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.

Adaptive evolution of malaria parasites in French Guiana: Reversal of chloroquine resistance by acquisition of a mutation in pfcrt

“…French Guianan culture-adapted clones selected for whole-genome sequencing were assayed in a controlled gas atmosphere with 5% (vol/vol) CO 2 , 10% (vol/vol) O 2 , and 85% (vol/vol) N 2 . For 7G8 and D10 clones, in vitro 72-h drug dose-response assays were performed, and parasite growth was determined using flow cytometry with SYBR Green I and MitoTracker Deep Red (42). Mean IC 50 ± SEM values and numbers of repeats are listed in Table S1.…”

Subtle Changes in Endochin-Like Quinolone Structure Alter the Site of Inhibition within the Cytochrome bc 1 Complex of Plasmodium falciparum

“…Upon recrudescence, the population of parasites was cloned by limiting dilution (0.8 infected red blood cell [RBC]/ well) at 1.8% hematocrit in a 96-well flat-bottom tissue culture plate in the presence of 70 nM ELQ-300. On day 21 of cloning, 5 l of parasite culture from each well was mixed with a solution containing 0.1 l/ml SYBR green I and 0.1 M MitoTracker Deep Red (Life Technologies) and incubated for 20 min prior to an analysis of parasitemia on an Accuri C6 flow cytometer (26).…”

Flow Cytometry-Based Analysis of Artemisinin-Resistant Plasmodium falciparum in the Ring-Stage Survival Assay

“…1C) made it impossible to accurately identify viable parasites using SYBR alone. More recently, the combination of SYBR and MTDR was used to quantify viable parasites in erythrocytes following exposure to chloroquine and azithromycin in conventional drug assays (14).…”