Newly Identified Antibacterial Compounds Are Topoisomerase Poisons in African Trypanosomes

Abstract

Human African trypanosomiasis, caused by the Trypanosoma brucei protozoan parasite, is fatal when left untreated. Current therapies are antiquated, and there is a need for new pharmacologic agents against T. brucei targets that have no human ortholog. Trypanosomes have a single mitochondrion with a unique mitochondrial DNA, known as kinetoplast DNA (kDNA), a topologically complex network that contains thousands of interlocking circular DNAs, termed minicircles (ϳ1 kb) and maxicircles (ϳ23 kb). Replication of kDNA depends on topoisomerases, enzymes that catalyze reactions that change DNA topology. T. brucei has an unusual type IA topoisomerase that is dedicated to kDNA metabolism. This enzyme has no ortholog in humans, and RNA interference (RNAi) studies have shown that it is essential for parasite survival, making it an ideal drug target. In a large chemical library screen, two compounds were recently identified as poisons of bacterial topoisomerase IA. We found that these compounds are trypanocidal in the low micromolar range and that they promote the formation of linearized minicircles covalently bound to protein on the 5 end, consistent with the poisoning of mitochondrial topoisomerase IA. Surprisingly, however, band depletion studies showed that it is topoisomerase II mt , and not topoisomerase IA mt , that is trapped. Both compounds are planar aromatic polycyclic structures that intercalate into and unwind DNA. These findings reinforce the utility of topoisomerase II mt as a target for development of new drugs for African sleeping sickness.Human African trypanosomiasis, also known as African sleeping sickness, is a protozoal infection caused by Trypanosoma brucei that is fatal without treatment (3). Parasites are transmitted to humans via the tsetse fly, which is found exclusively in sub-Saharan Africa and thus limits the geographic distribution of the disease. Currently, there are an estimated 50,000 to 70,000 cases (25). Despite the lethality of sleeping sickness, the drugs currently available for its treatment are toxic and they require parenteral administration (10). In addition to these obstacles, resistance has been a growing concern, and availability and cost are major problems for the population affected. In light of these issues, new drugs for African trypanosomiasis are in demand. The recently completed T. brucei genome sequence has made possible the identification of potential drug targets which are essential to trypanosomes and have no human ortholog (2).Trypanosomes have a single mitochondrion with an unusual mitochondrial DNA, known as kinetoplast DNA (kDNA). The kinetoplast is a massive and topologically complex structure, comprised of thousands of interlocked ϳ1-kb DNA minicircles and a few dozen ϳ23-kb maxicircles (structure and replication of kDNA are reviewed in reference 16). This intricate network is in striking contrast with the monomeric circular DNA molecule found in several copies in each human mitochondrion. Replication of kDNA requires the release of individual covalently closed m...

Independence from Kinetoplast DNA Maintenance and Expression Is Associated with Multidrug Resistance in Trypanosoma brucei In Vitro

“…Other compounds have been proposed to act, at least in part, by disrupting the replication of kDNA through the inhibition of key enzymes, such as the topoisomerases (14–18). Until now, attempts to quantify the degree to which compounds target the kinetoplast have relied upon comparing the sensitivities of divergent strains and species (16, 19–21).…”

6-Arylpyrazine-2-carboxamides: A New Core for Trypanosoma brucei Inhibitors

“…This enzyme has no orthologue in humans, and RNA interference studies have shown that it is essential for parasite survival, making it an ideal drug target. These compounds were found to be trypanocidal in the low micromolar range (175).…”

3,4-Dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I

“…32 These compounds have also been shown to be poison inhibitors of the mitochondrial type II topoisomerase in African trypanosomes. 33 Recently, phenanthrene analogues were reported to target Streptococcus pneumoniae topoisomerase I without affecting human cell viability. 34 These inhibitors of S. pneumoniae topoisomerase I were found to be DNA intercalators and did not affect the growth of E. coli significantly.…”