High-throughput Plasmodium falciparum growth assay for malaria drug discovery

Abstract

New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in Plasmodium falciparum, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [3H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a P. falciparum growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC50s) of a diverse set of known antimalarials. The resultant IC50s compared favorably with those obtained in the [3H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI P. falciparum growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.

FIG. 1.

DAPI P. falciparum growth assay design. Compounds and DMSO mock-treatment controls are added to 384-well microtiter plates containing complete medium. Next, synchronized ring-stage cultures of P. falciparum are added to the microtiter plates in the presence and absence of compound, and the plates are incubated under a standard gas environment at 37°C for 72 h. Following the incubation period, DAPI DNA stain is added to detect the parasite DNA in infected erythrocytes. Stained parasite DNA is detected using a fluorescence plate reader or an automated microscope, as described in Materials and Methods. (A) Imaging-based analysis image showing parasite growth for a control well with no drug added. (B) Image showing the effect of a small molecule on parasite growth, resulting in an LD_90.

FIG. 2.

Assessment of the percentage of P. falciparum-infected erythrocytes with fluorescence intensity, using the fluorimetry-based DAPI P. falciparum growth assay. A serial twofold dilution of synchronized ring-stage culture (6% parasitemia) of P. falciparum strain 3D7 was performed, as described in Materials and Methods. The inset shows the assessment of the percentage of P. falciparum-infected erythrocytes with fluorescence intensity, using the imaging-based DAPI P. falciparum growth assay.

FIG. 3.

Determination of the IC₅₀s for chloroquine against 3D7, HB3, and Dd2 by the DAPI P. falciparum fluorimetry-based growth assay in 384-well format. Synchronized ring-stage cultures of erythrocytes infected by P. falciparum strains 3D7 (▪), HB3 (•), and Dd2 (▾) were incubated with different concentrations of chloroquine. Parasite growth was measured at 72 h as described in Materials and Methods. The IC₅₀s are reported in Table 2.

FIG. 4.

Distribution of identified screening positives against P. falciparum strains 3D7, HB3, and Dd2. The Venn diagram shows the number of screening positives that were unique to each strain, and the overlapping regions illustrate the number of screening positives shared by each of the two strains and the number of screening positives shared by all three strains. A screening positive was defined as a compound that inhibited parasite growth to greater than 90% (LD₉₀) of that observed in the no-drug control well. A compound was identified to be selective for a specific strain or strains if it inhibited parasite growth at an LD₉₀ for one strain and an LD₁₀ for another strain(s). The HTS DAPI P. falciparum fluorimetry-based growth assay was performed in which ∼20,000 small molecules at a drug concentration of 10 mM in DMSO were dispensed using a Visio compound transfer robot equipped with a 20-nl, 384-pin head array into 384-well microtiter plates containing 30 μl of medium. Synchronized ring-stage cultures of erythrocytes infected by P. falciparum strains 3D7, HB3, and Dd2 were dispensed in each well, and parasite growth was measured at 72 h as described in Materials and Methods.