract

Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5′ UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs.

Monepantel is a member of the recently identified class of anthelmintics known as the amino-acetonitrile derivatives (AADs). Monepantel controls all major gastro-intestinal nematodes in sheep including those that are resistant to the classical anthelmintics. Previous studies have shown that the Caenorhabditis elegans acr-23 and the Haemonchus contortus Hco-mptl-1 genes may be prominent targets of monepantel. With this discovery it became possible to investigate the mode of action of monepantel in nematodes at the molecular level. In the present study, we show that a C. elegans mutant acr-23 strain is fully rescued by expressing the wild-type acr-23 gene. Moreover, we present a new mutant allele, and characterize acr-23 alleles genetically. We also show that acr-23 is expressed in body wall muscle cells, and provide therefore a possible explanation for the paralysis caused by monepantel. Furthermore, genetic evidence suggests that the chaperone RIC-3 is required for expression of full monepantel resistance. Finally, we present reconstitution of the C. elegans ACR-23 receptor in Xenopus laevis oocytes and provide direct evidence of its modulation by monepantel. Conversely, co-injection of the chaperone RIC-3 had no impact for channel reconstitution in X. laevis oocytes. These results reinforce the involvement of the ACR-23 family in the mode of action of monepantel and advance our understanding of this new class of anthelmintics.

The recently launched veterinary anthelmintic drench for sheep (Novartis Animal Health Inc., Switzerland) containing the nematocide monepantel represents a new class of anthelmintics: the amino-acetonitrile derivatives (AADs), much needed in view of widespread resistance to the classical drugs. Recently, it was shown that the ACR-23 protein in Caenorhabditis elegans and a homologous protein, MPTL-1 in Haemonchus contortus, are potential targets for AAD action. Both proteins belong to the DEG-3 subfamily of acetylcholine receptors, which are thought to be nematode-specific, and different from those targeted by the imidazothiazoles (e.g. levamisole). Here we provide further evidence that Cel-ACR-23 and Hco-MPTL-1-like subunits are involved in the monepantel-sensitive phenotype. We performed comparative genomics of ligand-gated ion channel genes from several nematodes and subsequently assessed their sensitivity to anthelmintics. The nematode species in the Caenorhabditis genus, equipped with ACR-23/MPTL-1-like receptor subunits, are sensitive to monepantel (EC50<1.25 µM), whereas the related nematodes Pristionchus pacificus and Strongyloides ratti, which lack an ACR-23/MPTL-1 homolog, are insensitive (EC50>43 µM). Genome sequence information has long been used to identify putative targets for therapeutic intervention. We show how comparative genomics can be applied to predict drug sensitivity when molecular targets of a compound are known or suspected.

Monepantel is the first drug of a new family of anthelmintics, the amino acetonitrile derivatives (AAD), presently used to treat ruminants infected with gastrointestinal nematodes such as Haemonchus contortus. Monepantel shows an excellent tolerability in mammals and is active against multidrug-resistant parasites, indicating that its molecular target is absent or inaccessible in the host and is different from those of the classic anthelmintics. Genetic approaches with mutant nematodes have suggested acetylcholine receptors of the DEG-3 subfamily as the targets of AADs, an enigmatic clade of ligand-gated ion channels that is specific to nematodes and does not occur in mammals. Here we demonstrate direct interaction of monepantel, its major active metabolite monepantel sulfone, and other AADs with potential targets of the DEG-3 subfamily of acetylcholine receptors. H. contortus DEG-3/DES-2 receptors were functionally expressed in Xenopus laevis oocytes and were found to be preferentially activated by choline, to permeate monovalent cations, and to a smaller extent, calcium ions. Although monepantel and monepantel sulfone did not activate the channels by themselves, they substantially enhanced the late currents after activation of the channels with choline, indicating that these AADs are type II positive allosteric modulators of H. contortus DEG-3/DES-2 channels. It is noteworthy that the R-enantiomer of monepantel, which is inactive as an anthelmintic, inhibited the late currents after stimulation of H. contortus DEG-3/DES-2 receptors with choline. In summary, we present the first direct evidence for interaction of AADs with DEG-3-type acetylcholine receptors and discuss these findings in the context of anthelmintic action of AADs.

No abstract

BackgroundThe isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls). Lotilaner (Credelio™), a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties.MethodsIn this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the successful gene identification, cDNA cloning and functional expression in Xenopus oocytes of Drosohpila melanogaster (wild type and dieldrin/fipronil-resistant forms), Lepeophtheirus salmonis (an ectoparasite copepod crustacean of salmon), Rhipicephalus microplus and Canis lupus familiaris GABACls. Automated Xenopus oocyte two-electrode voltage clamp electrophysiology was used to assess GABACls functionality and to compare ion channel inhibition by lotilaner with that of established insecticides addressing GABACls as targets.ResultsIn these assays, we demonstrated that lotilaner is a potent non-competitive antagonist of insects (fly) GABACls. No cross-resistance with dieldrin or fipronil resistance mutations was detected, suggesting that lotilaner might bind to a site at least partly different from the one bound by known GABACl blockers. Using co-application experiments, we observed that lotilaner antagonism differs significantly from the classical open channel blocker fipronil. We finally confirmed for the first time that isoxazoline compounds are not only powerful antagonists of GABACls of acari (ticks) but also of crustaceans (sea lice), while no activity on a dog GABAA receptor was observed up to a concentration of 10 μM.ConclusionsTogether, these results demonstrate that lotilaner is a non-competitive antagonist specific to invertebrate’s γ-aminobutyric acid-gated chloride channels (GABACls). They contribute to our understanding of the mode of action of this new ectoparasiticide compound.Electronic supplementary materialThe online version of this article (10.1186/s13071-017-2470-4) contains supplementary material, which is available to authorized users.