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

Abstract Historically, type II topoisomerases have yielded clinically useful drugs for the treatment of bacterial infections and cancer, but the corresponding enzymes from malaria parasites remain unexplored. This is due to the general challenges of producing malaria proteins in functional forms in heterologous expression systems. Here, we express full-length Plasmodium falciparum topoisomerase II (PfTopoII) in a wheat-germ in vitro 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 magnesium and ATP. 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-malaria indications inhibited PfTopoII as well as malaria parasites in culture at sub-uM concentrations. Even before optimization, inhibitors of bacterial gyrase, GSK299423, ciprofloxacin and etoposide exhibited 15, 57 and 3-fold selectively for the malarial enzyme over human 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.