Development and optimization of a novel 384-well anti-malarial imaging assay validated for high-throughput screening

Abstract

With the increasing occurrence of drug resistance in the malaria parasite, Plasmodium falciparum, there is a great need for new and novel anti-malarial drugs. We have developed a 384-well, high-throughput imaging assay for the detection of new anti-malarial compounds, which was initially validated by screening a marine natural product library, and subsequently used to screen more than 3 million data points from a variety of compound sources. Founded on another fluorescence-based P. falciparum growth inhibition assay, the DNA-intercalating dye 4',6-diamidino-2-phenylindole, was used to monitor changes in parasite number. Fluorescent images were acquired on the PerkinElmer Opera High Throughput confocal imaging system and analyzed with a spot detection algorithm using the Acapella data processing software. Further optimization of this assay sought to increase throughput, assay stability, and compatibility with our high-throughput screening equipment platforms. The assay typically yielded Z'-factor values of 0.5-0.6, with signal-to-noise ratios of 12.

Figure 1.

Representative images of wells containing the 3D7 parasite and varying concentrations of artemisinin. Images obtained on the Opera with the number of spots determined using the Acapella software with a spot detection algorithm. Relative % inhibition values were also calculated.

Figure 2.

Comparison of minimum inhibition (0.4% DMSO) and maximum inhibition (2 μM artemisinin) control responses at varying concentrations of Dd2 parasites (infected red blood cells) using 0.25%, 0.5%, and 1.0% hematocrit. Each point represents the mean ± SEM of eight wells. Each data point represents the mean ± SEM of 16 wells.

Figure 3.

Comparison of permeabilization buffer and paraformaldehyde fixation on minimum inhibition response (solid squares), maximum inhibition response (solid triangles), and Z' factor (red circles) of Plasmodium falciparum Dd2 strain parasites at varying parasitemia and 0.3% hematocrit. Combinations of Tris-HCl buffer with fixation (A), Tris-HCl buffer without fixation (B), phosphate-buffered saline (PBS) buffer with fixation (C), and PBS buffer without fixation (D) are shown. Where a linear relationship between parasitemia and minimum inhibition response was observed, linear regression (dotted) line and r² values were included.

Figure 4.

Stability of signal after 4′,6-diamidino-2-phenylindole (DAPI) staining of assay plates containing the 3D7 strain of parasite. The response to in-plate controls of plates (mean ± SEM of 16 wells) read up to 24 hours after DAPI staining is shown in (A). Dose response curves of artemisinin read 24 and 48 hours after DAPI staining (B). Each point represents the mean ± SEM of 4 wells.

Figure 5.

Evaluation of in-plate minimum inhibition (0.4% dimethylsulphoxide [DMSO]) and maximum inhibition (2 μM artemisinin) controls during a 7-day screening run using 3D7 parasites. In-plate controls (mean ± SEM of 16 wells) within a 48-plate batch as measured with image analysis were compared (A). Average in-plate control values (mean ± SEM of 256 wells) for each screening day as measured using spot detection image analysis (B) or total well fluorescence (C). Average signal-to-background ratio for each screening day using both detection methods were also calculated (D).

Figure 6.

Comparison of dose response curves of a representative marine natural product against 3D7 parasites as measured using spot detection image analysis (solid squares) or total well fluorescence (open triangles).