Antiparasitic agents produced by microorganisms

Microorganisms produce many useful antiparasitic agents. Antiparasitic activities and biochemical targets of antiprotozoal and anthelmintic antibiotics are reviewed. Antimalarial apicidin, thiolactomycin, fosmidomycin, and borrelidin, antitrypanosomal ascofuranone, and nematocidal emodepside, 2-deoxoparaherquamide A, and nafuredin are recently discovered antibiotics, and they have potential as useful drugs.

suggesting that tetracyclines may be beneficial for treatment of humans infected with filarial nematodes. 8) A chloroplast-like organelle (apicoplast) is present in many species of the phylum Apicomplexa, containing Plasmodium, Toxoplasma, Eimeria, and Cryptosporidium. The organelle is essential for bloodstage development of Plasmodium and the formation of the parasitophorous vacuole of Toxoplasma. Tetracyclines are protein synthesis inhibitors that block binding of aminoacyl-tRNA to the A site of 16S rRNA on the 30S subunit of the prokaryotic ribosome. 9) Malarial parasites have two extrachromosomal DNAs with prokaryotic organelle-like characteristics, apicoplast and mitochondrial DNA. Tetracyclines are suggested to inhibit apicoplast and mitochondrial protein synthesis of Plasmodium. 10) Apicidin (3) is a cyclotetrapeptide produced by the fungus Fusarium pallidoroseum. It exhibits potent, broad-spectrum antiprotozoal activity in vitro against Apicomplexan parasites. It is orally and parenterally active against P. berghei malaria in mice. 11) Its antiparasitic activity appears to be due to low nanomolar inhibition of Apicomplexan histone deacetylase, which induces hyperacetylation of histones in treated parasites. Antimalarial activities have also been reported in other peptide antibiotics, such as cyclic peptide (takaokamycin), 12) cyclic depsipeptides (enniatins), 13) linear peptides (leucinostatin A 12) and efrapeptins 14) ), and peptaibols (antiamebin I and zervamicins). 14) A thiazole-containing cyclic depsipeptide, thiostrepton (4) (10) actinomycetes coccidioisis increase the influx of cations Spiramycin (13) S. ambofaciens cryptosporidiosis protein synthesis inhibitor Acetylspiramycin (14) semisynthetic toxoplasmosis protein synthesis inhibitor Clindamycin (15) semisynthetic toxoplasmosis & babesiosis protein synthesis inhibitor Paromomycin (16) S. rimosus cryptosporidiosis, amebiasis protein synthesis inhibitor & giardiasis Amphotericin B (17) S. nodosus leishmaniasis bind to membrane Trichomycin (19) S. hachijoensis trichomoniasis bind to membrane Fumagillin (21) Aspergillus fumigatus microsporidiasis Met aminopeptidase 2 inhibitor

Anthelmintics
Ivermectin (24) semisynthetic nematode infections Glu-gated Cl channel agonist Milbemycin D (29) S. hycroscopicus nematode infections Glu-gated Cl channel agonist Destomycin A (38) S. rimofaciens nematode infections protein synthesis inhibitor Hygromycin B (39) S. hygroscopicus nematode infections protein synthesis inhibitor Paromomycin (16) S. rimosus cestode infections protein synthesis inhibitor Plasmodium berghei in mice. It interacts with the GTPase binding domain of the apicoplast large subunit rRNA and inhibits protein synthesis, as it does in bacteria. 15) It has recently been demonstrated that type II fatty acid biosynthesis occurs in the apicoplast, and an inhibitor of this pathway, thiolactomycin (5), produced by the actinomycete Nocardia sp., restricts the growth of P. falciparum and Toxoplasma gondii. 16) The type II pathway is common to plant chloroplasts and bacteria but distinct from the type I pathway of mammals. Furthermore, Trypanosoma brucei, which belongs to the phylum Sarcomastigophora, also uses type II fatty acid biosynthesis for myristate synthesis, and is susceptible to thiolactomycin.
In some eubacteria, algae, and plants, the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway was described as an alternative nonmevalonate pathway for the early steps in the biosynthesis of isoprenoids. This pathway is present in Plasmodium, and its DOXP reductoisomerase is inhibited by fosmidomycin (6) produced by Streptomyces lavendulae. 17) Treatment with fosmidomycin for acute uncomplicated P. falciparum infection has been effective in clinical trials. 18) P. falciparum phospholipase C hydrolyzes sphin-gomyelin and lysocholine phospholipids, and is inhibited by a mammalian neutral sphingomyelinase inhibitor, scyphostatin (7), produced by the fungus Trichopeziza mollissima.

19)
It also inhibits the intraerythrocytic proliferation of P. falciparum.
Borrelidin (8) is produced by Streptomyces spp., 20) and it is a nonglycosidic 18-membered ring macrolide with a side chain of cyclopentanecarboxylic acid. It has various biological activities, such as antimicrobial, antiviral, herbicidal, insecticidal, and antitumor activities. The activities may be due to the inhibition of threonyl-tRNA synthetase and the activation of caspase-3 and caspase-8. 21) Recently, our group found that borrelidin shows excellent antimalarial activity against both chloroquine-sensitive and -resistant P. falciparum in vitro. 22) It also has antimalarial activity against chloroquine-sensitive and -resistant strains in mice. The effective dose is lower than that of artemether, artesunate, and chloroquine in each experiment.
In the same screening, we found that polyether antibiotics have a high selective inhibitory effect against P. falciparum in vitro. Among these polyethers, X-206 (9), produced by an actinomycete, 23) shows the highest selectivity and potency. 24) Although, it has potent in vivo antimalarial activity in mice, the therapeutic window is narrow compared with its selective toxicity in vitro.
(b) Anticoccidials, anticryptosporidials & antitoxoplasmals. Polyether antibiotics such as monensin (10, Fig. 3), 25) lasalocid A, and salinomycin are produced by actinomycetes and are used as feed additives for the prevention of poultry coccidiosis. They are ionophore antibiotics that increase the influx of monovalent cations through the membrane, causing swelling and death of the parasites. The development of resistance to anticoccidial agents is now posing similar problems to those encountered with malaria. 26) No active member from a new class of chemical substances had been developed for more than 10 years, so our group screened anticoccidial antibiotics using monensin-resistant Eimeria tenella in vitro. As a result, we isolated several new antibiotics, including xanthoquinodins, 27) diolmycins, 28) hynapenes, 29) arohynapenes, 30) cytosaminomycins, 31), 32) and fudecalone. 33) Xanthoquinodin A1 (11), produced by a fungus (Humicola sp.), showed anticoccidial activity against a monensin-resistant strain in vivo. Frenolicin B (12) was originally isolated as an antimycoplasmal antibiotic from Streptomyces roseofulvus by our group, 34) and later was revealed to show potent anticoccidial activity in vivo. 35) Spiramycin (13) is a 16-membered macrolide produced by Streptomyces ambofaciens and used as an antibacterial in the veterinary field. 36) It is also effective in treating patients with cryptosporidiosis. 37) Its acetylated derivative, acetylspiramycin (14), is used as an antibacterial antibiotic for humans, and also used to treat toxoplasmosis. 38) Clindamycin (15) is a semi-synthetic antibiotic prepared from lincomycin, which is produced by Streptomyces lincolnensis. 39) It is effective against toxoplasmosis and babesiosis. 6) Spiramycins and clindamycin inhibit protein synthesis by binding to 23S rRNA on the 50S subunit of the prokaryotic ribosome. They bind adjacent to the peptidyltransferase center of the 50S subunit, and their binding sites partially overlap. 9) They do not inhibit chromosomal or mitochondrial ribosomes. 40) Clindamycin-resistant mutants of Toxoplasma gondii have a point mutation in the apicoplast large-subunit rRNA, and the position of the mutation corresponds to the position in Escherichia coli predicted to bind clindamycin. The mutants show cross-resistance to macrolides. Therefore, spiramycins and clindamycin are believed to target the apicoplast ribosome. 41) Paromomycin (16), produced by Streptomyces rimosus, is a member of the aminoglycoside antibiotics. 42) It binds to 16S rRNA on the 30S subunit of the ribosome and inhibits protein synthesis by increasing the affinity of the A-site for tRNA, inducing errors in translation. 43) Paromomycin, an anthelmintic for cestode infection, is also used for cryptosporidiosis, amebiasis, and giardiasis. 44) Fig. 4), produced by Streptomyces nodosus, is a widely used 38-membered ring heptaene macrolide with antifungal properties. 45) Amphotericin B or its lipid formulations (liposomal amphotericin B) are also used for visceral leishmaniasis (kala-azar) and mucocutaneous leishmaniasis unresponsive to pentavalent antimony compounds. 6), 46) The cell membrane of Leishmania parasites are composed of ergosterol-related sterols similar to those of fungi. Therefore, amphotericin B can bind to the membrane and kill the parasites. Although the growth of Leishmania parasites is susceptible to azoles and other ergosterol biosynthesis inhibitors, they can survive with greatly altered sterol profiles induced by continuous treatment with low concentration of the inhibitors, and they also have some ability to utilize and metabolize host sterol. 16) Therefore, ergosterol biosynthesis inhibitors are not used in practice for leishmaniasis but are still in clinical studies.
Ascofuranone (18) was originally isolated from the culture broth of the fungus Ascochyta viciae as an antibiotic that reduced serum lipid levels of rats. 47) Recently, it was revealed to inhibit the trypanosome alternative oxidase, which is the terminal oxidase of the respiratory chain of long slender bloodstream forms of Trypanosoma brucei. 48) Ascofuranone shows good therapeutic effects for African trypanosomiasis in mice.
(d) Antitrichomonals. Trichomycin is used as an antifungal and antitrichomonal antibiotic. It is a mixture of polyene macrolides produced by Streptomyces hachijoensis. 49) The major component is a 38-membered ring heptaene macrolide, trichomycin A (19). 50) Hitachimycin (20) isolated by our group is a 19-membered ring lactam produced by Streptomyces sp. 51) It shows antitrichomonal and antitumor activities.
(e) Antimicrosporidial. Fumagillin (21) was isolated from the culture broth of the fungus Aspergillus fumigatus as an antiphage and amebicidal antibiotic. 52) It was used to control microsporidial disease due to Nosema apis in honeybees for more than 40 years. 44) Microsporidia are also responsible for opportunistic infections in patients with AIDS. Recently, fumagillin has been used topically to treat ocular microsporidiosis caused by Encephalitozoon hellem or E. intestinalis. 53) Antiangiogenic activity of fumagillin is also known, and its target was revealed as methionine aminopeptidase 2 (MetAP2). 54) Since microsporidia appear to lack MetAP1 and to carry only the gene for MetAP2, fumagillin should be particularly promising because the absence of MetAP1 would prevent microsporidia from compensating for the loss of MetAP2 activity when exposed to fumagillin. 55) Anthelmintics. Helminths include nematodes (roundworms), trematodes (flukes), and cestodes (tapeworms) as shown in Fig. 1. Nematodes have a cylindrical or filamentous shape and belong to the phylum Aschelminthes. Trematodes and cestodes have a flat shape and belong to the phylum Platyhelminthes. Trematodes have a digestive tract, but cestodes do not, and are composed of many segments. We describe some important and interesting anthelmintic antibiotics below.   61) It is also used against a number of arthropod parasites, including grubs, lice, mites, and screw worms. In companion animals, it provides highly effective control of Dirofilaria immitis (heartworm). Ivermectin is also used clinically for controlling some human nematode infections. Onchocerca volvulus causes human onchocerciasis (river blindness), an infection characterized by chronic skin and eye lesions. Ivermectin is highly efficacious against the microfilariae of O. volvulus and is used in programs dedicated to controlling onchocerciasis in the endemic areas of Africa and the Americas. 64) Recently, it has also become an invaluable tool for a control program against lymphatic filariasis (elephantiasis) caused by Wuchereria bancrofti and Brugia malayi. 65) Strongyloidiasis, distributed in the tropics and subtropics, is an intestinal disease caused by the nematode Strongyloides stercoralis. Ivermectin is currently the most useful agent against S. stercoralis. 66) In addition to the control of nematode infections, ivermectin shows efficacy for human scabies, a skin disease caused by infection with the ectoparasitic mite Sarcoptes scabiei.
Avermectin B 1 , under the non-proprietary name abamectin, is used as a gastrointestinal nematocide through subcutaneous injection for cattle and also as an agricultural miticide. 61) Doramectin (25) is a C-25 substituted avermectin B 1 produced by mutational biosynthesis. 67) Cyclohexane carboxylic acid was fed to a mutant strain of S. avermectinius lacking branchedchain 2-oxo acid dehydrogenase activity. Doramectin was developed for uses similar to those of ivermectin in the veterinary field. The other semisynthetic avermectins are commercially available as nematocides or insecticides. 68 Milbemycins are originally isolated from the culture broth of Streptomyces hycroscopicus. 69) Milbemycin D (29) is used as a nematocide in the veterinary field, and milbemectin, a mixture of milbemycins A 3 (30) and A 4 (31), is used as an agricultural acaricide. 68) Milbemycin oxime (32), a 5-oxoimino derivative of milbemectin, has been launched as a nematocide for dogs. Nemadectin (33) is a milbemycin group antibiotic produced by Streptomyces cyaneogriseus. 70) Moxidectin (34) is a 23methyloxime derivative of nemadectin, used for control of nematodes and arthropods in the veterinary field.
The major target of avermectins is a glutamategated chloride channel, which does not exist in vertebrates. 71) Avermectins activate the channel, causing neurological effects. Although avermectins also bind to GABA-and glycine-gated chloride channels in mammals, their affinity for invertebrate receptors is approximately 100-fold higher. Knock-out mice of the P-glycoprotein gene showed increased sensitivity to ivermectin. 72) This result established an important role for the P-glycoprotein in the maintenance of blood-brain barrier, and suggests that the safety of avermectins is due to the bloodbrain barrier, dependent on P-glycoprotein, in addition to the low affinity of the receptors.
The fungal toxins cochlioquinone A (35, Fig. 6) and ophiobolins were isolated as nematocidal compounds. Cochlioquinone A, produced by the fungi Cochliobolus miyabeanus and Helminthosporium sativum, showed competitive inhibition against [ 3 H]ivermectin binding to membranes prepared from the free-living nematode Caenorhabditis elegans. 73) On the other hand, ophiobolin M (36) and its analogs, produced by Bipolaris spp. fungi, are noncompetitive inhibitors of ivermectin binding. 74) It has been suggested that cochlioquinone A and ophiobolins may have a similar target to ivermectin. Recently, a potent insecticidal antibiotic, nodulisporic acid A (37), and its analogs were isolated from the culture broth of a endophytic fungus, Nodulisporium sp. 75) Nodulisporic acid A activates a glutamate-gated chloride channel like ivermectin, but lacks activity at GABA-and glycine-gated chloride channels. However, no useful anthelmintic activity was observed in vitro or in vivo. 76) (b) Aminoglycosides. Aminoglycoside antibiotics are protein synthesis inhibitors, and some of them are used as anthelmintics. Paromomycin (16) is used for treatment of human and animal cestode infections. 77) It is also useful for some protozoan infections as mentioned above. Destomycin A (38, Fig. 7) and hygromycin B (39) are produced by Streptomyces rimofaciens and S. hygroscopicus, respectively. 78),79) They are aminoglycosides used as feed additives for treatment of nematode infections by Ascaris, Oesophagostomum, Heterakis, Trichuris, and Capillaria in the veterinary field. 80) Antiparasitic agents produced by microorganisms No. 6] 251 Fig. 6. Inhibitors of ivermectin binding to its binding site.
Our group found that hygromycin A coproduced with 39 shows good therapeutic effect for swine dysentery caused by a spirochete Treponema hyodysenteriae. 81),82) (c) Emodepside. PF1022A (40) is a cyclic octadepsipeptide nematocide produced by a fungus, Rosellinia sp. 83)-85) Emodepside (BAY 44-4400, 41) is a semisynthetic derivative of PF1022A with a morpholine ring at each of the two D-phenyllactic acids in the para position. Its target was identified as a novel 110 kDa heptahelical transmembrane receptor, named HC-110R, which is similar to mammalian latrophilins. 86) Latrophilins are latrotoxin (black widow spider venom) receptors and G protein-coupled receptors implicated in the regulation of exocytosis. Latrotoxin also binds to HC110-R and causes influx of external Ca 2+ . Emodepside acts as an antagonist to latrotoxin signaling by impairing the influx of Ca 2+ . Emodepside exerts a high efficacy against a wide variety of nematodes in different animal species, and it possesses resistance-breaking properties against the usual classes of anthelmintics. 84) It will be commercially available as a veterinary nematocide soon.
(d) Paraherquamide group compounds. Brevianamides, paraherquamides, and marcfortines are a group of oxindole alkaloids produced by Penicillium spp. fungi. 87)-89) Their nematocidal activity was initially detected in a gerbil and Trichostrongylus colubriformis model using paraherquamide A (42). 90) A semisynthetic analog, 2-deoxoparaherquamide A (PNU-141962, 43), has an excellent nematocidal activity and safety profile, and is currently under development. 91) Although they are nematocidals, they do not have insecticidal activity. They are antagonists of nicotinic acetylcholine receptors and block cholinergic neuromuscular transmission. 92) There are differences between the sensitivity of nematodes and mammals to  paraherquamides due to intrinsic differences in receptor affinity between these phyla. (e) Nafuredin. Differences in energy metabolisms between the host and helminths are attractive targets for treatment of helminthiasis (Fig. 8). The NADHfumarate reductase system is part of a special respiratory system in parasitic helminths, which is found in many anaerobic organisms. The system is composed of complex I (NADH-rhodoquinone oxidoreductase) and complex II (rhodoquinol-fumarate reductase). Electrons from NADH are accepted by rhodoquinone through complex I, and then transferred to fumarate through complex II (Fig. 7). This anaerobic electron transport system can provide ATP in the absence of oxygen. We screened for inhibitors of NADH-fumarate reductase using Ascaris suum (roundworm) mitochondria, and obtained a novel antibiotic, nafuredin (44), produced by an Aspergillus niger strain isolated from a marine sponge. 93)- 95) Nafuredin inhibits NADH-fumarate reductase and NADH-rhodoquinone oxidoreductase of A. suum at nanomolar concentrations, while it shows only weak inhibition of rhodoquinol-fumarate reductase (Table II). Therefore, nafuredin is a complex I inhibitor. However, it inhibits rat liver complex I (NADH-ubiquinone oxidoreductase) at very high concentration. It is interesting that nafuredin inhibits not only anaerobic adult complex I of A. suum but also larval complex I, which has an aerobic energy metabolism like that of mammals. Thus, nafuredin is a selective inhibitor of helminth complex I.
Nafuredin shows anthelmintic activity against Haemonchus contortus (barberpole worm) in in vivo trials using sheep. This anthelmintic activity may be due to the inhibition of complex I because nafuredin also inhibits the enzyme of H. contortus (Table II). Nafuredin is a novel anthelmintic lead compound, and we have accomplished its total synthesis. 96) Recently, we found that nafuredin is converted to a novel γ-lactone derivative named nafuredin-γ (45) under mild basic conditions, and this derivative has the same enzyme inhibitory and anthelmintic activity. Since the lactone moiety synthesis of nafuredin-γ is simpler than that of nafuredin, nafuredin-γ is useful for the structure-activity relationship study. We have also achieved its total synthesis and are studying its analogs. 97) In the same screening, we rediscovered atpenin A5 (46) and related compounds as NADH-fumarate reductase inhibitors. Atpenins were originally isolated from the culture broth of Penicillium sp. as antifungal antibiotics by our group. 98) In contrast to nafuredin, atpenins inhibit complex II (succinate-ubiquinone oxidoreductase), and the inhibition is nonselective between helminths and mammals. 99) Although there are potent inhibitors of complexes I, III, and IV, no very potent inhibitors of complex II have been described. The IC 50 value of atpenin A5 is 300-fold lower than that for carboxin, the most potent known complex II inhibitor. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II. Recently, the X-ray crystallographic structure of Escherichia coli complex II was elucidated. 100) Eukaryotic helminth and mammalian complex II structures may be determined soon. This information may clarify the interaction of atpenins and complex II, and give a clue to design of helminth-specific atpenin analogs.
(f) Other anthelmintics. Avermectin and milbemycin group macrolides are used as nematocides as described above. Some other macrolides are also reported to have nematocidal activities. We showed that the 26-membered ring macrolides, oligomycins A and G, produced by Streptomyces sp. have nematocidal activity in vitro, but they are also cytotoxic. 101) Oligomycins are complex V (H + -transporting ATP synthase) inhibitors. A 14-membered ring macrodiolide, clonostachydiol, produced by the fungus Clonostachys Antiparasitic agents produced by microorganisms No. 6] 253 cylindrospora, and a 16-membered ring macrodiolide, pyrenophorol (helmidiol), produced by some fungi were active against Haemonchus contortus in vivo. 102), 103) Axenomycins are 34-membered ring macrolides with sugar and naphthoquinone moieties produced by Streptomyces lysandri. They show in vivo cestocidal activity. 104), 105) Jietacins A (47) and B (48), which were isolated from the culture broth of Streptomyces sp. by our group, have unique vinylazoxy moieties. 106) They were isolated based on screening using the pine wood nematode Bursaphelenchus lignicolus, 107) and they show ten times higher nematocidal activity than avermectin B 1a against B. lignicolus in vitro.
Antiparasitic antibiotics with a variety of structures have been discovered by the efforts of many researchers, mainly as metabolites of actinomycetes and fungi. Our group alone has isolated about fifty new antibiotics. These compounds affect various biological targets, such as protein synthesis, energy metabolism, lipid metabolism, neurotransmitters, and cellular integrity, and have selectivity against susceptible parasites. Although not many antiparasitic antibiotics are currently used clinically, some are commercially available as tools for biochemical studies. We hope that further work in this field shall yield effective antiparasitic remedies.