Topoisomerase II from XXXX Malaria Parasites: EXPRESSION, PURIFICATION, AND SELECTIVE INHIBITION.

@entity70665 @entity1 @entity5 @entity2177 @entity2879 @entity3964 @entity3505 @entity311 @entity855 @entity1760
Historically, type II topoisomerases have yielded clinically useful drugs for the treatment of @entity5 , but the corresponding enzymes from @entity311 parasites remain understudied. This is due to the general challenges of producing @entity311 proteins in functional forms in heterologous expression systems. Here, we express full-length @entity3964 topoisomerase II (PfTopoII) in a @entity3505 germ cell-free transcription-translation system. Functional activity of soluble PfTopoII from the translation lysates was confirmed through both a plasmid relaxation and a DNA decatenation activity that was dependent on @entity1760 and @entity855 . To facilitate future drug discovery, a convenient and sensitive fluorescence assay was established to follow DNA decatenation, and a stable, truncated PfTopoII was engineered for high level enzyme production. PfTopoII was purified using a DNA affinity column. Existing TopoII inhibitors previously developed for other non- @entity311 indications inhibited PfTopoII, as well as @entity311 parasites in culture at submicromolar concentrations. Even before optimization, inhibitors of bacterial gyrase, @entity70665 , @entity2177 , and @entity2879 exhibited 15-, 57-, and 3-fold selectivity for the malarial enzyme over @entity1 TopoII. Finally, it was possible to use the purified PfTopoII to dissect the different modes by which these varying classes of TopoII inhibitors could trap partially processed DNA. The present biochemical advancements will allow high throughput chemical screening of compound libraries and lead optimization to develop new lines of antimalarials.
