title: Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches.
abstract: Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for "delayed death" compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC(50) in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.
authors: Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, 701 W. 168th St., New York, NY 10032, USA. ee2196@columbia.edu
authors: Eric H
authors: EH
authors: Ekland
authors: Jessica
authors: J
authors: Schneider
authors: David A
authors: DA
authors: Fidock
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000962:Antimalarials / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D051379:Mice; D015388:Organelles / Q000187:drug effects*; D018512:Parasitemia; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010963:Plasmodium falciparum / Q000166:cytology / Q000187:drug effects; D010967:Plasmodium yoelii / Q000166:cytology / Q000187:drug effects; D015203:Reproducibility of Results
chemical_list: D000900:Anti-Bacterial Agents; D000962:Antimalarials