Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway

One of the most promising new compound classes in clinical development for the treatment of malaria is the imidazolopiperazines (IZPs) class. Human trials have demonstrated that members of the IZP series, which includes KAF156 (Ganaplacide) and GNF179, are potent and effective against Plasmodium symptomatic asexual blood stage infections. Unlike other commonly used antimalarials, they also prevent transmission and block future infection in animal models. Despite the identification of several Plasmodium falciparum resistance mechanisms including mutations in ER-localized PfCARL (PfEMP65), Acetyl-coA transporter, and PfUGT transporter, IZPs mechanism of action remains unknown. To investigate, we combined in vitro evolution and whole-genome analysis in the model organism Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods, in P. falciparum. S. cerevisiae clones that resist IZP activity carry multiple mutations in genes that encode endoplasmic reticulum(ER)-based lipid homeostasis and autophagy including elo2, elo3, sur2, atg15 and lcb4, as well as ER-based sec66. In Plasmodium, IZPs cause inhibition of protein trafficking, block the establishment of new permeation pathways and result in ER expansion. We also observe sensitization with other secretion inhibitors such as brefeldin A and golgicidin as well as synthetic lethality with PfSEC62. Our data show that IZPs target the secretory pathway and highlight a novel mechanism for blocking parasite growth and development that is distinct from those of standard compounds used to treat malaria. In addition, we provide physiological signatures and hallmarks for inhibitors that work through this mechanism of action and show that IZPs are tool compounds for studying ER-dependent protein processing in different species.