P. falciparum Growth Inhibition.

The in vitro effects of reported compounds were evaluated by the SYBR Green I assay. Briefly, ring stage parasite cultures (100 μL per well, at 1% hematocrit and 1% parasitemia) were grown for 72 h in the presence of increasing concentrations of the inhibitor under reduced oxygen conditions (5% CO₂, 5% O₂, and 90% N₂) at 37 °C. After 72 h in culture, growth was determined by DNA quantitation using SYBR Green I as described previously.⁴¹ The half-maximal effective concentration (EC₅₀) values were calculated with GraphPad Prism (GraphPad Software, Inc.) using nonlinear regression curve fitting. The reported values represent averages of at least three independent experiments performed in triplicate, using 10-point serial dilutions, with standard errors of the mean (SEM). The range for serial dilutions was adjusted accordingly for each test compound after the first screening to set the EC₅₀ value in the middle of the concentration range. The final concentration of DMSO (vehicle) did not exceed 0.02%.

In vitro potencies of the Malaria Box and Pathogen Box compounds were assessed by measuring growth inhibition against P. falciparum 3D7-WT and 3D7-R1 strains at 1 μM. Chlorajaponilide C (20 nM) was used as a control for the reduction of in vitro potency while artemisinin (200 nM) was used as a control for growth inhibition since it is not affected by the presence of resistance to chlorajaponilide C. All conditions were set in 96-well half-area dark plates (100 μL/well at 1% hematocrit and 1% parasitemia), incubated for 72 h, and analyzed by the SYBR Green I assay as described above. The percentage of growth was normalized to untreated control parasites. Uninfected erythrocytes were used for background determination. Data were analyzed using GraphPad Prism (GraphPad Software, Inc.).