Plasmodium falciparum: Differential Sensitivity In Vitro to E‐64 (Cysteine Protease Inhibitor) and Pepstatin A (Aspartyl Protease Inhibitor)

Abstract

We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.

Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems

“…We noted a discrepancy between the potency of 5-year-old pepstatin A (from Streptomyces sp., Sigma) and fresh pepstatin A (from Streptomyces sp., Sigma) or synthetic (Calbiochem). The IC 50 value for RPMI medium 1640-cultured 3D7 parasites with old pepstatin A (7 M) was in line with published reports (5,15,17), whereas the IC 50 of both new preparations was much higher (Ϸ60 M; Table 2). Inhibition curves of the various knockout clones were quite similar with old pepstatin A (Fig.…”

“…Knockouts of plasmepsins individually or in combination (plasmepsin IV͞I double knockout) give parasites with only slightly impaired growth (5,16), and a falcipain-2 knockout shows normal growth (17). The effect of aspartic protease inhibitors is potentiated when combined with cysteine protease inhibitors or used in falcipain-2 knockout parasites (15,17). Therefore the evidence suggests that there is overlap in and between the food vacuole proteolytic families; their relative roles remain unclear.…”

“…All of them are capable of degrading hemoglobin or globin in biochemical assays using recombinant enzyme, although their order of action is unclear (9)(10)(11)(12)(13)(14). Inhibitors of aspartic and cysteine proteases kill parasites in culture and animal models, although their specificity is not strict (13)(14)(15). Knockouts of plasmepsins individually or in combination (plasmepsin IV͞I double knockout) give parasites with only slightly impaired growth (5,16), and a falcipain-2 knockout shows normal growth (17).…”

Characterization of Native and Recombinant Falcipain-2, a Principal Trophozoite Cysteine Protease and Essential Hemoglobinase ofPlasmodium falciparum

“…A critical role for a cysteine protease hemoglobinase was suggested when it was demonstrated that cultured malaria parasites failed to develop when incubated with cysteine protease inhibitors (8,11,12). Morphological examination of cysteine protease inhibitor-treated parasites revealed abnormally swollen, dark-staining food vacuoles, and biochemical evaluation indicated that the abnormality was caused by an accumulation of undigested hemoglobin in the food vacuole (8,(12)(13)(14)(15). These results suggested a central role for a cysteine protease in early steps in hemoglobin degradation by P. falciparum.…”

Evaluation of novel 1-(4-(substituted)piperazin-1-yl)- 2-(phenylamino)ethanone derivatives as Falcipain-2 inhibitors

“…[10][11][12] Studies affirmed that when falcipain-2 gene is disrupted, undegraded hemoglobin accumulates in the food vacuole, confirming that this enzyme participates in digestion of hemoglobin in the acidic food vacuole. 13,14 Various research groups have investigated the cysteine protease inhibitors, which are mainly originated from peptides derivatives 15,16 and having nanomolar IC 50 values, due to the formation of covalent bond with thiol of active site Cys42, which behaves as a Michael acceptor. 17 The poor selectivity for parasitic cysteine proteases over the human cysteine proteases remains a noteworthy concern.…”