Human dihydroorotate dehydrogenase (HsDHODH) is a key enzyme of pyrimidine de novo biosynthesis pathway. It is located on the mitochondrial inner membrane and contributes to the respiratory chain by shuttling electrons to the ubiquinone pool. We have discovered ascofuranone (1), a natural compound produced by Acremonium sclerotigenum, and its derivatives are a potent class of HsDHODH inhibitors. We conducted a structure–activity relationship study and have identified functional groups of 1 that are essential for the inhibition of HsDHODH enzymatic activity. Furthermore, the binding mode of 1 and its derivatives to HsDHODH was demonstrated by co-crystallographic analysis and we show that these inhibitors bind at the ubiquinone binding site. In addition, the cytotoxicities of 1 and its potent derivatives 7, 8, and 9 were studied using human cultured cancer cells. Interestingly, they showed selective and strong cytotoxicity to cancer cells cultured under microenvironment (hypoxia and nutrient-deprived) conditions. The selectivity ratio of 8 under this microenvironment show the most potent inhibition which was over 1000-fold higher compared to that under normal culture condition. Our studies suggest that under microenvironment conditions, cancer cells heavily depend on the pyrimidine de novo biosynthesis pathway. We also provide the first evidence that 1 and its derivatives are potential lead candidates for drug development which target the HsDHODH of cancer cells living under a tumor microenvironment.

Accurate sorting of proteins to the three types of parasite-specific secretory organelles namely rhoptry, microneme and dense granule in Toxoplasma gondii is crucial for successful host cell invasion by this obligate intracellular parasite. Despite its tiny body architecture and limited trafficking machinery, T. gondii relies heavily on transport of vesicles containing proteins, lipids and important virulence-like factors that are delivered to these secretory organelles. However, our understanding on how trafficking of vesicles operates in the parasite is still limited. Here, we show that the T. gondii vacuolar protein sorting 9 (TgVps9), has guanine nucleotide exchange factor (GEF) activity towards Rab5a and is crucial for sorting of proteins destined to secretory organelles. Our results illuminate features of TgVps9 protein as a key trafficking facilitator that regulates protein maturation, secretory organelle formation and secretion, thereby ensuring a primary role in host infection by T. gondii.

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC50 of 0.822 μM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites.

The endoperoxide artemisinin is a current first-line antimalarial and a critical component of the artemisinin-based combination therapies (ACT) recommended by WHO for treatment of Plasmodium falciparum, the deadliest of malaria parasites. However, recent emergence of the artemisinin-resistant P. falciparum urged us to develop new antimalarial drugs. We have shown that synthetic endoperoxides N-89 and its hydroxyl derivative N-251 had high antimalarial activities both in vivo and in vitro. However, the mechanisms including the cellular targets of the endoperoxide antimalarials are not well understood. Thus, in this study, we employed chemical proteomics to survey potential molecular targets of endoperoxides by evaluating P. falciparum proteins capable to associate with endoperoxide structure (N-346, a carboxyamino derivative of N-89). We also analyzed the protein expression profiles of malaria parasites treated with N-89 or N-251 to explore possible changes associated with the drug action. From these experiments, we found that P. falciparum endoplasmic reticulum-resident calcium binding protein (PfERC) had high affinity to the endoperoxide structure (N-346) and was decreased by treatment with N-89 or N-251. PfERC is a member of CREC protein family, a potential disease marker and also a potential target for therapeutic intervention. We propose that the PfERC is a strong candidate of the endoperoxide antimalarial's target.

Toxoplasma gondii is classified into 16 haplogroups based on a worldwide genotyping study of the parasite. However, only a few isolates from Japan were included in this analysis. To conduct more precise genotyping of T. gondii, we examined the genotypes of Japanese isolates in this study. DNA sequences of 6 loci were determined in 17 Japanese isolates and compared with those of strains of 16 haplogroups. As a result, Japanese isolates were classified into four groups. We investigated the virulence of some Japanese isolates and found a highly virulent strain in mice, comparable to that of RH strain, although this Japanese isolate was sister to strains of haplogroup 2, which show moderate virulence in mice. We further investigated whether this high virulence isolate had different virulence mechanism and strategy to adapt to Japanese host from other strains by comparing the virulence-related genes, ROP5, 18 and the immunomodulatory gene, ROP16 of the isolate with those of archetypical strains (GT1, ME49 and VEG). This analysis indicated the high virulence of the isolate in mice was partly explained by gene sequences of ROP5 and ROP16. These findings lead to the elucidation of biodiversity of T. gondii and have potential to optimize the diagnostic protocol.

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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.

Strongyloides stercoralis infection is caused by skin penetration of third-stage larvae (L3s). We studied skin penetration of L3s of Strongyloides ratti using an in vitro assay that has been used previously to study Angiostrongylus cantonensis, an agarose membrane with a temperature gradient, and scanning electron microscopy. Our results revealed that skin penetration of L3s depended on host skin temperature. When the target temperature of the outer liquid was 37°C, more than 80% of L3s penetrated the skin, but penetration was only 60% when the target temperature was 20°C. Thirdstage larvae moved rapidly on the agarose membrane toward optimum temperature area for this parasite, which indicates that L3 has a sensor that is sensitive to temperature changes. Penetration rate for hosts such as cat (36%), dog (32%), and bird (13%) were significantly lower than that for rat (82%). Although we could not establish the reason, L3s seemed to have an ability to differentiate these hosts at the time of penetration. By using scanning electron microscopy, penetration of L3s could be observed within 10 min. We demonstrated thermotaxis of L3 of S. ratti, and this peculiar characteristic seemed to have a close relationship with the process of searching for the host.