Ann. Parasitol. Hum. Comp., Key-words: Antimonials. Ascaris. Ascaris suum. ATP. Caeno- 1990, 65, Suppl. I : 99-102. rhabditis elegans. Calcium. Cholinergic receptors. Closantel. DNA. Fasciola hepatica. Fumarate reductase. GABA. Hycanthone. Hyme- nolepis diminuta. Ivermectin. Levamisole. Mebendazole. Micro­ tubule. Mitochondria. Nitrobenzylthioinosine. Oxamniquine. Oxi­ dative phosphorylation. Permease. Phosphofructokinase. Praziquantel. Protofilament. Rafoxanide. Schistosoma. Tubercidin. Tubulin. Mots-clés : Antimonies. Ascaris. ATP. Caenorhabditis elegans. Calcium. Récepteurs cholinergiques. Closantel. ADN. Fasciola hepa­ tica. Fumarate réductase. GABA. Hycanthone. Hymenolepis dimi­ nuta. Ivermectine. Levamisole. Mebendazole. Microtubule. Mito­ chondrie. Nitrobenzylthioinosine. Oxamniquine. Phosphorylation oxydative. Perméase. Phosphofructokinase. Praziquantel. Proto­ filament. Rafoxamide. Schistosoma. Tubercidine. Tubuline. STUDIES OF THE MODE OF ACTION OF ANTHELMINTIC DRUGS: TOOLS TO INVESTIGATE THE BIOCHEMICAL PECULARITIES OF HELMINTHS H. VANDEN BOSSCHE Summary -------------------------------------------------------------------------------_ _ An overview is given of the results from mode of action studies zine, levamisole, ivermectine, salicylanilides, praziquantel, benzi- which improved our knowledge of some biochemical and/or elec- midazolecarbamates, oxamniquine and hycanthome offered tools trophysiological aspects of parasitic helminths. Studies of the mole­ to learn more of the parasites and their hosts. cular mechanism of action of for example, antimonials, pipera- R ésumé : Les études sur les mécanismes d’action des anthelmintiques : instruments pour l’analyse des particularités des helminthes. Un certain nombre d’acquisitions anciennes et nouvelles dans dérivés de l’antimoine, la pipérazine, le lévamisole, l’ivermectine, la connaissance de la biochimie et de T électro-physiologie des hel­ les salicylanilides, le praziquantel, les bezimidazole-carbamates, minthes sont passées en revue à partir des données obtenues lors l’oxamniquine et l’hycanthone, pour l’analyse des particularités des études sur les mécanismes d’action. Cette revue sommaire a des helminthes. permis de souligner l’importance des anthelmintiques tels que les Mode of action studies might tell us how a chemical are of great help in the study of the distribution and role compound interferes with a target in the parasite and of sterols in fungal and protozoan membranes (Bolard, changes the host-parasite interplay. These studies also might 1986; Vanden Bossche, 1990). Azole antifungals triggered highlight differences between the organisms and thus the studies on fungal cytochrome P450 and contributed to improve our knowledge of the biochemical systems in host the identification of new opportunities for prostate and and parasite. Already in 1878, Claude Bernard considered breast cancer therapy (Vanden Bossche et al., 1990). pharmacologically active compounds as « instruments well What do we know on the mode of action of anthelmin­ suited for dissecting one by one the properties of the ele­ tics? Do these studies also provide guidance for future ments of the living organisms ». research? The battery of anti-microbial agents that became avai­ lable opened the way to a better understanding of binding sites and active targets (Gale et al., 1981). Antifungal agents Interference with carbohydrate metabolism such as the polyenes and imidazole and triazole derivatives Investigations using trivalent antimonials, the oldest group of compounds used in antischistosomal therapy, have been of great help in elucidating schistosomal glycolysis. Already Department of Comparative Biochemistry, Janssen Research in the early fifties Ernest Bueding and Tag Mansour showed Foundation, Turnhoutseweg, 30, B 2340 Beerse, Belgium. that the activity of schistosomal phosphofructokinase (PFK) 99 Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/1990651099 H. VANDEN BOSSCHE is inhibited by low concentrations of antimonials (Bueding, It is of interest that piperazine does not act on rat sympa­ 1972). Of great interest is the fact that the reduced glycolytic thetic neurons. rate, resulting from inhibition of PFK, could be reversed A more recently developed nematocide, ivermectin, also by the addition of purified mammalian phosphofructoki- paralyzes nematodes by activating membrane chloride con­ nase. These studies proved that PFK is a key regulatory ductance in neurons of the nerve cord (see Rew and Fet­ enzyme in glycolysis, that the mammalian enzyme is much terer, 1986). This might originate from stimulation of the less sensitive (70-80 times) to the antimonials and that schis­ presynaptic release of GABA. The selectivity might result tosomal PFK might be an excellent target for the develop­ from the inability of ivermectin to reach its target in the ment of new antischistosomal drugs. Flowever, none of host central nerves system. This highlights the importance the currently used modern anthelmintics has PFK as target. of pharmacokinetic studies in both parasite and host. Although levamisole inhibits the fumarate reductase in Levamisole causes a spastic contraction of nematode nematodes at relatively high concentrations only, the results muscle by acting as a potent agonist at acetylcholine recep­ obtained pinpoint the fumarate reductase complex as an­ tors on muscle bag membranes of Ascaris suum. Lewis other possible target for anthelmintics. et al. (1980) showed that levamisole-resistant strains of Cae- Salicylanilides such as, closantel and rafoxanide, are norhabditis elegans lack normal cholinergic receptors. The potent fasciolicides. Their activity has been linked to their latter studies not only suggest that nematodal acetylcho­ capacity to uncouple mitochondrial electron-transport asso­ line receptors are targets for levamisole but also focus atten­ ciated phosphorylation. For example, 12 hours after i.m. tion on the small free-living nematode C. elegans. This treatment of the sheep host with 5 mg closantel/kg body nematode and levamisole have contributed to genetic stu­ weight the ATP content of Fasciola hepatica was decreased dies of the nervous system in nematodes (Lewis et al., 1980). by more than 60 % and the adenylate energy charge was 0.53 instead of the 0.84 found in liver flukes from control Interaction with lipid membranes sheep (Vanden Bossche, 1985a). No effect on the oxida­ tive phosphorylation was found in mitochondria isolated Praziquantel has broad spectrum activity against cestodes from livers of uninfected rats and from hearts of rats, and many trematodes. This pyrazinoisoquinoline deriva­ infected and uninfected, 4 and 16 hours after i.m. injec­ tive also induces next to a rapid muscle contraction, vacuo­ tion with 5 mg closantel per kg (Vanden Bossche et al., lization of the tegument, followed by a pronounced struc­ 1980). Studies with closantel offered examples of possibili­ tural disruption of the parasite’s tegument (for a review ties to learn more about Fasciola hepatica and Schisto­ see Vanden Bossche, 1985a). The molecular mechanism soma mansoni. Indeed, studies to measure the effects of underlaying both the tegumental alterations and muscle closantel on liver mitochondria from rats infected with Fas­ contraction are not well understood. However, from the ciola revealed that the mitochondria from untreated rats mode of action studies we learned that muscle contraction were uncoupled (Vanden Bossche et al., 1983). This uncou­ in Hymenolepis diminuta muscle depends on endogenous pling might be induced by a product(s) excreted by the Ca2+, whereas muscle contraction in schistosomes is liver fluke (Vanden Bossche, 1985b). Treatment of the rats dependent on the influx of external Ca2+. The combined with closantel resulted in a normalization of the mitochon­ use of a theoretical approach and experimental procedures drial activity. These Fasciola-induced alterations of liver (such as IR spectroscopy) (Schepers et al., 1988) established mitochondria might be involved in the pathology of fas- the lipid destabilizing capacity of praziquantel. This capa­ cioliasis. Studies on the effects of closantel on S. mansoni city has been explained in terms of the high praziquantel were suggestive of a role for aerobic metabolism in the interaction and the large area occupied per drug molecule generation of energy required by S. mansoni for motility in the lipid layer. It should be mentioned that praziquantel (Vanden Bossche, 1985b). These findings indicate that does not modify the lipid structure but act as a spacer S. mansoni might derive a considerable part of their energy between lipid molecules. This opens new perspectives in from mitochondrial oxidative phorphorylation instead of the search for anthelmintics. However, it might be diffi­ from glycolysis only. cult to find compounds that insert selectively into parasite membranes. Effects on neuromuscular systems As already mentioned, one of the early morphological results of praziquantel treatment is disruption of the inte­ Piperazine, one of the first effective anthelmintics, ini­ grity of the tegument. The membrane over tubercles on tiated important studies on the neuromuscular system of the S. mansoni male dorsal surface is damaged and an Ascaris (for a review see Martin, 1987). It has been shown increase in parasite-specific antigenicity is observed after that piperazine acts as an agonist at the extrasynaptic GABA in vitro treatment (Harnet and Kusel, 1986). Studies