Identification of new inhibitors for alternative NADH dehydrogenase (NDH-II).

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans, we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.

Synthesis and biological evaluation of potential modulators of malarial glutathione-S-transferase(s).

Glutathione-S-transferase(s) (E.C.2.5.1.18, GSTs) have been investigated in parasitic protozoans with respect to their biochemistry and they have been identified as potential vaccine candidates in protozoan parasites and as a target in the synthesis of new antiparasitic agents. In a search towards the identification of novel biochemical targets for antimalarial drug design, the area of Plasmodium glutathione metabolism provides a number of promising chemotherapeutic targets. GST activity was determined in various subcellular fractions of malarial parasites Plasmodium yoelii and was found to be localized mainly in the cytosolic fraction (specific activity, c. 0.058 +/- 0.016 micromol/min/mg protein). Hemin, a known inhibitor of mammalian GST(s), maximally inhibited this enzyme from P. yoelii to nearly 86%. In a search towards synthetic modulators of malarial GST(s), 575 compounds belonging to various chemical classes were screened for their effect on crude GST from P. yoelii and 92 compounds belonging to various chemical classes were studied on recombinant GST from P. falciparum. Among all the compounds screened, 83 compounds inhibited/stimulated the enzyme from P. yoelii/P. falciparum to the extent of 40% or more.

Increase in glutathione peroxidase activity in malaria parasite after selenium supplementation.

Glutathione peroxidase (GPx), a key enzyme involved in the detoxification of many peroxides, has been investigated in two malaria parasite species: P. yoelii in vivo (murine malaria) and P. falciparum in vitro (human malaria). We demonstrate the presence of an endogenous GPx activity in these two Plasmodia species. Enzymatic assays and the use of specific substrates and inhibitors allowed us to determine that the activity is selenium dependent. As this activity was shown to be lower in P. falciparum than in P. yoelii, and selenium levels were found to be low in culture medium and culture red blood cells, we hypothesized that a severe selenium deficiency could be responsible for this difference. After selenium supplementation, with either sodium selenite or selenocystine, we observed an increase in growth of P. falciparum only in with sodium selenite, whereas higher GPx activities were noted in parasites grown in media supplemented with both. An increase in GPx activities was also observed in parasites that had undergone an experimental oxidative stress with TBOOH. As the erythrocyte is unable to synthesize new proteins, these results provide further evidence for the existence of an endogenous parasitic selenium-dependent glutathione peroxidase.