AID	Name	Type	Comment
488774	Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: Summary	summary

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

The goal of this screen is to measure the ability of a set of anti-malarial compounds to inhibit the Dd2 strain of P. falciparum. These compounds have been screened in a delayed death phenotype assay and have been identified as inhibitors of the delated death phenotype, by inhibiting the development of the apicoplast in the malarial parasite Plasmodium falciparum (described below)

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

3 ul culture medium was dispensed into black clear-bottom plates (aurora) with multidrop Combi followed by a pin-transfer of 23 ul compounds and positive control into assay plates. (Controls: 1 mM Artemisinin 1:2 dilution in duplicate Multi-Drop dispense of P. falciparum infected RBCs (0.3% parasitemia, 2.5% hematocrit final concentration) followed by 72 hr incubation (37C, 5% CO2). Multi-drop dispense 2 ul 10 X SYBR Green lysis buffer (20 M Tris-HCl, 10 mM EDTA, 0.16% saponin (w/v), 1.6% Triton-X (v/v), 10 x SYBR Green I supplied as 10,000 x final concentration by Invitrogen) into assay plates. Incubated at Room temperature, overnight in the dark and then read using Envision (Perkin Elmer) with fluorescence intensity at 485 nM excitation and 535 nM emission wavelengths.

Compound Ranking:

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.

Grant Number: R21 NS059500