We observed no new genetic changes [either single nucleotide polymorphisms (SNPs) or copy number variations (CNVs)] inpfmdr1,pfcrt, or any other knownPlasmodiumdrug resistance gene. malariae,Plasmodium ovale,Plasmodium vivaxand the simian parasitePlasmodium knowlesi(2).Plasmodiumis naturally transmitted by the bite of an infected femaleAnophelesmosquito. During the bite, the sporozoites are injected with the mosquitos saliva and find their way to the host liver. There the parasites multiply asexually as exo-erythrocytic forms (EEFs) during an asymptomatic incubation period of ~1 week prior to emerging into the blood stream. This initiates the asexual erythrocytic cycle that is responsible for Uramustine disease manifestations. While the EEFs of somePlasmodiumspecies have a limited life span, in the case ofP. ovaleandP. vivaxthe parasite can persist within the liver as dormant hypnozoite for several months to years (3). Upon hypnozoite reactivation via an unknown mechanism, parasites can Uramustine re-populate the blood with rapidly multiplying parasites that can Uramustine cause pathology. As a consequence, the persistence of hypnozoites represents a formidable barrier to the eradication of malaria. The only drugs with significant activity against proliferating EEFs and hypnozoites are the 8-aminoquinolines, including primaquine, pamaquine and tafenoquine (4,5). Primaquine is the only treatment recommended by the World Health Organization to Uramustine eliminate liver stages (4). However, 8-aminoquinolines can all cause dangerous levels of methemoglobinemia as a side effect in patients with glucose-6-phosphate dehydrogenase deficiency (6,7), a common adaptive genetic condition in malaria-endemic regions. Resistance to this chemical class has also been reported, further increasing the need to find alternative drugs (8). To identify leads with liver stage activity, we refined anin vitroassay withPlasmodium yoeliisporozoites and HepG2-A16-CD81EGFPcells (9) to screen a library of compounds known to be active againstP. falciparumblood stages. The data revealed substantial differences in activity between blood and liver stages of the parasite and identified a chemically diverse group of molecules active against both. == Measuring sporozoite infection ratios and schizont size using high-content imaging == Only 1% of liver cells typically become infected by sporozoitesin vitro, and because we needed to screen Rabbit Polyclonal to Smad2 (phospho-Thr220) many thousands of compounds, we created a robust, reproducible assay and image analysis to automate data collection. Eight thousand sporozoites freshly dissected from infectedAnopheles stephensimosquitoes were added to each well of a 384-well plate, containing ~15,000 hepatoma cells per well. After a two-day incubation in the presence of compound the host cell and parasite nuclei were labeled with Hoechst 33342 and the parasites were labeled with PyHSP70 antibodies. A Perkin Elmer high-content imaging system captured 25 images (Fig. 1) for each well of the 384-well plate. A custom Acapella(PerkinElmer) script parameterized for this assay was used to analyze the images on the basis of morphology and fluorescence intensity for each liver cell nucleus and each parasite. The infection ratio was the ratio between parasite number (PyHSP70 positive) and the number of host nuclei per well. == Fig. 1. == Images of high-content screening of parasite hepatic schizonts and parasite growth dynamics.(A)P. yoeliisporozoites were stained 48 hours post infection (hpi) with (PyHSP70/goat anti-mouse antibodies. The parasites areas were defined by outlines generated by a custom Acapella script (31), indicating separate objects.(B)Hoechst 33342 nuclear staining of HepG2-A16-CD81EGFPcells. 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 IC50of 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. yoeliiEEF growth approximates a linear rate with anR2of 0.98 in Uramustine the HepG2-A16-CD81EGFPcell line when no test compounds are added. Data are mean +/ SD based on four experimental replicates of approx. 50 infected cells per time point. To quantify the effects of the test compounds, we determined median parasite size to be preferable to the infection ratio, because infection ratio may be affected by drug toxicity to host cells and host cell division during the incubation (e.g., a 1% infection ratio may drop to 0.30.6% after 48 hours exposure to a cell-toxic compound). Measuring parasite size also enabled the identification of compounds that arrest parasite growth without reducing the infection ratio. For example, the compound ovalicin reduced schizont size (Fig. 1D) with an IC50of 736 pM, but had no.