Where are all the anthelmintics? Challenges and opportunities on the path to new anthelmintics.

Control of helminth parasites is a key challenge for human and veterinary medicine. In the absence of effective vaccines and adequate sanitation, prophylaxis and treatment commonly rely upon anthelmintics. There are concerns about the development of drug resistance, side-effects, lack of efficacy and cost-effectiveness that drive the need for new classes of anthelmintics. Despite this need, only three new drug classes have reached the animal market since 2000 and no new classes of anthelmintic have been approved for human use. So where are all the anthelmintics? What are the barriers to anthelmintic discovery, and what emerging opportunities can be used to address this? This was a discussion group focus at the 2019 8th Consortium for Anthelmintic Resistance and Susceptibility (CARS) in Wisconsin, USA. Here we report the findings of the group in the broader context of the human and veterinary anthelmintic discovery pipeline, highlighting challenges unique to antiparasitic drug discovery. We comment on why the development of novel anthelmintics has been so rare. Further, we discuss potential opportunities for drug development moving into the 21st Century.

Jietacins, azoxy antibiotics with potent nematocidal activity: Design, synthesis, and biological evaluation against parasitic nematodes.

Jietacins, an azoxy antibiotic class of chemicals, were isolated from the culture broth of Streptomyces sp. KP-197. They have a unique structural motif, including a vinyl azoxy group and a long acyclic aliphatic chain, which is usually branched but non-branched in the case of jietacin C. During a drug discovery program, we found that jietacins display potent anthelmintic activity against parasitic nematodes and that jietacin A has a moderate or low acute toxicity (LD50 > 300 mg/kg) and no mutagenic potential in a mini Ames screen. This suggests that jietacins have potential for drug discovery research. In order to create a novel anthelmintic agent, we performed design, synthesis, and biological evaluation of jietacin derivatives against parasitic nematodes. Of these derivatives, we found that a fully synthesized simplified derivative exhibited better anthelmintic activity against three parasitic nematodes than natural jietacins. In addition, it had a better efficacy in vivo through oral administration against a mouse nematode. This indicated that the azoxy motif could prove useful as a template for anthelmintic discovery, possibly creating a class of anthelmintic with novel skeletons, a potential new mode of action, and providing further insight for rational drug design.

Anthelmintic cyclcooctadepsipeptides: complex in structure and mode of action.

The broad-spectrum anthelmintic cyclooctadepsipeptide PF1022A is a fungal metabolite from Rosellinia sp. PF1022, which is a Mycelia sterilia found on the leaves of Camellia japonica. A broad range of structurally related cyclooctadepsipeptides has been characterized and tested for anthelmintic activities. These metabolites have been used as starting points to generate semisynthetic derivatives with varying nematocidal capacity. Predominant among these compounds is emodepside, which exhibits a broad nematocidal potential against gastrointestinal and extraintestinal parasites. Here we review the chemical biology and mode of action of cyclooctadepsides with particular attention to PF1022A and emodepside. We illustrate how they target nematode neuromuscular function, opening up new avenues for antiparasitic treatments with potential capability for important selective toxicity.

Worms take to the slo lane: a perspective on the mode of action of emodepside.

The cyclo-octapdepsipeptide anthelmintic emodepside exerts a profound paralysis on parasitic and free-living nematodes. The neuromuscular junction is a significant determinant of this effect. Pharmacological and electrophysiological analyses in the parasitic nematode Ascaris suum have resolved that emodepside elicits a hyperpolarisation of body wall muscle, which is dependent on extracellular calcium and the efflux of potassium ions. The molecular basis for emodepside's action has been investigated in forward genetic screens in the free-living nematode Caenorhabditis elegans. Two screens for emodepside resistance, totalling 20,000 genomes, identified several mutants of slo-1, which encodes a calcium-activated potassium channel homologous to mammalian BK channels. Slo-1 null mutants are more than 1000-fold less sensitive to emodepside than wild-type C. elegans and tissue-specific expression studies show emodepside acts on SLO-1 in neurons regulating feeding and motility as well as acting on SLO-1 in body wall muscle. These genetic data, combined with physiological measurements in C. elegans and the earlier physiological analyses on A. suum, define a pivotal role for SLO-1 in the mode of action of emodepside. Additional signalling pathways have emerged as determinants of emodepside's mode of action through biochemical and hypothesis-driven approaches. Mutant analyses of these pathways suggest a modulatory role for each of them in emodepside's mode of action; however, they impart much more modest changes in the sensitivity to emodepside than mutations in slo-1. Taken together these studies identify SLO-1 as the major determinant of emodepside's anthelmintic activity. Structural information on the BK channels has advanced significantly in the last 2 years. Therefore, we rationalise this possibility by suggesting a model that speculates on the nature of the emodepside pharmacophore within the calcium-activated potassium channels.

In vivo efficacy of the anthelmintic tribendimidine against the cestode Hymenolepis microstoma in a controlled laboratory trial.

Tribendimidine has been registered for the treatment of human soil transmitted helminthiases in China. In the model nematode Caenorhabditis elegans it is an agonist of L-subtype nicotinic acetylcholine receptors and therefore shares its mode of action with levamisole and pyrantel. Besides its broad spectrum of nematicidal efficacy, tribendimidine is efficacious against several trematodes and has been attributed to have anti-cestodal effects. However, there are few published data available for the latter. The efficacy of tribendimidine and its nematicidal metabolite deacylated amidantel against Hymenolepis microstoma were examined for their anti-cestodal potential. Doses of 50 and 100mg/kg body weight deacylated amidantel and 10, 25, 50, and 100mg/kg tribendimidine were administered orally on three consecutive days to mice experimentally infected with eight cysticercoids. Necropsy was performed and the worm burdens were determined one day after the last treatment. Furthermore, levamisole was used in combination with tribendimidine (100mg/kg levamisole plus 10 and 25mg/kg tribendimidine, respectively) and alone (50 and 100mg/kg) to investigate any possible interactions of the partner compounds against cestodes. Tribendimidine showed a very high efficacy at dosages of 50mg/kg or higher. Surprisingly, deacylated amidantel led to no reduction of the worm burden in any of the treatments. Combinations of levamisole with tribendimidine did not augment the effects of tribendimidine alone and as expected levamisole alone also showed no anti-cestodal activity. To our knowledge, this study shows for the first time activity of tribendimidine against a cestode in a controlled laboratory study. Due to the excellent cure rates observed here, multiple tribendimidine treatments might be considered as useful scheme for treatments of cestode, nematode and trematode infections although this would significantly increase both costs and management efforts. Moreover, the differences between tribendimidine and deacylated amidantel indicate at least a strong difference in sensitivity of H. microstoma or a strong difference in drug availability.

Strongyloides papillosus: changes in transcript levels of lysozyme and aspartic protease 2 in percutaneously migrated larvae.

The infection of the host is the crucial event in the life-cycle of parasites. To understand the molecular mechanisms of this important step, different methods are used in present studies. For analysis of changes in transcript levels the most sensitive method is the quantitative real-time PCR (qPCR). For an accurate analysis the evaluation of a set of adequate reference genes is necessary. The present study aimed to analyse the transcriptional levels of two genes of interest, the putative aspartic protease Spa-asp-2 and the putative lysozyme Spa-lys, in infective, free-living larvae of Strongyloides papillosus at different ages and from long-term and short-term infections and percutaneously migrated ("parasitic") larvae. Percutaneously migrated larvae were collected using the PERL chamber system and ovine skin in vitro. Reference genes identified as most suitable for transcriptional analysis according to geNorm analysis were genes for the eukaryotic translation elongation factor 1 alpha (Spa-eft-2), actin variation 2 (Spa-act-v2) and beta tubulin (Spa-tbb-1). Transcriptional analysis of the genes in percutaneously migrated larvae showed an upregulation of Spa-asp-2, while Spa-lys was downregulated. Data from the presented study provide a first glance into the changes of transcript levels of S. papillosus induced by percutaneous migration.

SLO-1-channels of parasitic nematodes reconstitute locomotor behaviour and emodepside sensitivity in Caenorhabditis elegans slo-1 loss of function mutants.

The calcium-gated potassium channel SLO-1 in Caenorhabditis elegans was recently identified as key component for action of emodepside, a new anthelmintic drug with broad spectrum activity. In this study we identified orthologues of slo-1 in Ancylostoma caninum, Cooperia oncophora, and Haemonchus contortus, all important parasitic nematodes in veterinary medicine. Furthermore, functional analyses of these slo-1 orthologues were performed using heterologous expression in C. elegans. We expressed A. caninum and C. oncophora slo-1 in the emodepside-resistant genetic background of the slo-1 loss-of-function mutant NM1968 slo-1(js379). Transformants expressing A. caninum slo-1 from C. elegans slo-1 promoter were highly susceptible (compared to the fully emodepside-resistant slo-1(js379)) and showed no significant difference in their emodepside susceptibility compared to wild-type C. elegans (p = 0.831). Therefore, the SLO-1 channels of A. caninum and C. elegans appear to be completely functionally interchangeable in terms of emodepside sensitivity. Furthermore, we tested the ability of the 5' flanking regions of A. caninum and C. oncophora slo-1 to drive expression of SLO-1 in C. elegans and confirmed functionality of the putative promoters in this heterologous system. For all transgenic lines tested, expression of either native C. elegans slo-1 or the parasite-derived orthologue rescued emodepside sensitivity in slo-1(js379) and the locomotor phenotype of increased reversal frequency confirming the reconstitution of SLO-1 function in the locomotor circuits. A potent mammalian SLO-1 channel inhibitor, penitrem A, showed emodepside antagonising effects in A. caninum and C. elegans. The study combined the investigation of new anthelmintic targets from parasitic nematodes and experimental use of the respective target genes in C. elegans, therefore closing the gap between research approaches using model nematodes and those using target organisms. Considering the still scarcely advanced techniques for genetic engineering of parasitic nematodes, the presented method provides an excellent opportunity for examining the pharmacofunction of anthelmintic targets derived from parasitic nematodes.

Selective toxicity of the anthelmintic emodepside revealed by heterologous expression of human KCNMA1 in Caenorhabditis elegans.

Emodepside is a resistance-breaking anthelmintic of a new chemical class, the cyclooctadepsipeptides. A major determinant of its anthelmintic effect is the calcium-activated potassium channel SLO-1. SLO-1 belongs to a family of channels that are highly conserved across the animal phyla and regulate neurosecretion, hormone release, muscle contraction, and neuronal network excitability. To investigate the selective toxicity of emodepside, we performed transgenic experiments in which the nematode SLO-1 channel was swapped for a mammalian ortholog, human KCNMA1. Expression of either the human channel or Caenorhabditis elegans slo-1 from the native slo-1 promoter in a C. elegans slo-1 functional null mutant rescued behavioral deficits that otherwise resulted from loss of slo-1 signaling. However, worms expressing the human channel were 10- to 100-fold less sensitive to emodepside than those expressing the nematode channel. Strains expressing the human KCNMA1 channel were preferentially sensitive to the mammalian channel agonists NS1619 and rottlerin. In the C. elegans pharyngeal nervous system, slo-1 is expressed in neurons, not muscle, and cell-specific rescue experiments have previously shown that emodepside inhibits serotonin-stimulated feeding by interfering with SLO-1 signaling in the nervous system. Here we show that ectopic overexpression of slo-1 in pharyngeal muscle confers sensitivity of the muscle to emodepside, consistent with a direct interaction of emodepside with the channel. Taken together, these data predict an emodepside-selective pharmacophore harbored by SLO-1. This has implications for the development of this drug/target interface for the treatment of helminth infections.

Larval migration in PERL chambers as an in vitro model for percutaneous infection stimulates feeding in the canine hookworm Ancylostoma caninum.

BACKGROUND: Ancylostoma caninum third-stage larvae are the non-feeding infective stage of this parasite and are able to infect potential hosts via different infection routes. Since percutaneous infection is one of the most important routes and skin penetration is the first step into parasitic life, an existing in vitro model for percutaneous migration was modified and evaluated. The main parameter used to evaluate migration was the migration ratio (migrated larvae as a percentage of total number of larvae recovered). Additionally, the skin lag was calculated, expressing the percentage of larvae remaining in the skin and therefore not being recovered. Since initiation of feeding is proposed to be an important step in the transition from free-living to parasitic A. caninum larvae, feeding assays were performed with in vitro percutaneously migrated larvae. Additionally, infective larvae of A. caninum were activated via serum-stimulation and feeding behaviour was analysed and compared between percutaneously migrated and serum-stimulated larvae. RESULTS: Maximum skin migration levels of infective larvae were observed at temperatures above 32°C when larvae were placed on the epidermal side of skin for more than 12 hours. The medium beneath the skin had no effect on migration ratio, and no significant difference between the migration ratios through fresh and frozen/thawed skin was observed. Maximum feeding levels of 93.2% were observed for percutaneously migrated larvae after 48 h incubation, whereas serum-stimulated larvae reached the maximum of 91.0% feeding larvae after 24 h. CONCLUSIONS: The PERL chamber system was optimised and standardised as an in vitro model for percutaneous migration. The larvae recovered after percutaneous migration showed characteristic signs of activation similar to that of serum-stimulated larvae. The observed difference in time course of resumption of feeding indicates that percutaneously migrated larvae are not identical to serum-stimulated larvae, which are currently representing the model for early parasitic stages.

Larval development of Toxocara canis in dogs.

The parasitic roundworm Toxocara canis is present in dog populations all over the world. Due to its zoonotic potential, this roundworm is of special interest not only for veterinarians, but also for medical practitioners. In the present review, current knowledge of infection routes and the subsequent development of larvae within the canine host is summarised. Furthermore, information about the clinical, pathological, enzymatic, haematological and histopathological changes was collected, giving a broad overview of current knowledge of the infection. Although the data collected over the years give an idea of what happens during the larval development of T. canis, many questions remain open. Nevertheless, it is important that we continue our efforts to further understand the biology of this versatile and compelling parasite and try to improve and optimise strategies to prevent the infection in dogs and thereby to protect humans from this infection.