qHTS profiling of the MIPE4 collection as inhibitors of Plasmodium falciparum (HB3) proliferation
Plasmodium falciparum is a virulent malarial parasite that kills 1-2 million people worldwide each year. Transmitted by mosquitoes, the sporozoite form of the parasite enters the blood stream to infect hepatocytes, where they become merozoites that infect red blood cells (erythrocytes). Within erythrocytes, merozoites begin a 72-hour cycle of growth and division, after which the parasite is more ..
Plasmodium falciparum is a virulent malarial parasite that kills 1-2 million people worldwide each year. Transmitted by mosquitoes, the sporozoite form of the parasite enters the blood stream to infect hepatocytes, where they become merozoites that infect red blood cells (erythrocytes). Within erythrocytes, merozoites begin a 72-hour cycle of growth and division, after which the parasite is released to infect new red blood cells. Gametocytes, which form within erythrocytes as well, circulate in the bloodstream and are ingested by the mosquito, thereby completing the life cycle. To profile malarial isolates for differential chemical responses and to identify inhibitors of malarial growth, an assay that measures P. falciparum growth and replication within erythrocytes was employed. This assay uses a DNA binding fluorescent dye (SYBR Green) to detect parasite DNA following cell lysis. Because mature erythrocytes lack DNA, only Plasmodium-derived DNA is detected.
The SYBR Green viability assay was adapted from a method described previously (Plouffe et al., 2008). For screening 3 ul culture medium was dispensed into 1536 well black clear bottom plates (Aurora Biotechnologies) using a Multidrop Combi (Thermo Fisher Scientific Inc.), 23 nL compounds in DMSO were added by a pin tool (Kalypsys), and 5 ul of erythrocytes infected with P. falciparum (0.3% parasitemia, 2.5% hematocrit final concentration) were added. The plates were incubated at 37 C in a humidified incubator in 5% CO2 for 72 h, and 2 ul lysis buffer (20 mM Tris HCl, 10 mM EDTA, 0.16% saponin, 1.6% triton X, 10X SYBR Green I supplied as 10,000X final concentration by Invitrogen) was added to each well. The plates were mixed for 25 sec with gentle shaking and incubated overnight at room temperature in the dark. The following morning, fluorescence intensity at 485(14) nm excitation and 535(25) nm emission wavelengths was measured on an EnVision (Perkin Elmer) plate reader. Plate reads were normalized relative to the control inhibitor (0.29 uM artemisinin) and vehicle (DMSO) wells present on each plate and then corrected by an algorithm using vehicle-only control plates at the beginning and end of the compound plate stack.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
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* Activity Concentration. ** Test Concentration.
Data Table (Concise)