| Human African trypanosomiasis, caused by the Trypanosoma brucei protozoan parasite, is fatal when left untreated. Malaria, caused by Plasmodium parasites, kills nearly 1 million people annually. Resistance against current chemotherapies and the lack of effective vaccines have made eradication difficult to achieve. Consequently, it has been important to identify new targets for drug development. Topoisomerases, enzymes that catalyze reactions that change DNA topology, are excellent targets in bacteria. Topoisomerase poisons, a special class of inhibitors, bind to the enzyme-DNA Michaelis complex, preventing the enzyme from dissociating from its substrate and promoting "cleavable complex" formation. DNA breaks are formalized when replication machinery collides into the topoisomerase or when a rapid denaturant is added in vitro. Poisons allow us to study the function of topoisomerases within the cell.;Trypanosomes have a single mitochondrion with a unique mitochondrial DNA, known as kinetoplast DNA (kDNA), a topologically complex network. T. brucei has an unusual type IA topoisomerase, dedicated to kDNA metabolism. We found that two compounds, recently identified as poisons of bacterial topoisomerase IA, are trypanocidal in the low micromolar range and promote the formation of linearized minicircles. Surprisingly, however, band depletion studies showed that it is topoisomerase IImt, and not topoisomerase IAmt, that is trapped.;The malaria parasite contains an organelle known as the apicoplast, which harbors a 35-kb circular plastid DNA molecule (p1DNA) that is served by the gyrase enzyme. With our new robust methods, we identified four forms of p1DNA: negatively supercoiled covalently closed, linear, nicked and a newly identified form IV. Exposure of parasites to ciprofloxacin (a specific gyrase poison) leads to an increase in 35-kb protein-bound linear forms. There is a dose-dependent increase in the linears with either ciprofloxacin or etoposide, a general type II topoisomerase poison, and nearly 1/3 of the p1DNA molecules are linearized at high concentrations. Quinolones, a class of gyrase poisons, had a range of antimalarial activity and gyrase poisoning activity, and these activities generally correlated with each other, with one exception. Ciprofloxacin, one of the most potent compounds against the parasite, was not one of the most potent in generating cleavable complexes. |
