A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3′,4′-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

Abstract

Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca 2؉ . This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3=,4=-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.

<strong>A portable image-based cytometer for rapid malaria detection and quantification</strong>

“…Next, dose-response curves for chloroquine, artemisinin and cycloheximide estimated through image-based cytometer and standard flow cytometer are shown in Fig 8. Despite minor differences, IC 50 values defined as half maximal inhibitory concentration calculated through these methods were similar (and mostly in agreement with reported values elsewhere [57][58][59]) as listed in Table 3. These results demonstrate the feasibility of our proposed system for inexpensive, highly sensitive and reliable, high throughput screening of antimalarials.…”

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

“…To study if DV membrane permeabilization upon heat shock is an early cellular event associated with death, we measured mitochondrial accumulation of JC-1 as a proxy of parasite survival ( Paloque et al, 2016 ; Peatey et al, 2015 ; Lee et al, 2014 ; Rathore et al, 2011 ). JC-1 produces green fluorescence in the cytosol, while mitochondrial accumulation is associated with an increase in red fluorescence, which requires maintenance of the mitochondrial membrane potential.…”

Imaging flow cytometry analysis of intracellular pathogens

“…Recently, Lee et al [56; 128] provided a detailed description of phenotype-based IFC assays to analyze a library of chemicals disrupting the integrity of the digestive vacuole of Plasmodium falciparum; these positive results of drug screening were further validated by confocal microscopy. It should be noted that these studies used colocalization of Fluo-4-AM and DNA dye-stained P. falciparum by IFC and demonstrated the capabilities of the method.…”