Quantitative high throughput screen to test cell viability of anti-malarial compounds targeting the delayed death phenotype
The biochemical processes that make this organelle essential for erythrocytic stage parasites are not well understood. However, antibiotics, such as azithromycin and tetracycline, which target the apicoplast translational machinery, have a potent antimalarial effect. The killing caused by these drugs is unusual in that it does not appear to affect the first generation of parasites that are exposed to the drug, but rather manifests itself in their progeny. ..more
BioActive Compound: 1
Depositor Specified Assays
The biochemical processes that make this organelle essential for erythrocytic stage parasites are not well understood. However, antibiotics, such as azithromycin and tetracycline, which target the apicoplast translational machinery, have a potent antimalarial effect. The killing caused by these drugs is unusual in that it does not appear to affect the first generation of parasites that are exposed to the drug, but rather manifests itself in their progeny.
We have developed a cell-based assay that measures parasite growth based on the expression of an integrated copy of a firefly luciferase reporter. To detect small molecules that cause this 'delayed death' phenotype, erythrocytes infected with the luciferase-expressing parasites were incubated with compounds for either one or two generations, corresponding to 48 and 96 hours, respectively. Compounds that inhibit parasite growth in the second generation, but not the first, should be enriched in antimalarials that target the apicoplast. Growth inhibition is detected by a decrease in luciferase activity.
The goal of this counterscreen is to measure the cytoxoxicity (against HepG2 cell line) of a set of anti-malarial compounds that target the delayed death pheonotype as identified by the qHTS assay that inhibit the development of the apicoplast in the malarial parasite Plasmodium falciparum.
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: R21 NS059500
Assay Provider: David Fidock and Eric Ekland, Columbia University
Cells were plated at a density of 2000 cells/well/5uL in 1536 well white tissue culture plate with culture medium. Triplicate plates were prepared. Plates were incubated at 37C, 5% CO2 for 5 hours and then 23nL of compounds and positive control were transferred to the assay plates. Tetraoctylammonium bromide was added as a positive control. Plates were then incubated at 37C, 5% CO2 for 48 hours. 5 uL of CellTitler-Glo buffer was added to each well and then incubated for 30 min at room temperature. Luminescence was measured using ViewLux (1 second, slow, 2X).
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)