title: 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 understudied. 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 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 submicromolar concentrations. Even before optimization, inhibitors of bacterial gyrase, GSK299423, ciprofloxacin, and etoposide exhibited 15-, 57-, and 3-fold selectivity 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.
authors.affiliation: From the Department of Chemistry, University of Washington, Seattle, Washington 98195.
authors.forename: Devaraja G
authors.initials: DG
authors.lastname: Mudeppa
authors.affiliation: From the Department of Chemistry, University of Washington, Seattle, Washington 98195.
authors.forename: Shiva
authors.initials: S
authors.lastname: Kumar
authors.affiliation: From the Department of Chemistry, University of Washington, Seattle, Washington 98195.
authors.forename: Sreekanth
authors.initials: S
authors.lastname: Kokkonda
authors.affiliation: From the Department of Chemistry, University of Washington, Seattle, Washington 98195.
authors.forename: John
authors.initials: J
authors.lastname: White
authors.affiliation: From the Department of Chemistry, University of Washington, Seattle, Washington 98195 rathod@chem.washington.edu.
authors.forename: Pradipsinh K
authors.initials: PK
authors.lastname: Rathod
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology*; D014908:Triticum / Q000235:genetics
chemical_list: D059005:Topoisomerase II Inhibitors; D004250:DNA Topoisomerases, Type II
keywords: DNA enzyme; cell-free; drug discovery; fluorescence; plasmodium; translation; wheat germ