Anthelmintic Targets Research Articles

Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi.

Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi) is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA) and double stranded RNA (dsRNA) into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1) is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed.

A glycine residue essential for high ivermectin sensitivity in Cys-loop ion channel receptors

Ivermectin exerts its anthelmintic effect by activating nematode Cys-loop glutamate-gated receptors. Here we show that a glycine residue at a specific transmembrane domain location is essential for high ivermectin sensitivity in both glycine- and glutamate-gated Cys-loop receptors. We also show that ivermectin sensitivity can be conferred on an ivermectin-insensitive receptor by introducing a glycine at this position. Furthermore, comparison of amino acid sequences of ivermectin-sensitive and -resistant receptors reveals that the presence of a glycine reliably predicts ivermectin sensitivity. By providing a means of identifying ivermectin-sensitive receptors, this finding should help in characterising ivermectin-resistance mechanisms and identifying new anthelmintic targets.

Modulation of aSchistosoma mansonimultidrug transporter by the antischistosomal drug praziquantel

P-glycoprotein (Pgp) is an ATP-dependent efflux pump involved in transport of xenobiotics from cells that, when overexpressed, can mediate multidrug resistance in mammalian cells. Pgp may be a candidate target for new anthelmintics, as it plays critical roles in normal cell physiology, in removal of drugs from cells, and potentially in the development of drug resistance. Schistosomes are parasitic flatworms that cause schistosomiasis, which affects hundreds of millions of people worldwide. Here, we express SMDR2, a Pgp homologue from Schistosoma mansoni (Platyhelminthes), in Chinese hamster ovary (CHO) cells and use fluorescence-based assays to examine the functional and pharmacological properties of this transporter. Membrane vesicles from stably transfected CHO cells expressing recombinant SMDR2 show significant increases in rhodamine transport and ATP hydrolysis compared with those from control cells or cells transfected with empty vector. SMDR2-mediated transport is inhibited by the Pgp modulators verapamil (IC(50)=12.1 muM) and nifedipine, and also by praziquantel, the current drug of choice against schisotosomiasis (IC(50)=17.4 muM). Efflux measurements of a fluorescent analog of praziquantel indicate that it is also a substrate for SMDR2. The interaction of praziquantel with SMDR2 may offer new strategies for potentiating the action of praziquantel and possibly overcoming drug resistance.

Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30 h and adult worms for 4–6 h. Ammonia was the main excretory product, with about 20% urea. For the first 4–6 h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30 h. An initial external ammonia concentration of 600 μM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.

Advances in the sequencing of the genome of the adenophorean nematodeTrichinella spiralis

The adenophorean nematodes are evolutionarily distant from other species in the phylum Nematoda. Interspecific comparisons of predicted proteins have supported such an ancient divergence. Accordingly, Trichinella spiralis represents a basal nematode representative for genome sequencing focused on gaining a deeper insight into the evolutionary biology of nematodes. In addition, molecular characteristics that are conserved across the phylum could be of great value for control strategies with broad application. In this review, we describe and summarize progress that has been made on the sequencing and analysis of the T. spiralis genome. The genome sequence was used in preliminary analyses for the investigation of specific questions relating to the biology of T. spiralis and, more generally, to parasitic nematodes. For instance, we evaluated an unusually large DNase II-like protein family, predicted proteins of prospective interest in the parasite-host muscle cell interaction, anthelmintic targets and prospective intestinal genes, the encoded proteins (potentially) linked to immunological control against other nematodes. The results are discussed in relation to characteristics that are broadly conserved among evolutionary distant nematodes. The results lead to expectations that this genome sequence will contribute to advances in research on T. spiralis and other parasitic nematodes.

A novel member of the ligand-gated chloride channel gene family from Haemonchus contortus

Ligand-gated chloride channels (LGCCs) are key components of the nervous system of parasitic nematodes and important targets for anthelmintics. Here, we describe the isolation and characterization of a novel member of the LGCC gene family (HcLGCC1) from the parasitic nematode Haemonchus contortus. Sequence analysis revealed that the channel subunit encoded by HcLGCC1 is anion selective and a member of a group of channels characterized as having two Cys-loops in the N-terminal ligand-binding domain. Although the overall function of HcLGCC1 is presently unknown, the gene may play a key role in the early developmental stages of the parasite. Further investigations into the function of LGCCs, such as HcLGCC1, in parasitic nematodes should have implications for the discovery of new anthelmintic targets.

SLO, SLO, quick, quick, slow: calcium-activated potassium channels as regulators of Caenorhabditis elegans behaviour and targets for anthelmintics

Large-conductance calcium and voltage-activated potassium channels, termed SLO-1 (or BK), are pivotal players in the regulation of cell excitability across the animal phyla. Furthermore, emerging evidence indicates that these channels are key mediators of a number of neuroactive drugs, including the most recent new anthelmintic, the cyclo-octadepsipeptide emodepside. Detailed reviews of the structure, function and pharmacology of BK channels have recently been provided (Salkoff et al. in Nat Rev Neurosci 7:921-931, 2006; Ghatta et al. in Pharmacol Ther 110:103-116, 2006) and therefore these aspects will only briefly be covered here. The purpose of this review is to discuss how SLO-1 channels might function as regulators of neural transmission and network activity. In particular, we focus on the role of SLO-1 in the regulation of Caenorhabditis elegans behaviour and highlight the role of this channel as an effector for pleiotropic actions of neuroactive drugs, including emodepside. On the premise that C. elegans is a 'model nematode' with respect to many aspects of neural function, the intention is that this might inform a broader understanding of the role of these channels in the nematodes and their potential as novel anthelmintic targets.

The cys-loop ligand-gated ion channel gene family of Brugia malayi and Trichinella spiralis: a comparison with Caenorhabditis elegans

Nematode cys-loop ligand gated ion channels (CLGIC) mediate neurotransmission and are important targets for anthelmintics in parasitic nematodes. The CLGIC superfamily in nematodes includes ion channels gated by acetylcholine, gamma-amino butyric acid (GABA), glutamate, glycine and 5-HT. The macrocyclic lactones and the nicotinic agonists are important groups of anthelmintics that target the glutamate gated chloride channels and the nicotinic acetylcholine receptors, respectively. The model organism Caenorhabditis elegans has the most diverse families of cys-loop LGIC known in any organism. Many parasitic nematodes have homologues of C. elegans receptors but to date no genome wide investigations have been done. The genome sequencing projects of Brugia malayi (clade III) and Trichinella spiralis (clade I) have allowed us to characterise the CLGIC families in these species. Although the main groups of CLGICs targeted by anthelmintics are represented in both the nematode genomes investigated here, the CLGIC family is much smaller in B. malayi and T. spiralis, suggesting that care must be taken when using C. elegans as a model organism for distantly related nematodes.

Relationships between sterol/phospholipid composition and xenobiotic transport in nematodes

Therapeutic failure limits prophylaxis of nematode diseases and has been mainly attributed to mutations in cellular targets of anthelmintics. Besides these specific mechanisms, alterations of drug transport also occur in parasites resistant to anthelmintics and depend on both the presence of membrane pumps such as P-glycoprotein (Pgp) and on the lipid composition of membranes. We recently showed in the nematode Haemonchus contortus, using eggs as a model, that the total cholesterol (TC) concentration alters the transport of lipophilic molecules due to membrane pumps such as P-glycoprotein and the resistance to anthelmintics. The effect of TC may depend on the presence of other lipids interacting with TC. Therefore, we analysed the lipid composition and its relationship with Pgp and resistance to anthelmintics. Better correlations were found between Pgp and free cholesterol (FC) than with TC. We also showed that the relationships between lipid composition and resistance to anthelmintics or Pgp depended on the equilibrium between FC and phospholipids (PLs), mainly PLs known to be present primarily in either the external leaflets of cell membranes or the internal leaflets. The PLs phosphatidylcholine and phosphatidylethanolamine played the most significant role, but phosphatidic acid also influenced drug resistance.

526 POSTER A prostate-Specific Antigen (PSA) activated channel-forming toxin as therapy for prostatic disease

Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 μM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.

525 POSTER Targeting Doxorubicin to LHRH-receptor positive tumors by the cytotoxic hybrid ZEN-008 (AN-152)

Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 μM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.

The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.

Neuropeptide signalling systems in flatworms

Two distinct families of neuropeptides are known to endow platyhelminth nervous systems - the FMRFamide-like peptides (FLPs) and the neuropeptide Fs (NPFs). Flatworm FLPs are structurally simple, each 4-6 amino acids in length with a carboxy terminal aromatic-hydrophobic-Arg-Phe-amide motif. Thus far, four distinct flatworm FLPs have been characterized, with only one of these from a parasite. They have a widespread distribution within the central and peripheral nervous system of every flatworm examined, including neurones serving the attachment organs, the somatic musculature and the reproductive system. The only physiological role that has been identified for flatworm FLPs is myoexcitation. Flatworm NPFs are believed to be invertebrate homologues of the vertebrate neuropeptide Y (NPY) family of peptides. Flatworm NPFs are 36-39 amino acids in length and are characterized by a caboxy terminal GRPRFamide signature and conserved tyrosine residues at positions 10 and 17 from the carboxy terminal. Like FLPs, NPF occurs throughout flatworm nervous systems, although less is known about its biological role. While there is some evidence for a myoexcitatory action in cestodes and flukes, more compelling physiological data indicate that flatworm NPF inhibits cAMP levels in a manner that is characteristic of NPY action in vertebrates. The widespread expression of these neuropeptides in flatworm parasites highlights the potential of these signalling systems to yield new targets for novel anthelmintics. Although platyhelminth FLP and NPF receptors await identification, other molecules that play pivotal roles in neuropeptide signalling have been uncovered. These enzymes, involved in the biosynthesis and processing of flatworm neuropeptides, have recently been described and offer other distinct and attractive targets for therapeutic interference.

Caenorhabditis elegans ivermectin receptors regulate locomotor behaviour and are functional orthologues of Haemonchus contortus receptors

The target site for the anthelmintic action of ivermectin is a family of nematode glutamate-gated chloride channel alpha subunits (GluClα) that bind the drug with high affinity and mediate its potent paralytic action. Whilst the action of ivermectin on the pharyngeal muscle of nematodes is relatively well understood, its effect on locomotor activity is less clear. Here we use RNAi and gene knockouts to show that four GluClα subunits are involved in regulating the pattern of locomotor activity in Caenorhabditis elegans. A Haemonchus contortus orthologue of these subunits, HcGluClα3, has been shown to be expressed in the motor nervous system and here we have shown that it is a functional, as well as a structural, orthologue by virtue of the observation that it can restore normal motor movement in the C. elegans GluClα mutant, avr-14( ad1032), when expressed under the control of the avr-14 promoter. This supports the contention that ivermectin exerts its paralytic action on parasitic nematodes through activation of GluCl channels in the motor nervous system. Furthermore, functional complementation in C. elegans provides a method to further the understanding of this important class of anthelmintic targets.

A High-Throughput Assay for Modulators of Ligand-Gated Chloride Channels

Invertebrate glutamate-gated chloride channels (GluCls) are important targets for anthelmintics and insecticides such as ivermectin. To facilitate screening for novel GluCl modulators, the Caenorhabditis elegans GluCl alpha2beta channel was chosen as a surrogate for parasite channels not yet cloned, and an inducible stable human embryonic kidney cell line was generated. Functional expression of the alpha2 and beta subunits was confirmed by whole-cell voltage clamp assays. Using this cell line, a high-throughput assay was developed that detects membrane potential changes associated with the activation of GluCls. In this assay, membrane depolarization was quantified via changes in fluorescence resonance energy transfer between two membrane-associated dyes. Robust and reproducible signals were detected in response to addition of glutamate or ivermectin. This assay was used for the screening of over 180,000 samples from natural and synthetic sources.

Screening for microtubule-disrupting antifungal agents by using a mitotic-arrest mutant of Aspergillus nidulans and novel action of phenylalanine derivatives accompanying tubulin loss.

The microtubule, which is one of the major targets of anthelmintics, anticancer drugs, and fungicides, is composed mainly of alpha- and beta-tubulins. We focused on a unique characteristic of an Aspergillus nidulans benA33 mutant to screen for microtubule-disrupting antifungal agents. This mutant, which has a beta-tubulin with a mutation of a single amino acid, undergoes mitotic arrest due to the formation of hyperstable microtubules at 37 degrees C. The heat sensitivity of the mutant is remedied by some antimicrotubule agents. We found that an agar plate assay with the mutant was able to distinguish three types of microtubule inhibitors. The growth recovery zones of the mutant were formed around paper disks containing microtubule inhibitors, including four benzimidazoles, ansamitocin P-3, griseofulvin, and rhizoxin, on the agar plate at 37 degrees C. Nocodazole, thiabendazole, and griseofulvin reversed the mitotic arrest of the mutant and promoted its hyphal growth. Ansamitocin P-3 and rhizoxin showed growth recovery zones around the growth-inhibitory zones. Benomyl and carbendazim also reversed mitotic arrest but produced weaker growth recovery than the aforementioned drugs. Other microtubule inhibitors, such as colchicine, Colcemid, paclitaxel, podophyllotoxin, TN-16, vinblastine, and vincristine, as well as some cytoskeletal inhibitors tested, did not show such activity. In our screening, we newly identified two mycotoxins, citrinin and patulin, two sesquiterpene dialdehydes, polygodial and warburganal, and four phenylalanine derivatives, arphamenine A, L-2,5-dihydrophenylalanine (DHPA), N-tosyl-L-phenylalanine chloromethylketone, and N-carbobenzoxy-L-phenylalanine chloromethyl ketone. In a wild-type strain of A. nidulans, DHPA caused selective losses of microtubules, as determined by fluorescence microscopy, and of both alpha- and beta-tubulins, as determined by Western blot analysis. This screening method involving the benA33 mutant of A. nidulans is useful, convenient, and highly selective. The phenylalanine derivatives tested are of a novel type of microtubule-disrupting antifungal agents, producing an accompanying loss of tubulins, and are different from well-known tubulin inhibitors affecting the assembly of tubulin dimers into microtubules.

Tubulin as an antiprotozoal drug target.

Since tubulin is a known anticancer and anthelmintic drug target, the investigation of protozoal tubulin could lead to the development of new antiparasitic drugs. This review outlines the current state of knowledge concerning drug-mammalian tubulin interactions, the effects of antimicrotubule agents on parasites and parasite tubulin, and our current hypotheses regarding the development of selective ligands for protozoal tubulin as antiparasitic drug candidates.

Characterisation of a β-tubulin gene from the liver fluke, Fasciolahepatica

This study represents the first β-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one β-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. β-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke β-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.

Molecular cloning and in vitro expression of C. elegans and parasitic nematode ionotropic receptors.

The free living nematode, C. elegans is understood at a level of detail equalled by few other organisms, and much of the cell biology and sequence information is proving of considerable utility in the study of parasitic nematodes. Already, C. elegans provides a convenient vehicle for investigating anthelmintic drug action and resistance mechanisms. Among the ionotropic receptors, with their important roles in the behaviour and development of the organism, are targets for anthelmintics. The subunits of nicotinic acetylcholine receptors of C. elegans form a large and diverse multigene family. Members of this family are among the 11 genes associated with resistance to the anthelmintic drug levamisole.

The γ-aminobutyric acid receptor of Ascaris as a target for anthelmintics

The <i>γ</i>-aminobutyric acid receptor of <i>Ascaris</i> as a target for anthelmintics