Role of Plasmodium falciparum digestive vacuole plasmepsins in the specificity and antimalarial mode of action of cysteine and aspartic protease inhibitors.
Hemoglobin (Hb) degradation is essential for the growth of the intraerythrocytic stages of malarial parasites. This process, which occurs inside an acidic digestive vacuole (DV), is thought to involve the action of four aspartic proteases, termed plasmepsins (PMs). These enzymes have received considerable attention as potential antimalarial drug targets. Leveraging the availability of a set of PM-knockout lines generated in Plasmodium falciparum, we report here that a wide range of previously characterized or novel aspartic protease inhibitors exert their antimalarial activities independently of their effect on the DV PMs. We also assayed compounds previously shown to inhibit cysteine proteases residing in the DV. The most striking observation was a ninefold increase in the potency of the calpain inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) against parasites lacking all four DV PMs. Genetic ablation of PM III or PM IV also decreased the level of parasite resistance to the beta-hematin binding antimalarial chloroquine. On the basis of the findings of drug susceptibility and isobologram assays, as well as the findings of studies of the inhibition of Hb degradation, morphological analyses, and stage specificity, we conclude that the DV PMs and falcipain cysteine proteases act cooperatively in Hb hydrolysis. We also identify several aspartic protease inhibitors, designed to target DV PMs, which appear to act on alternative targets early in the intraerythrocytic life cycle. These include the potent diphenylurea compound GB-III-32, which was found to be fourfold less potent against a P. falciparum line overexpressing plasmepsin X than against the parental nontransformed parasite line. The identification of the mode of action of these inhibitors will be important for future antimalarial drug discovery efforts focusing on aspartic proteases.
Moura
Pedro A
PA
Department of Microbiology, Columbia University, New York, NY 10032, USA.
Dame
John B
JB
Fidock
David A
DA
D000818:Animals; D000962:Antimalarials / Q000494:pharmacology* / Q000627:therapeutic use; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000502:physiology*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D006454:Hemoglobins / Q000378:metabolism; D006868:Hydrolysis; D007976:Leupeptins / Q000494:pharmacology / Q000627:therapeutic use; D016778:Malaria, Falciparum / Q000188:drug therapy; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000472:pathogenicity; D011480:Protease Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use
D000962:Antimalarials; D015853:Cysteine Proteinase Inhibitors; D006454:Hemoglobins; D007976:Leupeptins; D011480:Protease Inhibitors; C063261:acetylleucyl-leucyl-norleucinal; D003546:Cysteine Endopeptidases; C099607:falcipain; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin