In Vitro and in Vivo Study of a Photostable Quinone Compound with Enhanced Therapeutic Efficacy against Chagas Disease.

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi poses a significant health challenge in rural areas of Latin America. The current pharmacological options exhibit notable side effects, demand prolonged administration, and display limited efficacy. Consequently, there is an urgent need to develop drugs that are safe and clinically effective. Previously, we identified a quinone compound (designated as compound 2) with potent antiprotozoal activity, based on the chemical structure of komaroviquinone, a natural product renowned for its antitrypanosomal effects. However, compound 2 was demonstrated considerably unstable to light. In this study, we elucidated the structure of the light-induced degradation products of compound 2 and probed the correlation between the quinone ring's substituents and its susceptibility to light. Our findings led to the discovery of quinones with significantly enhanced light stability, some of which exhibiting antitrypanosomal activity. The most promising compound was evaluated for drug efficacy in a mouse model of Chagas disease, revealing where a notable reduction in blood parasitemia.

Discovery of a Hidden Trypanosoma cruzi Spermidine Synthase Binding Site and Inhibitors through In Silico, In Vitro, and X-ray Crystallography.

In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure. Molecular dynamics (MD) simulation is a method for predicting protein dynamics and can detect potential ensembles of protein binding sites and hidden sites unobservable in a single-point structure. In this study, to demonstrate the utility of virtual screening with protein dynamics, MD simulations were performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding sites with a high probability of existence. The structure of the binding site obtained through MD simulation revealed pockets in addition to the active site that was present in the initial structure. Using the obtained binding site structures, virtual screening of 4.8 million compounds by docking simulation, in vitro assays, and X-ray analysis was conducted, successfully identifying two hit compounds.

A versatile Plasmodium falciparum reporter line expressing NanoLuc enables highly sensitive multi-stage drug assays.

Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial drugs effective against multiple stages of the parasite, we generate a P. falciparum reporter parasite that constitutively expresses NanoLuciferase (NanoLuc) throughout its whole life cycle. The NanoLuc-expressing P. falciparum reporter parasite shows a quantitative NanoLuc signal in the asexual blood, gametocyte, mosquito, and liver stages. We also establish assay systems to evaluate the anti-malarial activity of compounds at the asexual blood, gametocyte, and liver stages, and then determine the 50% inhibitory concentration (IC50) value of several anti-malarial compounds. Through the development of this robust high-throughput screening system, we identify an anti-malarial compound that kills the asexual blood stage parasites. Our study highlights the utility of the NanoLuc reporter line, which may advance anti-malarial drug development through the improved screening of compounds targeting the human malarial parasite at multiple stages.

Biochemical characterization and identification of ferulenol and embelin as potent inhibitors of malate:quinone oxidoreductase from Campylobacter jejuni.

Campylobacter jejuni infection poses a serious global threat to public health. The increasing incidence and antibiotic resistance of this bacterial infection have necessitated the adoption of various strategies to curb this trend, primarily through developing new drugs with new mechanisms of action. The enzyme malate:quinone oxidoreductase (MQO) has been shown to be essential for the survival of several bacteria and parasites. MQO is a peripheral membrane protein that catalyses the oxidation of malate to oxaloacetate, a crucial step in the tricarboxylic acid cycle. In addition, MQO is involved in the reduction of the quinone pool in the electron transport chain and thus contributes to cellular bioenergetics. The enzyme is an attractive drug target as it is not conserved in mammals. As a preliminary step in assessing the potential application of MQO from C. jejuni (CjMQO) as a new drug target, we purified active recombinant CjMQO and conducted, for the first time, biochemical analyses of MQO from a pathogenic bacterium. Our study showed that ferulenol, a submicromolar mitochondrial MQO inhibitor, and embelin are nanomolar inhibitors of CjMQO. We showed that both inhibitors are mixed-type inhibitors versus malate and noncompetitive versus quinone, suggesting the existence of a third binding site to accommodate these inhibitors; indeed, such a trait appears to be conserved between mitochondrial and bacterial MQOs. Interestingly, ferulenol and embelin also inhibit the in vitro growth of C. jejuni, supporting the hypothesis that MQO is essential for C. jejuni survival and is therefore an important drug target.

Killing Two Birds with One Stone: Discovery of Dual Inhibitors of Oxygen and Fumarate Respiration in Zoonotic Parasite, Echinococcus multilocularis.

Ascofuranone (AF), a meroterpenoid isolated from various filamentous fungi, including Acremonium egyptiacum, has been reported as a potential lead candidate for drug development against parasites and cancer. In this study, we demonstrated that AF and its derivatives are potent anthelminthic agents, particularly against Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis. We measured the inhibitory activities of AF and its derivatives on the mitochondrial aerobic and anaerobic respiratory systems of E. multilocularis larvae. Several derivatives inhibited complex II (succinate:quinone reductase [SQR]; IC50 = 0.037 to 0.135 µM) and also complex I to III (NADH:cytochrome c reductase; IC50 = 0.008 to 0.401 µM), but not complex I (NADH:quinone reductase), indicating that mitochondrial complexes II and III are the targets. In particular, complex II inhibition in the anaerobic pathway was notable because E. multilocularis employs NADH:fumarate reductase (fumarate respiration), in addition to NADH oxidase (oxygen respiration), resulting in complete shutdown of ATP synthesis by oxidative phosphorylation. A structure-activity relationship study of E. multilocularis complex II revealed that the functional groups of AF are essential for inhibition. Binding mode prediction of AF derivatives to complex II indicated potential hydrophobic and hydrogen bond interactions between AF derivatives and amino acid residues within the quinone binding site. Ex vivo culture assays revealed that AF derivatives progressively reduced the viability of protoscoleces under both aerobic and anaerobic conditions. These findings confirm that AF and its derivatives are the first dual inhibitors of fumarate and oxygen respiration in E. multilocularis and are potential lead compounds in the development of anti-echinococcal drugs.

Effect of geranylated dihydrochalcone from Artocarpus altilis leaves extract on Plasmodium falciparum ultrastructural changes and mitochondrial malate: Quinone oxidoreductase.

Nearly half of the world's population is at risk of being infected by Plasmodium falciparum, the pathogen of malaria. Increasing resistance to common antimalarial drugs has encouraged investigations to find compounds with different scaffolds. Extracts of Artocarpus altilis leaves have previously been reported to exhibit in vitro antimalarial activity against P. falciparum and in vivo activity against P. berghei. Despite these initial promising results, the active compound from A. altilis is yet to be identified. Here, we have identified 2-geranyl-2', 4', 3, 4-tetrahydroxy-dihydrochalcone (1) from A. altilis leaves as the active constituent of its antimalarial activity. Since natural chalcones have been reported to inhibit food vacuole and mitochondrial electron transport chain (ETC), the morphological changes in food vacuole and biochemical inhibition of ETC enzymes of (1) were investigated. In the presence of (1), intraerythrocytic asexual development was impaired, and according to the TEM analysis, this clearly affected the ultrastructure of food vacuoles. Amongst the ETC enzymes, (1) inhibited the mitochondrial malate: quinone oxidoreductase (PfMQO), and no inhibition could be observed on dihydroorotate dehydrogenase (DHODH) as well as bc1 complex activities. Our study suggests that (1) has a dual mechanism of action affecting the food vacuole and inhibition of PfMQO-related pathways in mitochondria.

Fumarate respiration of Fasciola flukes as a potential drug target.

Fascioliasis is a neglected tropical zoonotic disease caused by liver flukes belonging to the genus Fasciola. The emergence of resistance to triclabendazole, the only World Health Organization-recommended drug for this disease, highlights the need for the development of new drugs. Helminths possess an anaerobic mitochondrial respiratory chain (fumarate respiration) which is considered a potential drug target. This study aimed to evaluate the occurrence of fumarate respiration in Fasciola flukes. We analyzed the properties of the respiratory chain of Fasciola flukes in both adults and newly excysted juveniles (NEJs). Fasciola flukes travel and mature through the stomach, bowel, and abdominal cavity to the liver, where oxygen levels gradually decline. High fumarate reductase activity was observed in the mitochondrial fraction of adult Fasciola flukes. Furthermore, rhodoquinone-10 (RQ10 Em'= -63 mV), a low-potential electron mediator used in fumarate respiration was found to be predominant in adults. In contrast, the activity of oxygen respiration was low in adults. Rotenone, atpenin A5, and ascochlorin, typical inhibitors of mitochondrial enzymes in complexes I, II, and III, respectively, inhibit the activity of each enzyme in the adult mitochondrial fraction. These inhibitors were then used for in vitro viability tests of NEJs. Under aerobic conditions, NEJs were killed by rotenone or ascochlorin, which inhibit aerobic respiration (complex I-III), whereas atpenin A5, which inhibits complex II involved in fumarate respiration, did not affect NEJs. Moreover, ubiquinone-10 (UQ10 Em'= +110 mV), which is used in oxidative respiration, was detected in NEJs, in addition to RQ10. In contrast, under anaerobic conditions, rotenone and atpenin A5, which inhibit fumarate respiration (complex I-II), were crucial for NEJs. These findings demonstrate that NEJs have active hybrid respiration, in which they can properly use both oxygen and fumarate respiration, depending on oxygen availability. Thus, fumarate respiration is a promising drug target for Fasciola flukes, because it plays an essential role in both adults and NEJs.

Data on the combined effect of atovaquone, mefloquine, and 3-bromopyruvic acid against Echinococcus multilocularis protoscoleces.

The dataset presented here is related to a previous research article titled "Mitochondrial Complex III in Larval Stage of Echinococcus multilocularis as a Potential Chemotherapeutic Target and in vivo Efficacy of Atovaquone Against Primary Hydatid Cysts"[1]. In this report, data were collected from aerobic and anaerobic culture assays of E. multilocularis protoscoleces in the presence of three anti-echinococcal drug candidates (atovaquone, mefloquine, and 3-bromopyruvic acid). The data were analyzed for viability of the protoscoleces between day 0 and day 7 upon adding drug candidates. In aerobic condition, all drug candidates caused damage to the protoscoleces, as described previously [1], [2], [3], [4], [5], [6]. Mefloquine, alone as well as in combination with atovaquone, immediately eliminated the protoscoleces, whereas combination of atovaquone with 3-bromopyruvic acid did not show clear synergy. In anaerobic condition, mefloquine, alone as well as in combination with atovaquone, eliminated protoscoleces immediately. 3-Bromopyruvic acid showed stronger efficacy in anaerobic condition than in aerobic condition. Combination of atovaquone with 3-bromopyruvic acid eliminated the protoscoleces, indicating that synergy occurred only under anaerobic condition. The data clarified that combined use of the three drugs eliminated protoscoleces in both aerobic and anaerobic conditions, hence suggesting that these could inhibit aerobic and anaerobic respiration pathways of Echinococcus multilocularis in vivo. The obtained data would be useful for the development of new drug dosing method for alveolar echinococcosis.

Leishmania major Strain-Dependent Macrophage Activation Contributes to Pathogenicity in the Absence of Lymphocytes.

Infection of C57BL/6 wild-type mice with Leishmania major 5-ASKH or Friedlin strains results in relatively similar pathogenicity with self-healing lesions within weeks. Parasite clearance depends on nitric oxide production by activated macrophages in response to cytokines produced mainly by CD4+ Th1 cells. In contrast, C57BL/6 Rag2 knockout mice, which lack T and B lymphocytes, show distinct pathologies during infection with these strains. Despite of the similar parasite number, the 5-ASKH infection induced severe inflammation rather than the Friedlin. To determine the immunological factors behind this phenomenon, we infected C57BL/6 Rag2 knockout mice with these two strains and compared immune cell kinetics and macrophage activation status. Compared with the Friedlin strain, the 5-ASKH strain elicited increased pathology associated with the accumulation of CD11bhigh, Ly6Ghigh neutrophils by week four and increased the expression of macrophage activation markers. We then analyzed the differentially expressed transcripts in infected bone marrow-derived macrophages by RNA sequencing. It showed upregulation of multiple inflammatory transcripts, including Toll-like receptor 1/2 (TLR1/2), CD69, and CARD14, upon 5-ASKH infection. Our findings suggest that different L. major strains can trigger distinct macrophage activation, contributing to the disease outcome observed in the absence of lymphocytes but not in the presence of lymphocytes. IMPORTANCE Disease manifestations of cutaneous leishmaniasis (CL) range from self-healing cutaneous lesions to chronic forms of the disease, depending on the infecting Leishmania sp. and host immune protection. Previous works on mouse models of CL show the distinct pathogenicity of Leishmania major strains in the absence of lymphocytes. However, the mechanisms of this pathology remain uncovered. In the trial to understand the immunological process involved in lymphocyte-independent pathology, we have found a specific induction of macrophages by different L. major strains that affect their ability to mount innate responses leading to neutrophilic pathology when lymphocytes are ablated.

Siccanin Is a Dual-Target Inhibitor of Plasmodium falciparum Mitochondrial Complex II and Complex III.

Plasmodium falciparum contains several mitochondrial electron transport chain (ETC) dehydrogenases shuttling electrons from the respective substrates to the ubiquinone pool, from which electrons are consecutively transferred to complex III, complex IV, and finally to the molecular oxygen. The antimalarial drug atovaquone inhibits complex III and validates this parasite's ETC as an attractive target for chemotherapy. Among the ETC dehydrogenases from P. falciparum, dihydroorotate dehydrogenase, an essential enzyme used in de novo pyrimidine biosynthesis, and complex III are the two enzymes that have been characterized and validated as drug targets in the blood-stage parasite, while complex II has been shown to be essential for parasite survival in the mosquito stage; therefore, these enzymes and complex II are considered candidate drug targets for blocking parasite transmission. In this study, we identified siccanin as the first (to our knowledge) nanomolar inhibitor of the P. falciparum complex II. Moreover, we demonstrated that siccanin also inhibits complex III in the low-micromolar range. Siccanin did not inhibit the corresponding complexes from mammalian mitochondria even at high concentrations. Siccanin inhibited the growth of P. falciparum with IC50 of 8.4 µM. However, the growth inhibition of the P. falciparum blood stage did not correlate with ETC inhibition, as demonstrated by lack of resistance to siccanin in the yDHODH-3D7 (EC50 = 10.26 µM) and Dd2-ELQ300 strains (EC50 = 18.70 µM), suggesting a third mechanism of action that is unrelated to mitochondrial ETC inhibition. Hence, siccanin has at least a dual mechanism of action, being the first potent and selective inhibitor of P. falciparum complexes II and III over mammalian enzymes and so is a potential candidate for the development of a new class of antimalarial drugs.

5-Aminolevulinic acid antiviral efficacy against SARS-CoV-2 omicron variant in vitro.

The coronavirus disease 2019 (COVID 19) pandemic continues to pose a threat to global health. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529) has spread rapidly worldwide and became dominant in many countries. A natural 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) has demonstrated antiviral activity in Wuhan, Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 infections in vitro. In this study, we report antiviral activity of 5-ALA, 5-ALA with SFC led to IC50 of 329 and 765/191, respectively after infection with Omicron variant of SARS-CoV-2 in vitro. Our finding suggests that 5-ALA could be used as antiviral drug candidate to treat Omicron variant infected patients.

Antiviral activity of 5-aminolevulinic acid against variants of severe acute respiratory syndrome coronavirus 2.

BACKGROUND: Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began to emerge in 2020 and have been spreading globally during the coronavirus disease 2019 (COVID-19) pandemic. Despite the presence of different COVID-19 vaccines, the discovery of effective antiviral therapeutics for the treatment of patients infected with SARS-CoV-2 are still urgently needed. A natural amino acid, 5-aminolevulinic acid (5-ALA), has exhibited both antiviral and anti-inflammatory activities. In a previous study, we demonstrated an in vitro antiviral effect of 5-ALA against SARS-CoV-2 infection without significant cytotoxicity. In the present study, we sought to investigate whether 5-ALA with or without sodium ferrous citrate (SFC) can inhibit in vitro both the original SARS-CoV-2 Wuhan strain and its variants, including the Alpha, Beta, Gamma and Delta strains. METHODS: The antiviral activity of ALA with or without SFC was determined in Vero-E6 cell. The virus inhibition was quantified by real time RT-PCR. RESULTS: Co-administration of 5-ALA and SFC inhibited the Wuhan, Alpha and Delta variants of SARS-CoV-2 with IC50 values of 235, 173 and 397 µM, respectively, and the Beta and Gamma variants with IC50 values of 1311 and 1516 µM. CONCLUSION: Our study suggests that 5-ALA with SFC warrants accelerated clinical evaluation as an antiviral drug candidate for treating patients infected with SARS-CoV-2 variants.

Exploring natural microbial resources for the discovery of anti-malarial compounds.

Microorganisms in nature are highly diverse biological resources, which can be explored for drug discovery. Some countries including Brazil, Columbia, Indonesia, China, and Mexico, which are blessed with geographical uniqueness with diverse climates and display remarkable megabiodiversity, potentially provide microorganismal resources for such exploitation. In this review, as an example of drug discovery campaigns against tropical parasitic diseases utilizing microorganisms from such a megabiodiversity country, we summarize our past and on-going activities toward discovery of new antimalarials. The program was held in a bilateral collaboration between multiple Indonesian and Japanese research groups. In order to develop a new platform of drug discovery utilizing Indonesian bioresources under an international collaborative scheme, we aimed at: 1) establishment of an Indonesian microbial depository, 2) development of robust enzyme-based and cell-based screening systems, and 3) technology transfer necessary for screening, purification, and identification of antimalarial compounds from microbial culture broths. We collected, characterized, and deposited Indonesian microbes. We morphologically and genetically characterized fungi and actinomycetes strains isolated from 5 different locations representing 3 Indonesian geographical areas, and validated genetic diversity of microbes. Enzyme-based screening was developed against two validated mitochondrial enzymes from Plasmodium falciparum, dihydroorotate dehydrogenase and malate:quinone oxidoreductase, while cell-based proliferation assay was developed using the erythrocytic stage parasite of 3D7 strain. More than 17 thousands microbial culture extracts were subjected to the enzyme- and cell-based screening. Representative anti-malarial compounds discovered in this campaign are discussed, including a few isolated compounds that have been identified for the first time as anti-malarial compounds. Our antimalarial discovery campaign validated the Indonesian microbial library as a powerful resource for drug discovery. We also discuss critical needs for selection criteria for hits at each stage of screening and hit deconvolution such as preliminary extraction test for the initial profiling of the active compounds and dereplication techniques to minimize repetitive discovery of known compounds.

Mitochondria as a Potential Target for the Development of Prophylactic and Therapeutic Drugs against Schistosoma mansoni Infection.

The emergence of parasites resistant to praziquantel, the only therapeutic agent, and its ineffectiveness as a prophylactic agent (inactive against the migratory/juvenile Schistosoma mansoni), make the development of new antischistosomal drugs urgent. The parasite's mitochondrion is an attractive target for drug development, because this organelle is essential for survival throughout the parasite's life cycle. We investigated the effects of 116 compounds against Schistosoma mansoni cercaria motility that have been reported to affect mitochondrion-related processes in other organisms. Next, eight compounds plus two controls (mefloquine and praziquantel) were selected and assayed against the motility of schistosomula (in vitro) and adults (ex vivo). Prophylactic and therapeutic assays were performed using infected mouse models. Inhibition of oxygen consumption rate (OCR) was assayed using Seahorse XFe24 analyzer. All selected compounds showed excellent prophylactic activity, reducing the worm burden in the lungs to less than 15% of that obtained in the vehicle control. Notably, ascofuranone showed the highest activity, with a 98% reduction of the worm burden, suggesting the potential for the development of ascofuranone as a prophylactic agent. The worm burden of infected mice with S. mansoni at the adult stage was reduced by more than 50% in mice treated with mefloquine, nitazoxanide, amiodarone, ascofuranone, pyrvinium pamoate, or plumbagin. Moreover, adult mitochondrial OCR was severely inhibited by ascofuranone, atovaquone, and nitazoxanide, while pyrvinium pamoate inhibited both mitochondrial and nonmitochondrial OCRs. These results demonstrate that the mitochondria of S. mansoni are a feasible target for drug development.

Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

Plasmodium falciparum's resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place.

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line (RNAiUGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle (RNAiUGP/EXPrUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

Gentisyl alcohol and homogentisic acid: Plasmodium falciparum dihydroorotate dehydrogenase inhibitors isolated from fungi.

Two Indonesian fungi Aspergillus assiutensis BioMCC-f.T.7495 and Penicillium pedernalense BioMCC-f.T.5350 along with a Japanese fungus Hypomyces pseudocorticiicola FKI-9008 have been found to produce gentisyl alcohol (1), which inhibits Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with an IC50 value of 3.4 µM. Another Indonesian fungus, Penicillium citrinum BioMCC-f.T.6730, produced an analog of 1, homogentisic acid (4), which also inhibits PfDHODH with an IC50 value of 47.6 µM.

In vivo efficacy of combination therapy with albendazole and atovaquone against primary hydatid cysts in mice.

Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis. Chemotherapy for AE involves albendazole (ABZ), which has shown insufficient efficacy. More effective chemotherapy for AE is needed. Previously, we have demonstrated that atovaquone (ATV), an antimalarial, inhibits mitochondrial complex III of E. multilocularis and restricts the development of larval cysts in in vivo experiments. Therefore, in this study, we evaluated the efficacy of ABZ and ATV combination therapy on E. multilocularis in culture and in vivo experiments. Protoscoleces were treated with 50 µM ABZ and/or ATV in the medium; the duration of parasite elimination was determined under aerobic and anaerobic culture. In the in vivo experiment, the effects of ABZ and ATV combination treatment in BALB/c mice infected orally with eggs from the feces of an adult-stage E. multilocularis-infected dog were compared with those of standard oral ABZ therapy. In the culture assay, the duration of elimination associated with ABZ and ATV combination treatment was shorter than that associated with ATV alone under aerobic conditions. Protoscolex viability progressively reduced owing to the combination treatment under anaerobic conditions; however, either drug used singly did not exhibit antiparasitic effects under hypoxia. Furthermore, compared with ABZ alone, the combination treatment significantly reduced the growth of the primary cyst in the liver of mice infected orally with parasite eggs (P = .011). ATV enhances the effect of ABZ in the treatment of AE in mice.

Characterization of Plasmodium falciparum Pantothenate Kinase and Identification of Its Inhibitors From Natural Products.

Coenzyme A (CoA) is a well-known cofactor that plays an essential role in many metabolic reactions in all organisms. In Plasmodium falciparum, the most deadly among Plasmodium species that cause malaria, CoA and its biosynthetic pathway have been proven to be indispensable. The first and rate-limiting reaction in the CoA biosynthetic pathway is catalyzed by two putative pantothenate kinases (PfPanK1 and 2) in this parasite. Here we produced, purified, and biochemically characterized recombinant PfPanK1 for the first time. PfPanK1 showed activity using pantetheine besides pantothenate, as the primary substrate, indicating that CoA biosynthesis in the blood stage of P. falciparum can bypass pantothenate. We further developed a robust and reliable screening system to identify inhibitors using recombinant PfPanK1 and identified four PfPanK inhibitors from natural compounds.

The ubiquinone synthesis pathway is a promising drug target for Chagas disease.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi). It was originally a Latin American endemic health problem, but now is expanding worldwide as a result of increasing migration. The currently available drugs for Chagas disease, benznidazole and nifurtimox, provoke severe adverse effects, and thus the development of new drugs is urgently required. Ubiquinone (UQ) is essential for respiratory chain and redox balance in trypanosomatid protozoans, therefore we aimed to provide evidence that inhibitors of the UQ biosynthesis have trypanocidal activities. In this study, inhibitors of the human COQ7, a key enzyme of the UQ synthesis, were tested for their trypanocidal activities because they were expected to cross-react and inhibit trypanosomal COQ7 due to their genetic homology. We show the trypanocidal activity of a newly found human COQ7 inhibitor, an oxazinoquinoline derivative. The structurally similar compounds were selected from the commercially available compounds by 2D and 3D ligand-based similarity searches. Among 38 compounds selected, 12 compounds with the oxazinoquinoline structure inhibited significantly the growth of epimastigotes of T. cruzi. The most effective 3 compounds also showed the significant antitrypanosomal activity against the mammalian stage of T. cruzi at lower concentrations than benznidazole, a commonly used drug today. We found that epimastigotes treated with the inhibitor contained reduced levels of UQ9. Further, the growth of epimastigotes treated with the inhibitors was partially rescued by UQ10 supplementation to the culture medium. These results suggest that the antitrypanosomal mechanism of the oxazinoquinoline derivatives results from inhibition of the trypanosomal UQ synthesis leading to a shortage of the UQ pool. Our data indicate that the UQ synthesis pathway of T. cruzi is a promising drug target for Chagas disease.