High content live cell imaging for the discovery of new antimalarial marine natural products

Abstract

Background: The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum.

Methods: A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope.

Results: Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts.

Conclusion: Collectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials.

Figure 1

A schematic diagram of natural products antimalarial screen. Marine organisms were collected from the reef off the Fijian Islands. Extracts were aliquoted into 96-well microtiter plates and cultures were subsequently added. A SYBR Green I based assay was used as a primary screen of 2685 extracts. Extracts that inhibited P. falciparum growth were subjected to a high-content live cell imaging secondary screen.

Figure 2

IC₅₀ graph of potent antimalarial extracts. An inhibitory concentration at 50% growth (IC₅₀) graph of extracts inhibiting parasites at concentrations 6.65 ng/ml to 0.85 μg/ml.

Figure 3

Morphological analysis of P. falciparum with IC₈₀ concentrations of extracts. Infected erythrocytes were synchronized and live parasites were stained with RNA probe 132A. Figure 3a are images of the P. falciparum 3D7 strain without any drug treatment at the ring and mature stage (control). Figure 3b displays the phenotype of extract G-0490-3, stunted growth of parasites at the trophozoite stage. Figure 3c displays the phenotype of extract G-0580-3, arrowheads indicate the enlarged food vacuoles at the early stages of the cell cycle. Figure 3d and Figure 3e, extract G-0005-4 and G-0049-4 respectively, inhibit parasite life cycle progression, decrease hemozoin, and hinder egression.

Figure 4

Morphological analysis of various strains of P. falciparum. Treatments with extract G-0021-3 on synchronized parasites at the ring and mature stage. A) chloroquine sensitive strain, HB3; and B) chloroquine resistant strain, Dd2.

Figure 5

Morphological analysis of P. falciparum. Images of synchronized parasites at the ring and mature stage with bromophycolide A treatment at IC₈₀ concentrations. Untreated control sample is shown in Figure 3a.