RESUMO
The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new therapeutic agent. From our screening, phebestin stood out with nanomolar efficacy against Plasmodium falciparum 3D7. Phebestin was initially identified as an aminopeptidase N inhibitor. Phebestin inhibited the in vitro multiplication of the P. falciparum 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains at IC50 values of 157.90 ± 6.26 nM and 268.17 ± 67.59 nM, respectively. Furthermore, phebestin exhibited no cytotoxic against human foreskin fibroblast cells at 2.5 mM. In the stage-specific assay, phebestin inhibited all parasite stages at 100 and 10-fold its IC50 concentration. Using 72-h in vitro exposure of phebestin at concentrations of 1 µM on P. falciparum 3D7 distorted the parasite morphology, showed dying signs, shrank, and prevented reinvasion of RBCs, even after the compound was washed from the culture. An in silico study found that phebestin binds to P. falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP), as observed for bestatin. In vivo evaluation using P. yoelii 17XNL-infected mice with administrations of 20 mg/kg phebestin, once daily for 7 days, resulted in significantly lower parasitemia peaks in the phebestin-treated group (19.53%) than in the untreated group (29.55%). At the same dose and treatment, P. berghei ANKA-infected mice showed reduced parasitemia levels and improved survival compared to untreated mice. These results indicate that phebestin is a promising candidate for development as a potential therapeutic agent against malaria.
Assuntos
Antimaláricos , Malária Falciparum , Malária , Humanos , Animais , Camundongos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Aminopeptidases/uso terapêutico , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Cloroquina/farmacologia , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum , Plasmodium bergheiRESUMO
The natural polyether ionophore antibiotics may be important chemotherapeutic agents. Among them, kijimicin represents an important type of ionophore compound because it inhibits Eimeria tenella and human immunodeficiency virus. The ionophore monensin displays potent activities against several coccidian parasites including the opportunistic pathogen of humans, Toxoplasma gondii. At first, we evaluated the anti-Toxoplasma activity of kijimicin, monensin as a reference control, and anti-Toxoplasma drugs such as clindamycin, in vitro. The half inhibitory concentrations (IC50) for the anti-Toxoplasma activities of kijimicin, monensin, and clindamycin were 45.6 ± 2.4 nM, 1.3 ± 1.8 nM, and 238.5 ± 1.8 nM, respectively. Morphological analyses by electron microscopy revealed cellular swelling and multiple intracellular vacuole-like structures in the T. gondii tachyzoites after treatment with kijimicin and monensin. Kijimicin and monensin also inhibited the invasion of extracellular parasites (IC50 = 216.6 ± 1.9 pM and 531.1 ± 1.9 pM, respectively). Importantly, kijimicin treatment resulted in decreased mitochondrial membrane potential and generation of reactive oxygen species in T. gondii as monensin did. Furthermore, mice treated with kijimicin at 10 mg/kg/day and 3 mg/kg/day showed 91.7% and 66.7% survival rates, respectively, 30 days after infection with T. gondii. The control mice all died within 18 days of infection. The present study shows that kijimicin inhibits T. gondii growth and changes the ultrastruct of the parasites. This finding may lead to validation of kijimicin as new drug to control T. gondii growth.
Assuntos
Toxoplasma , Toxoplasmose , Animais , Ionóforos , Camundongos , PiranosRESUMO
BACKGROUND: Toxoplasmosis, a parasitic disease caused by Toxoplasma gondii, is an important cause of miscarriage or adverse fetal effects, including neurological and ocular manifestations in humans. Current anti-Toxoplasma drugs have limited efficacy against toxoplasmosis and also have severe side effects. Therefore, novel efficacious drugs are urgently needed. Here, we identified metacytofilin (MCF) from a fungal Metarhizium species as a potential anti-Toxoplasma compound. METHODS: Anti-Toxoplasma activities of MCF and its derivatives were evaluated in vitro and in vivo using nonpregnant and pregnant mice. To understand the mode of action of MCF, the RNA expression of host and parasite genes was investigated by RNAseq. RESULTS: In vitro, MCF inhibited the viability of intracellular and extracellular T. gondii. Administering MCF intraperitoneally or orally to mice after infection with T. gondii tachyzoites increased mouse survival compared with the untreated animals. Remarkably, oral administration of MCF to pregnant mice prevented vertical transmission of the parasite. Interestingly, RNA sequencing of T. gondii-infected cells treated with MCF showed that MCF inhibited DNA replication and enhanced RNA degradation in the parasites. CONCLUSIONS: With its potent anti-T. gondii activity, MCF is a strong candidate for future drug development against toxoplasmosis.
Assuntos
Antiparasitários/uso terapêutico , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Oxazinas/uso terapêutico , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Toxoplasmose/mortalidade , Administração Intravenosa , Administração Oral , Animais , Antiparasitários/administração & dosagem , Antiparasitários/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA de Protozoário , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxazinas/administração & dosagem , Oxazinas/farmacologia , Gravidez , Taxa de Sobrevida , Toxoplasma/genética , Toxoplasmose/parasitologia , Toxoplasmose/transmissão , Resultado do TratamentoRESUMO
As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS.
Assuntos
Encéfalo/metabolismo , Capilares/metabolismo , Células Endoteliais/metabolismo , Poliovirus/metabolismo , Receptores da Transferrina/metabolismo , Ligação Viral , Internalização do Vírus , Animais , Encéfalo/patologia , Encéfalo/virologia , Capilares/patologia , Capilares/virologia , Proteínas do Capsídeo/metabolismo , Linhagem Celular Transformada , Células Endoteliais/patologia , Células Endoteliais/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Poliovirus/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores da Transferrina/genéticaRESUMO
p24 family proteins are evolutionarily conserved transmembrane proteins involved in the early secretory pathway. Saccharomyces cerevisiae has 8 known p24 proteins that are classified into four subfamilies (p24α, -ß, -γ, and -δ). Emp24 and Erv25 are the sole members of p24ß and -δ, respectively, and deletion of either destabilizes the remaining p24 proteins, resulting in p24 null phenotype (p24Δ). We studied genetic and physical interactions of p24α (Erp1, -5, and -6) and γ (Erp2, -3, and -4). Deletion of the major p24α (Erp1) partially inhibited p24 activity as reported previously. A second mutation in either Erp5 or Erp6 aggravated the erp1Δ phenotype, and the triple mutation gave a full p24Δ phenotype. Similar genetic interactions were observed among the major p24γ (Erp2) and the other two γ members. All the p24α/γ isoforms interacted with both p24ß and -δ. Interaction between p24ß and -δ was isoform-selective, and five major α/γ pairs were detected. These results suggest that the yeast p24 proteins form functionally redundant αßγδ complexes. We also identified Rrt6 as a novel p24δ isoform. Rrt6 shows only limited sequence identity (â¼15%) to known p24 proteins but was found to have structural properties characteristic of p24. Rrt6 was induced when cells were grown on glycerol and form an additional αßγδ complex with Erp3, Erp5, and Emp24. This complex was mainly localized to the Golgi, whereas the p24 complex containing Erv25, instead of Rrt6 but otherwise with the same isoform composition, was found mostly in the ER.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Complexos Multiproteicos/metabolismo , Multimerização Proteica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Crioprotetores/farmacologia , Retículo Endoplasmático/genética , Deleção de Genes , Glicerol/farmacologia , Complexo de Golgi/genética , Complexos Multiproteicos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
The emerging spread of drug-resistant malaria parasites highlights the need for new antimalarial agents. This study evaluated the growth-inhibitory effects of sparsomycin (Sm), a peptidyl transferase inhibitor, against Plasmodium falciparum 3D7 (chloroquine-sensitive strain), P. falciparum K1 (resistant to multiple drugs, including chloroquine), P. yoelii 17XNL (cause of uncomplicated rodent malaria) and P. berghei ANKA (cause of complicated rodent malaria). Using a fluorescence-based assay, we found that Sm exhibited half-maximal inhibitory concentrations (IC50) of 12.07 and 25.43 nM against P. falciparum 3D7 and K1, respectively. In vitro treatment of P. falciparum 3D7 with Sm at 10 or 50 nM induced morphological alteration, blocked parasites in the ring state and prevented erythrocyte reinvasion, even after removal of the compound. In mice infected with P. yoelii 17XNL, the administration of 100 µg/kg Sm for 7 days did not affect parasitemia. Meanwhile, treatment with 300 µg/kg Sm resulted in a significantly lower parasitemia peak (18.85%) than that observed in the control group (40.13%). In mice infected with P. berghei ANKA, both four and seven doses of Sm (300 µg/kg) prolonged survival by 33.33%. Our results indicate that Sm has potential antiplasmodial activities in vitro and in vivo, warranting its further development as an alternative treatment for malaria.
RESUMO
Membrane and secretory proteins are synthesized by ribosomes and then enter the endoplasmic reticulum (ER) where they undergo glycosylation and quality control for proper folding. Subsequently, proteins are transported to the Golgi apparatus and then sorted to the plasma membrane or intracellular organelles. Transport vesicles are formed at ER-exit sites (ERES) on the ER with several coat protein complexes. Cargo proteins loaded into the vesicles are selected by specific interactions with cargo receptors and/or adaptors during vesicle formation. p24 family and intracellular lectin ERGIC-53-membrane proteins are the known cargo receptors acting in the early secretory pathway (ER-Golgi). Oligomerization of the cargo receptors have been suggested to play an important role in cargo selection and sorting via posttranslational modifications in fungi and metazoans. On the other hand, the mechanisms involved in the early secretory pathway in protozoa remain unclear. In this review, we focus on Trypanosoma brucei as a representative of protozoan and discuss differences and commonalities in the molecular mechanisms of its early secretory pathway compared with other organisms.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma/metabolismo , África , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Via Secretória , Tripanossomíase Africana/metabolismoRESUMO
Metacytofilin (MCF) was isolated from the fungus Metarhizium sp. TA2759. Although MCF possesses anti-Toxoplasma activity, the effects of this compound against other parasites are unknown. Here, we evaluated the in vitro anti-malarial activity of MCF against the 3D7 strain and the chloroquine-resistant K1 strain of Plasmodium falciparum. The half maximal inhibitory concentrations (IC50) of MCF against the 3D7 and K-1 strains following culture for 48 h were 666 nM and 605 nM, respectively. Artemisinin was more potent than MCF against both strains (3D7 IC50: 17.4 nM; K-1 IC50: 18.3 nM), while chloroquine was ineffective against the chloroquine-resistant strain (3D7 IC50: 39.1 nM; K-1 IC50: 1.62 µM). MCF affected the ring stage of the parasites, resulting in their death as shown by spots within red blood cells. MCF also inhibited parasite growth following culture for 72 h (3D7 IC50, 285 nM). Four optical isomers of cyclo[Leu-Phe]-diketopiperazine derivatives with modified methoxy and/or hydroxyl groups lost anti-malarial activity, suggesting that the spatial positions of the methoxy and hydroxyl groups in MCF play an important role in its anti-malarial effects. Together, these data suggest that MCF may represent a promising lead compound for treatment of drug-resistant malarial parasites.
Assuntos
Antimaláricos/farmacologia , Oxazinas/farmacologia , Plasmodium falciparum/efeitos dos fármacosRESUMO
Natural resources are recognized as important sources of potential drugs for treating various infections, and microorganisms are a rich natural source of diverse compounds. Among the world's microorganisms, actinomycetes, which are abundant in soil and marine, are the well-known producers of a wide range of bioactive secondary metabolites and antibiotics. In the present study, four actinomycetes (samples N25, N6, N18, and N12) were isolated from soil samples in Mongolia. Phylogenetic analysis of these isolates revealed that they share the highest similarity with Streptomyces canus (N25), S. cirratus (N6), S. bacillaris (N18) and S. peucetius (N12), based on 16S rRNA gene sequencing. Crude extracts were obtained from them using ethyl acetate, and the crude fractions were separated by thin layer chromatography. The fractions were then evaluated for their cytotoxicities and their anti-Toxoplasma and antimalarial activities in vitro. The S. canus (N25) crude extract was selected for further chemical characterization based on its antiprotozoal activities. Using liquid chromatography-high resolution mass spectrometry, phenazine-1-carboxylic acid (PCA) was detected and identified in the active fractions of the metabolites from strain N25. We next confirmed that commercially available PCA possesses antiprotozoal activity against T. gondii (IC50: 55.5⯵g/ml) and Plasmodium falciparum (IC50: 6.4⯵g/ml) in vitro. The results of this study reveal that soil actinomycetes are potential sources of antiprotozoal compounds, and that PCA merits further investigation as an anti-protozoal agent.
Assuntos
Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Microbiologia do Solo , Streptomyces/química , Streptomyces/classificação , Mongólia , Filogenia , RNA Ribossômico 16S/genética , Streptomyces/isolamento & purificaçãoRESUMO
Succinate:ubiquinone oxidoreductase (SQR) was solubilized and purified from Escherichia coli inner membranes using several different detergents. The number of phospholipid molecules bound to the SQR molecule varied greatly depending on the detergent combination that was used for the solubilization and purification. Crystallization conditions were screened for SQR that had been solubilized and purified using 2.5%(w/v) sucrose monolaurate and 0.5%(w/v) Lubrol PX, respectively, and two different crystal forms were obtained in the presence of detergent mixtures composed of n-alkyl-oligoethylene glycol monoether and n-alkyl-maltoside. Crystallization took place before detergent phase separation occurred and the type of detergent mixture affected the crystal form.
Assuntos
Detergentes , Complexo II de Transporte de Elétrons/química , Complexo II de Transporte de Elétrons/isolamento & purificação , Escherichia coli/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Cristalização , Complexo II de Transporte de Elétrons/metabolismo , Escherichia coli/química , Proteínas de Membrana/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Solubilidade , Ácido Succínico/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
Ascofuranone, an antibiotic isolated from Ascochyta visiae, showed trypanocidal activity in Trypanosoma vivax-infected mice. A single dose of 50 mg/kg ascofuranone effectively cured the mice without the help of glycerol. Repeated administrations of this drug further enhanced its chemotherapeutic effect. After two, three, and four consecutive days treatment, the doses needed to cure the infection decreased to 25, 12, and 6 mg/kg, so that the total doses administered were 50, 36 and 24 mg/kg, respectively. Ascofuranone (50 mg/kg) also had a prophylactic effect against T. vivax infection within the first two days after administration. This prophylactic activity diminished to 80% by day 3 and completely disappeared four days after administration. Of particular interest in this study was that ascofuranone had trypanocidal activity in T. vivax-infected mice in the absence of glycerol, whereas co-administration of glycerol or repeated administrations of this drug are needed for Trypanosoma brucei brucei infection. Our present results strongly suggest that ascofuranone is also an effective tool in chemotherapy against African trypanosomiasis in domestic animals.
Assuntos
Sesquiterpenos/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma vivax , Tripanossomíase Africana/tratamento farmacológico , Animais , Modelos Animais de Doenças , Glicerol/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sesquiterpenos/administração & dosagem , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Resultado do Tratamento , Tripanossomicidas/administração & dosagem , Tripanossomicidas/farmacologia , Trypanosoma vivax/efeitos dos fármacos , Tripanossomíase Africana/parasitologiaRESUMO
Trypanosoma vivax causes nagana disease in cattle. Since T. vivax is transmitted not only by tsetse flies but also by other biting flies (non-cyclic transmission), the parasite has been distributed to and has had a significant economic impact on wide geographical areas, including Africa and South America. Our previous study on Trypanosoma brucei brucei showed that the trypanosome alternative oxidase (TAO, TbAOX) is a promising target of chemotherapy. For this reason, we also have cloned the T vivax AOX (TvAOX) gene and characterized the recombinant enzyme. The deduced amino acid sequence (328 a.a.) of TvAOX shares 76% identity with TbAOX and contains the diiron-coordination motifs (-E-, -EXXH-) that are conserved among AOXs. The Km of recombinant TvAOX (rTvAOX) expressed in Escherichia coli for ubiquinol (87.0 +/- 0.54 microM) was significantly lower than the value for recombinant TbAOX (rTbAOX) (714 +/- 4.5 microM). Ascofuranone, the most potent inhibitor of TbAOX, was a competitive inhibitor of rTvAOX with a Ki value (0.40 +/- 0.00 nM) significantly lower than that for rTbAOX (1.29 +/- 0.00 nM). The non-cyclic transmission ability of T. vivax and the in vivo chemotherapeutic efficacy of ascofuranone against T. vivax and T. b. brucei infection are discussed in terms of these Km and Ki values.
Assuntos
Clonagem Molecular , Oxirredutases/metabolismo , Sesquiterpenos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma vivax/enzimologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Bovinos , Proteínas Mitocondriais , Dados de Sequência Molecular , Oxirredutases/efeitos dos fármacos , Oxirredutases/genética , Proteínas de Plantas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Tripanossomíase Africana/veterinária , Tripanossomíase Bovina/parasitologiaRESUMO
Consecutive administration of ascofuranone without glycerol was found to have therapeutic efficacy against Trypanosoma brucei brucei infection in mice. A suspension of ascofuranone (25-100 mg/kg) was administrated intraperitoneally every 24 h for 1-4 consecutive days to trypanosome-infected mice and efficacy was compared with oral treatment. With intraperitoneal administration, all mice treated with 100 mg/kg ascofuranone for 4 consecutive days were cured. On contrary, with oral treatment a higher dose of ascofuranone (400 mg/kg) was needed for 8 consecutive days to cure the mice. With intraperitoneal treatment, parasitemia was strongly suppressed, with almost all long slender bloodstream forms of the parasite changed to short stumpy forms by day 3 and the parasites have been eliminated 4 days after the start of treatment. These ascofuranone-induced short stumpy forms were morphologically analogous to the stumpy forms 2 days after peak parasitemia of pleomorphic clone of T. b. brucei GUTat 3.1. However, the properties of ubiquinol oxidase activity, which is the target of ascofuranone, in mitochondria isolated from before and after treatment, were almost same. The enzymatic activities of ubiquinol oxidase were only decreased to approximately 30% within a day after treatment, and then kept at nearly the same level. In the present study, we have improved regimen for administration of ascofuranone without glycerol, and demonstrated that consecutively administrated ascofuranone showed trypanocidal effects in T. b. brucei infected mice. Our present results strongly suggest that consecutive administration of ascofuranone may be an effective chemotherapy for African trypanosomiasis.
Assuntos
Antiprotozoários/administração & dosagem , Sesquiterpenos/administração & dosagem , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Administração Oral , Animais , Antiprotozoários/sangue , Antiprotozoários/uso terapêutico , Esquema de Medicação , Feminino , Injeções Intraperitoneais/métodos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Oxirredutases/análise , Oxirredutases/metabolismo , Sesquiterpenos/sangue , Sesquiterpenos/uso terapêutico , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/sangue , Tripanossomíase Africana/metabolismoRESUMO
In humans, paralytic poliomyelitis results from the invasion of the central nervous system (CNS) by circulating poliovirus (PV) via the blood-brain barrier (BBB). After the virus enters the CNS, it replicates in neurons, especially in motor neurons, inducing the cell death that causes paralytic poliomyelitis. Along with this route of dissemination, neural pathway has been reported in humans, monkeys, and PV-sensitive human PV receptor (hPVR/CD155)-transgenic (Tg) mice. We demonstrated that a fast retrograde axonal transport process is required for PV dissemination through the sciatic nerve of hPVR-Tg mice and that intramuscularly inoculated PV causes paralysis in a hPVR-dependent manner. We also showed that hPVR-independent axonal transport of PV exists in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist in these mice. Circulating PV after intravenous inoculation in mice cross the BBB at a high rate in a hPVR-independent manner. We will implicate an involvement of a new possible receptor for PV to permeate the BBB based on our recent findings.
RESUMO
Cryptosporidium parvum is a parasitic protozoan that causes the diarrheal disease cryptosporidiosis, for which no satisfactory chemotherapy is currently available. Although the presence of mitochondria in this parasite has been suggested, its respiratory system is poorly understood due to difficulties in performing biochemical analyses. In order to better understand the respiratory chain of C. parvum, we surveyed its genomic DNA database in GenBank and identified a partial sequence encoding cyanide-insensitive alternative oxidase (AOX). Based on this sequence, we cloned C. parvum AOX (CpAOX) cDNA from the phylum apicomplexa for the first time. The deduced amino acid sequence (335 a.a.) of CpAOX contains diiron coordination motifs (-E-, -EXXH-) that are conserved among AOXs. Phylogenetic analysis suggested that CpAOX is a mitochondrial-type AOX, possibly derived from mitochondrial endosymbiont gene transfer. The recombinant enzyme expressed in Escherichia coli showed quinol oxidase activity. This activity was insensitive to cyanide and highly sensitive to ascofuranone, a specific inhibitor of trypanosome AOX.