Assessing antimalarial cidality in Plasmodium falciparum parasites.

摘要

The rise of resistance to commonly used antimalarial medications, such as chloroquine, has exacerbated a global health burden. Years of study of chloroquine resistance has focused on low concentrations of drug that result in an inhibition of parasite growth, known as cytostatic activity. In a clinical setting, the concentration of chloroquine peaks at much higher concentrations and kills parasites. Defined as "cytocidal" activity, the ability of higher concentrations of antimalarials to kill parasites has only recently been explored. Comparing in vitro chemical models of hemozoin inhibition (the predicted mechanism of action of drugs like chloroquine) to the in vivo activities supports that there is a different target at cytocidal concentrations. To probe these possible targets, a novel activity-based protein profiling probe was synthesized, relying on copper-catalyzed azide-alkyne cycloaddition to conjugate a photoaffinity probe of chloroquine to biotin, for mass-spectroscopy proteomics, and fluorophores, for subcellular-localization by fluorescence microscopy.;The rise of resistance to commonly used antimalarial medications, such as chloroquine, has exacerbated a global health burden. Years of study of chloroquine resistance has focused on low concentrations of drug that result in an inhibition of parasite growth, known as cytostatic activity. In a clinical setting, the concentration of chloroquine peaks at much higher concentrations and kills parasites. Defined as "cytocidal" activity, the ability of higher concentrations of antimalarials to kill parasites has only recently been explored. Comparing in vitro chemical models of hemozoin inhibition (the predicted mechanism of action of drugs like chloroquine) to the in vivo activities supports that there is a different target at cytocidal concentrations. To probe these possible targets, a novel activity-based protein profiling probe was synthesized, relying on copper-catalyzed azide-alkyne cycloaddition to conjugate a photoaffinity probe of chloroquine to biotin, for mass-spectroscopy proteomics, and fluorophores, for subcellular-localization by fluorescence microscopy. In addition to understanding the cellular effects of cytocidal drug activity, the concept of cytocidality is integrated into the search for new combination therapies. High throughput screening of combinations of known antimalarials and compounds currently under clinical review identified a series of compounds that showed possible synergistic activities with known antimalarials. One class of these compounds was found to be phosphitidylinositol 3-phosphate kinase (PI3K) inhibitors, known to bind to human PI3K and effect its action in autophagy and other signaling pathways. Autophagy has only recently been explored in Plasmodium falciparum,, and has been implicated in the cytocidal activity of chloroquine. Therefore, these PI3K inhibitors were paired with each of the drugs of the currently used therapy Coartem (artemether and lumefantrine), and their interaction was defined as synergistic, additive, or antagonistic under both the cytostatic and cytocidal conditions. Cytocidal, but not cystatic, synergy was observed when another PI3K inhibitor, GSK2126458, was paired with either artemether or lumefantrine in both chloroquine sensitive and chloroquine resistant strains. Understanding how drug activities change under cytostatic vs. cytocidal conditions will facilitate a better understanding of resistance and the identification of novel therapies.