| Assay Method Information | |
| | P. falciparum growth and inhibition assays |
| Description: | P. falciparum 3D7 cells were grown in RPMI media supplemented with 0.5% albuMAX I, human red blood cells to 0.5% hematocrit and 0.5% parasitemia as described (Coteron et al., 2011, J. Med. Chem., 54, 5540-5561). DSMO inhibitor stocks (as above) were diluted in DMSO using a 2 or 3-fold dilution series and then dispensed into media at 10× the final concentrations. A second step into media led to final inhibitor concentrations ranging from 0.001-30 μM and a final DMSO concentration of 0.2%. Parasites in the presence of either DMSO controls or DHODH inhibitors were grown at 37° C. for 72 h before growth was assessed using the SYBR Green method (as described in Bennett et al., 2004, Antimicrob Agents Chemother, 48 (5), 1807-10 with minor modifications as described in Deng, 2014, J Med Chem, 57 (12), 5381-94), which measures fluorescence (ex./em. 485/535 nm) as the output. Data were collected using triplicate technical replicates and where indicated additional biological replicates were collected (the number of biological replicates is provided in parenthesis). The 50% effective concentration (EC50) was determined by fitting data to log (inhibitor) vs. response equation Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((X−Log IC50))) equation in GraphPad Prism. |
| Affinity data for this assay | |
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