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| | Parasite Proliferation Assay |
| Description: | This parasite proliferation assay, a modification of published DNA intercalating fluorescent dye-based assay, was adapted to 384-well plate format and measures the increase in parasite DNA content using the DNA intercalating dye SYBR Green. Compound 1 exhibited strong potency in both the in vitro biochemical and parasite proliferation assays, which suggests that this compound is potentially acting against PfCDPK1 in vivo. Several other analogs, compounds 7 and 8, exhibited potent enzymatic activity but were inactive in parasite proliferation assays, whereas some analogs displayed low EC50 values in the parasite proliferation assay but were inactive against the enzyme. The EC50 values of all the compounds may be between four- and five-fold higher in the SYBR Green assay compared to the 3H-hypoxanthine-based assay, which measures incorporation of tritium into DNA, as the optimal fluorescent reading conditions required the use of albumax, a lipid-enriched bovine albumin to which small molecules bind. This binding effectively sequesters the drug, thereby reducing the compound concentration and shifting the EC50 to a higher value. A shift in the EC50 due to albumax was also observed for the reference inhibitors chloroquine and artemisinin, which yielded EC50 values of less than 10 nM with the 3D7 strain in the 3H-hypoxanthine-based assay under low protein condition but showed an EC50 in the same range as the best compound in the parasite proliferation assay (70 nM). |
| Affinity data for this assay | |
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