In vitro Evaluation of Antimalarial Activity

The activity of compounds against P. berghei-infected Huh7 cells was assessed in vitro by bioluminescence, as previously described^{58 (link)}. Briefly, Huh7 cells were seeded as indicated above on the day prior to infection. Compound stock solutions were prepared in DMSO. Test concentrations were obtained by serial dilution of compound stock solutions in infection medium, i.e. complete culture medium supplemented with 50 µg/mL of gentamicin and 0.8 µg/mL of fungizone. After 1 h of incubation with selected compound dilutions, 1 × 10⁴ luciferase-expressing P. berghei sporozoites were added per well. Plates were centrifuged and incubated for 46 h at 37 °C, 5% CO₂. At this timepoint, the impact of the compounds on cell viability was assessed by the AlamarBlue (Invitrogen) assay, according to the manufacturer’s recommendations. Next, compounds’ impact on parasite load was assessed by bioluminescence, employing the Firefly Luciferase Assay Kit 2.0 (Biotium).