Differential Effect of Atpenin A5 on ROS Production from Wild- Type Mitochondrial Complex II in Human Cancer Cells and Normal Cells

Human mitochondrial complex II is an intriguing enzyme, which has been the focus of medical research during the past few decades since it contributes to pathogenesis of mitochondrial diseases as well as a target for chemotherapy. Reactive oxygen species (ROS) produced by this enzyme has been implicated in both these conditions. While ROS produced from mutated mitochondrial complex II has been implicated in pathogenesis of mitochondrial diseases, ROS produced from pharmacologically inhibited mitochondrial complex II has been implicated in cancer cell death. In this chapter, we show that inhibition of mitochondrial complex II in human cancer cells with atpenin A5 produces detectable levels of ROS while normal cells do not. Thus, this enzyme may be used as a potential target for developing new anticancer drugs to trigger ROS-mediated selective death of cancer cells

Mitochondria of Malaria Parasites as a Drug Target

Mitochondria are organelle, which is found in most eukaryotic cells, and play an important roll in production of many biosynthetic intermediates as well as energy transduction. Recently, it has been reported that mitochondria contribute to cellular stress responses such as apoptosis and autophagy. These functions of mitochondria are known to be essential for survival and maintenance of homeostasis. The mitochondria of malaria parasites are quite different from those of their vertebrate hosts. Because these differences markedly contribute to drug selectivity, we have focused on the Plasmodium mitochondrion to develop antimalarial drugs. Here we summarize recent advances in our knowledge of the mitochondria of malaria parasites and discuss future prospective antimalarial drugs targeting the parasite mitochondrion

Gramicidin S identified as a potent inhibitor for cytochrome bd-type quinol oxidase

AbstractGramicidin S, a cationic cyclic decapeptide, exhibits the potent antibiotic activity through perturbation of lipid bilayers of the bacterial membrane. From the screening of natural antibiotics, we identified gramicidin S as a potent inhibitor for cytochrome bd-type quinol oxidase from Escherichia coli. We found that gramicidin S inhibited the oxidase with IC50 of 3.5μM by decreasing Vmax and the affinity for substrates but showed the stimulatory effect at low concentrations. Our findings would provide a new insight into the development of gramicidin S analogs, which do not share the target and mechanism with conventional antibiotics

Method for the separation of mitochondria and apicoplast from the malaria parasite Plasmodium falciparum

The growth and the survival of the human malaria parasite Plasmodium falciparum are critically dependent on the functions of the two organelles - the mitochondrion and the apicoplast. However, these two organelles have been known to be difficult to separate from each other when they are released from Plasmodium cell. We have been searching for the conditions with which separation of the mitochondrion and the apicoplast is achieved. In this study, we investigated how the two organelle's separation is affected when the pressure of the nitrogen gas to disrupt the Plasmodium cells by nitrogen cavitation method is lowered from the pressure regularly applied (1200 psi). The parasite cell was sufficiently disrupted even when nitrogen cavitation was carried out at 300 psi. The obtained mitochondrial sample was much less contaminated by DNA compared with the sample prepared using the gas at the regular pressure. After the fractionation by Percoll density gradient, the mitochondrion and the apicoplast from the 300 psi cell lysate exhibited different separation profiles. This is the first experimental evidence that indicates the mitochondrion and the apicoplast of P. falciparumare separable from each other

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

Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate–ubiquinone oxidoreductase

AbstractWe have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate–ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132–138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5′ promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions −137 and −104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome