Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma 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.

Two Plasmodium 6-Cys family-related proteins have distinct and critical roles in liver-stage development.

The 10 Plasmodium 6-Cys proteins have critical roles throughout parasite development and are targets for antimalaria vaccination strategies. We analyzed the conserved 6-cysteine domain of this family and show that only the last 4 positionally conserved cysteine residues are diagnostic for this domain and identified 4 additional "6-Cys family-related" proteins. Two of these, sequestrin and B9, are critical to Plasmodium liver-stage development. RT-PCR and immunofluorescence assays show that B9 is translationally repressed in sporozoites and is expressed after hepatocyte invasion where it localizes to the parasite plasma membrane. Mutants lacking B9 expression in the rodent malaria parasites P. berghei and P. yoelii and the human parasite P. falciparum developmentally arrest in hepatocytes. P. berghei mutants arrest in the livers of BALB/c (100%) and C57BL6 mice (>99.9%), and in cultures of Huh7 human-hepatoma cell line. Similarly, P. falciparum mutants while fully infectious to primary human hepatocytes abort development 3 d after infection. This growth arrest is associated with a compromised parasitophorous vacuole membrane a phenotype similar to, but distinct from, mutants lacking the 6-Cys sporozoite proteins P52 and P36. Our results show that 6-Cys proteins have critical but distinct roles in establishment and maintenance of a parasitophorous vacuole and subsequent liver-stage development.

Selective Cytotoxicity of Dihydroorotate Dehydrogenase Inhibitors to Human Cancer Cells Under Hypoxia and Nutrient-Deprived Conditions.

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.

Inhibition of trypanosome alternative oxidase without its N-terminal mitochondrial targeting signal (ΔMTS-TAO) by cationic and non-cationic 4-hydroxybenzoate and 4-alkoxybenzaldehyde derivatives active against T. brucei and T. congolense.

African trypanosomiasis is a neglected parasitic disease that is still of great public health relevance, and a severe impediment to agriculture in endemic areas. The pathogens possess certain unique metabolic features that can be exploited for the development of new drugs. Notably, they rely on an essential, mitochondrially-localized enzyme, Trypanosome Alternative Oxidase (TAO) for their energy metabolism, which is absent in the mammalian hosts and therefore an attractive target for the design of safe drugs. In this study, we cloned, expressed and purified the physiologically relevant form of TAO, which lacks the N-terminal 25 amino acid mitochondrial targeting sequence (ΔMTS-TAO). A new class of 32 cationic and non-cationic 4-hydroxybenzoate and 4-alkoxybenzaldehyde inhibitors was designed and synthesized, enabling the first structure-activity relationship studies on ΔMTS-TAO. Remarkably, we obtained compounds with enzyme inhibition values (IC50) as low as 2 nM, which were efficacious against wild type and multidrug-resistant strains of T. brucei and T. congolense. The inhibitors 13, 15, 16, 19, and 30, designed with a mitochondrion-targeting lipophilic cation tail, displayed trypanocidal potencies comparable to the reference drugs pentamidine and diminazene, and showed no cross-resistance with the critical diamidine and melaminophenyl arsenical classes of trypanocides. The cationic inhibitors 15, 16, 19, 20, and 30 were also much more selective (900 - 344,000) over human cells than the non-targeted neutral derivatives (selectivity >8-fold). A preliminary in vivo study showed that modest doses of 15 and 16 reduced parasitaemia of mice infected with T. b. rhodesiense (STIB900). These compounds represent a promising new class of potent and selective hits against African trypanosomes.