AID	Name	Type	Comment
624280	Broad Institute Inhibition of T.cruzi replication in culture Inhibitor Probe Project	summary	summary

Keywords: Plasmodium, malaria, Plasmodium falciparum


Assay Overview:
In vitro activity against erythrocytic stages of P. falciparum.

Expected Outcome: Compounds significantly suppressing Alamar blue, and P. falciparum proliferation, are considered active.

In vitro activity against erythrocytic stages of P. falciparum was determined using a 3H-hypoxanthine incorporation assay (Desjardins et al. 1979, Matiel and Pink. 1990), using the drug sensitive NF54 strain (Schipol Airport, The Netherlands, Ponnudurai et al. 1981) and the standard drug chloroquine (Sigma C6628). Compounds were dissolved in DMSO at 10 mg/ml and added to parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/l), NaHCO3 (2.1 g/l), neomycin (100 U/ml), AlbumaxR (5 g/l) and washed human red cells A+ at 2.5% haematocrit (0.3% parasitaemia). Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 ig/ml were prepared. The 96-well plates were incubated in a humidified atmosphere at 37 degrees C; 4% CO2, 3% O2, 93% N2. After 48 h 50 il of 3H-hypoxanthine (=0.5 iCi) was added to each well of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Betaplatetrade mark cell harvester (Wallac, Zurich, Switzerland), and the red blood cells transferred onto a glass fibre filter then washed with distilled water. The dried filters were inserted into a plastic foil with 10 ml of scintillation fluid, and counted in a Betaplatetrade mark liquid scintillation counter (Wallac, Zurich, Switzerland). IC50 values were calculated from sigmoidal inhibition curves by linear regression (Huber 1993) using Microsoft Excel. Chloroquine and artemisinin are used as control.


Desjardins, R. E., C. J. Canfield, J. D. Haynes, and J. D. Chulay. 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 16:710-718.
Matile, H., and J. R. L. Pink. 1990. Plasmodium falciparum malaria parasite cultures and their use in immunology. In I. Lefkovits and B. Pernis (ed.), Immunological Methods. Academic Press, San Diego.
Ponnudurai, T., Leeuwenberg, A. D. and Meuwissen, J. H. (1981) Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in vitro culture. Tropical and Geographical Medicine 33, 50-54.
Huber, W. Koella, J.C. 1993. A comparison of the three methods of estimating EC50 in studies of drug resistance of malaria parasites. Acta Trop. 55, 257-261.

Grant Number: 1 R03 MH085673-01