PMC

Images of high-content screening of parasite hepatic schizonts and parasite growth dynamics. (A) P. yoelii sporozoites were stained 48 hours post infection (hpi) with (αPyHSP70/goat anti-mouse antibodies. The parasites areas were defined by outlines generated by a custom Acapella script (), indicating separate objects. (B) Hoechst 33342 nuclear staining of HepG2-A16-CD81^EGFP cells. Nuclei are delineated using a script as in part A. (C) False color channel merge of A and B. (D and E) Measuring inhibition using parasite area. The IC₅₀ of ovalicin is 736 pM when measured by median parasite area (D), but no drug effect is seen when the overall infection ratio is measured in (E). (F) Correlation of incubation time and schizont size. P. yoelii EEF growth approximates a linear rate with an R² of 0.98 in the HepG2-A16-CD81^EGFP cell line when no test compounds are added. Data are mean +/− SD based on four experimental replicates of approx. 50 infected cells per time point.

SNPs identified in pfcarl by microarray analysis and whole genome sequencing analysis. (A) Stereochemical structures of three compounds (GNF452 (B1, B2, B3), GNF707 (C1, C2, C3), and Pf-5069 (A1, A2) from the IP series () used to generate resistant parasite clones. Detected SNP,s indicated below the compound IDs, are detailed in (SNPs identified by sequencing analysis are red and SNPs identified by both sequencing and microarray analysis are purple) (B) Schematic of pfcarl, showing the conserved regions, including the transmembrane domains. SNP color-coding is the same as in (A) Two regions of the protein are enlarged to show close SNPs and the amino acid sequence conservation of the region across parasite and other species (pfal, P. falciparum; pviv, P. vivax; pyoe, P. yoelii; pber, P. berghei; scer, S. cerevisiae; dmel, Drosophila melanogaster; mmus, Mus musculus; hsap, Homo sapiens).(C) Microarray SNP analysis across pfcarl from two GNF452-resistant clones (GNF452-1 in blue and GNF452-3 in red). The lines trace the p-values indicating whether mapped probes are different in the resistant clone compared with the Dd2 parental strain.

The effect of GNF179 on the liver stage parasite and a comparison with lasalocid, pyrimethamine and atovaquone. (A) High-resolution deconvolution microscopy of the GNF179-treated liver-stage parasites. Columns show Hoechst 33342 staining in blue, αPyHSP70 staining in red and a merge with the host plasma membrane marker CD81-GFP in green. Cultures were treated with 1 μM GNF179 for 48 hr. (Insets) DMSO-treated control parasite at the same scale and time point. Scale bar indicates 10 μm. (B) Chemical structure of GNF1.79. (C) The targets of GNF179 and lasalocid appear to be required throughout development. Compounds were added at 1 μM final concentration at the start (lightest shaded bars), 15 hpi (medium shaded bars) and 27 hpi (darkest shaded bars); all samples were incubated up to an end-point of 51 hpi. Dashed lines represent the control DMSO-treated growth levels at 15, 27 and 51 hpi. Data are mean +/− SD from 2 experimental replicats of approx. 50 infected cells per time point. Las, lasalocid; Pyr, pyrimethamine; Atov, atovaquone. (D) In vivo bioluminescence imaging of representative mice infected with P. berghei and treated with GNF179 (15 mg/kg) or vehicle (no compound), at 6 hpi. In the control, luminescence showing the developing parasite was detected in the liver area from 24 hpi and the lung and gastrointestinal track region from 72 hpi. No luminescence signal was detected from GNF179 or atovaquone (2.5 mg/kg) -treated mice even at the maximum sensitivity setting and no blood stage parasitemia was detected from later blood smear examinations. The luminescence intensity color-coding does not represent absolute values and is individually adjusted for each recording, which is why you can see background signal around the muzzle in some of the control pictures.