Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

Abstract

During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial drug development efforts. However, their expression during gametocytogenesis, which is required for malaria parasite transmission, has not been studied. Many of the available antimalarials do not inhibit development of sexual stage parasites, and therefore, the persistence of gametocytes after drug treatment allows continued transmission of the disease. In the work reported here, incubation of stage V gametocytes with membrane-permeant cysteine protease inhibitor E64d significantly inhibited oocyst production (80 to 100%). The same conditions inhibited processing of gametocyte-surface antigen Pfs230 during gametogenesis but did not alter the morphology of the food vacuole in gametocytes, inhibit emergence, or block male exflagellation. E64d reduced the level of oocyst production more effectively than that reported previously for falcipain 1-knockout parasites, suggesting that falcipains 2 and 3 may also be involved in malaria parasite transmission. However, in this study only falcipain 3 and not falcipain 2 was found to be expressed in stage V gametocytes. Interestingly, during gametocytogenesis falcipain 3 was transported into the red blood cell and by stage V was localized in vesicles along the RBC surface, consistent with a role during gamete emergence. The ability of a membrane-permeant cysteine protease inhibitor to significantly reduce malaria parasite transmission suggests that future drug design should include evaluation of gametogenesis and sporogonic development.The clinical symptoms of malaria are caused by asexually replicating parasites, but malaria parasite transmission requires that a subpopulation of parasites undergo sexual differentiation. In the species of parasite responsible for the most virulent form of malaria, Plasmodium falciparum, complete maturation into mature stage V gametocytes takes 10 to 12 days in the human host. Once stage V gametocytes are taken up by a mosquito, gametogenesis is stimulated and, after fertilization, the zygote differentiates into an ookinete. The ookinete then migrates to the basal surface of the midgut and forms an oocyst, where tens of thousands of infectious sporozoites are produced.Both asexual and sexual stage parasites are present concurrently in an infected individual and therefore would both be exposed to antimalarial drugs. However, gametocytes have been found to be resistant to many of the commonly used antimalarials, such as the 4-aminoquinolines; and sulfadoxinepyrimethamine has been reported to increase the production of gametocytes, which could enhance transmission (2, 12, 21, 36). Consequently, even after successful treatment for clinical symptoms, an individual can still transmit the malaria parasite for at least a week. Therefore, it is important to d...

The Proteasome Inhibitor Epoxomicin Has Potent Plasmodium falciparum Gametocytocidal Activity

“…In total, 341 oocysts were produced in the 70 mosquitoes fed with DMSO-treated parasites, while no oocysts were produced by epoxomicin-treated parasites and only a total of 3 oocysts were produced in the 43 mosquitoes fed ALLN-treated parasites. This is similar to the significant block found with E64d and suggests that both the cysteine proteases and the proteasome play important roles in sporogonic development (10).…”

“…5). In contrast, ALLN did effectively block Pfs230 processing, as expected from our previous work using the cysteine protease inhibitor E64d (10). The lack of effect of epoxomicin on Pfs230 processing is consistent with its reported specificity for the proteasome, not cysteine proteases.…”

“…The prolonged period required for P. falciparum gametocyte maturation in the human host suggests that malaria can be transmitted for several weeks after asexual parasites are eliminated (23). Thus, the development of drugs that are effective against both asexual-stage parasites and gametocytes may directly decrease malaria morbidity and mortality and reduce the spread of the disease.Cysteine protease and proteasome inhibitors have been found to affect asexual intraerythrocytic parasites and are being evaluated as possible antimalarial agents (4,7,10,15,(19)(20)(21)25). However, their effect on the 10-to 12-day course of intraerythrocytic gametocyte development has not been reported.…”

“…Cysteine protease and proteasome inhibitors have been found to affect asexual intraerythrocytic parasites and are being evaluated as possible antimalarial agents (4,7,10,15,(19)(20)(21)25). However, their effect on the 10-to 12-day course of intraerythrocytic gametocyte development has not been reported.…”

“…However, their effect on the 10-to 12-day course of intraerythrocytic gametocyte development has not been reported. Proteasome inhibitors also have not been tested on parasites taken up by a mosquito, while cysteine protease inhibitors have been shown to significantly decrease P. falciparum gamete surface antigen processing, oocyst production, and sporozoite maturation (7,10). The dual cysteine and serine protease inhibitors L-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK) also have been reported to reduce P. falciparum exflagellation and the transmission of Plasmodium berghei to mosquitoes (22,26).…”