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Topoisomerase II from Human Malaria Parasites: EXPRESSION, PURIFICATION, AND SELECTIVE INHIBITION.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2015 Aug 14; Vol. 290 (33), pp. 20313-24. Date of Electronic Publication: 2015 Jun 08. - Publication Year :
- 2015
-
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.<br /> (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- Amino Acid Sequence
Animals
Cell-Free System
DNA Topoisomerases, Type II chemistry
DNA Topoisomerases, Type II drug effects
DNA Topoisomerases, Type II isolation & purification
Molecular Sequence Data
Sequence Homology, Amino Acid
Triticum genetics
DNA Topoisomerases, Type II metabolism
Plasmodium falciparum enzymology
Topoisomerase II Inhibitors pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 290
- Issue :
- 33
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 26055707
- Full Text :
- https://doi.org/10.1074/jbc.M115.639039