Validation
of exoerythrocytic-stage activity using an established flow-cytometry-based
assay. (a) Flow cytometry plots measuring traversal and invasion (at
2 h postinfection) and EEF frequency and development (at 48 h postinfection)
of exoerythrocytic-stage malaria parasites in Huh7.5.1 cells as previously
described for three of the most potent compounds. Cytochalasin D was
used as a positive control for traversal and invasion; KDU691 was
used as a positive control for EEF frequency, and atovaquone was used
as a positive control for EEF frequency and development. While traversal
was measured by the percentage of rhodamine–dextran single-positive
cells, invasion was measured by the percentage of Pb-GFP single-positive
cells at 2 h postinfection. At 48 h postinfection, EEF frequency was
measured by the percentage of Pb-GFP-positive cells, and EEF development
was measured by the relative mean fluorescence intensity (MFI). Representative
flow cytometry plots are shown. Atovaquone was tested at 1 μM
(due to slight cytotoxicity at 10 μM in Huh7.5.1 cells, Figure S4), and cytochalasin D and KDU691 were
tested at 10 μM. (b) Mean and SEM are shown graphically from
the traversal/invasion, EEF frequency, and EEF size control experiments
shown in panel (a) (cytochalasin D, atovaquone, and KDU691, respectively).
Values are normalized to the DMSO control. (c) Exoerythrocytic-stage
traversal, invasion, EEF frequency, and EEF development are shown
for MMV666693, MMV007160, and MMV665916. Mean and SEM from three replicate
experiments are shown. MMV66693 was tested at 1 μM (due to slight
cytotoxicity at 10 μM in Huh7.5.1 cells, Figure S4), and MMV007160 and MMV665916 were tested at 10
μM.