Trophozoites of the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mM glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.

Tetracyclines are effective but slow-acting antimalarial drugs whose mechanism of action remains uncertain. To characterize the antimalarial mechanism of tetracyclines, we evaluated their stage-specific activities, impacts on parasite transcription, and effects on two predicted organelle targets, the apicoplast and the mitochondrion, in cultured Plasmodium falciparum. Antimalarial effects were much greater after two 48-h life cycles than after one cycle, even if the drugs were removed at the end of the first cycle. Doxycycline-treated parasites appeared morphologically normal until late in the second cycle of treatment but failed to develop into merozoites. Doxycycline specifically impaired the expression of apicoplast genes. Apicoplast morphology initially appeared normal in the presence of doxycycline. However, apicoplasts were abnormal in the progeny of doxycycline-treated parasites, as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogeny. Replication of the nuclear and mitochondrial genomes and mitochondrial morphology appeared normal. Our results demonstrate that tetracyclines specifically block expression of the apicoplast genome, resulting in the distribution of nonfunctional apicoplasts into daughter merozoites. The loss of apicoplast function in the progeny of treated parasites leads to a slow but potent antimalarial effect.Plasmodium falciparum causes an estimated half billion cases of malaria, resulting in over a million deaths, each year (5, 33). Tetracyclines are effective, albeit slow-acting, antimalarials that are used in combination with a more rapidly acting drug to treat malaria (1) and for antimalarial chemoprophylaxis (29). The antimalarial mechanism of action of tetracyclines remains undefined. Consistent with their slow clinical activity, tetracyclines exert in vitro antimalarial effects slowly, requiring incubation with cultured P. falciparum beyond a single 48-h asexual cycle for maximal effects (16). Tetracyclines exert antibacterial activity by inhibiting prokaryotic translation (41). However, in the eukaryote P. falciparum, protein synthesis is broadly inhibited by tetracyclines only at much higher concentrations than those required to block parasite growth (6), suggesting that their antimalarial effects are due to action against a target other than cytosolic ribosomes.Early studies of the mechanism of action of tetracyclines against P. falciparum focused on the parasite's single mitochondrion as the likely target (18, 26). However, these studies preceded the identification of the apicoplast, an organelle of uncertain function that is related to the chloroplast of plant cells (20,42). The mitochondrion and the apicoplast each contain their own genome, encoding prokaryote-like ribosomal RNAs, tRNAs, and some proteins (12, 45). In addition, several hundred nuclear genes encode proteins targeted to the apicoplast or the mitochondrion (2,14,15,28,39). The apicoplast houses enzy...

In the malaria parasite Plasmodium falciparum, erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis and development, indicating that cysteine proteases are required for these processes. Three papain-family cysteine protease sequences have been identified in the P. falciparum genome, but the specific roles of their gene products and other plasmodial proteases in haemoglobin hydrolysis are uncertain. Falcipain-2 was recently identified as a principal trophozoite cysteine protease and potential drug target. The present study characterizes the related P. falciparum cysteine protease falcipain-3. As is the case with falcipain-2, falcipain-3 is expressed by trophozoites and appears to be located within the food vacuole, the site of haemoglobin hydrolysis. Both proteases require a reducing environment and acidic pH for optimal activity, and both prefer peptide substrates with leucine at the P(2) position. The proteases differ, however, in that falcipain-3 undergoes efficient processing to an active form only at acidic pH, is more active and stable at acidic pH, and has much lower specific activity against typical papain-family peptide substrates, but has greater activity against native haemoglobin. Thus falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemoglobin in the acidic food vacuole. The redundancy of cysteine proteases may offer optimized hydrolysis of both native haemoglobin and globin peptides. Consideration of both proteases will be necessary to evaluate cysteine protease inhibitors as antimalarial drugs.

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 appear to be required for hemoglobin hydrolysis by intraerythrocytic malaria parasites. Previous studies showed that peptidyl vinyl sulfone inhibitors of falcipain-2 blocked the development of P. falciparum in culture and exerted antimalarial effects in vivo. We now report the structure-activity relationships for inhibition of falcipain-2, falcipain-3, and parasite development by 39 new vinyl sulfone, vinyl sulfonate ester, and vinyl sulfonamide cysteine protease inhibitors. Levels of inhibition of falcipain-2 and falcipain-3 were generally similar, and many potent compounds were identified. Optimal antimalarial compounds, which inhibited P. falciparum development at low nanomolar concentrations, were phenyl vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides with P 2 leucine moieties. Our results identify independent structural correlates of falcipain inhibition and antiparasitic activity and suggest that peptidyl vinyl sulfones have promise as antimalarial agents.Malaria is one of the most important infectious diseases in the world. Plasmodium falciparum, the most virulent human malaria parasite, is estimated to cause over 300 million new cases and 1 million deaths annually (33). Further complicating this grim scenario is the emergence of the widespread resistance of P. falciparum to available antimalarial drugs (25). New drugs to combat malaria are urgently needed.Among potential new targets for antimalarial chemotherapy are enzymes that mediate hemoglobin hydrolysis. Intraerythrocytic P. falciparum trophozoites derive amino acids for protein synthesis from the hydrolysis of host cell hemoglobin in an acidic food vacuole (12,20,27). Proteases that hydrolyze hemoglobin in the food vacuole include members of the aspartic protease (1), cysteine protease (38, 39), and metalloprotease (9) families. Cysteine protease inhibitors arrested the erythrocytic life cycle of P. falciparum (26). Examination of inhibitortreated parasites revealed abnormally swollen food vacuoles filled with undigested hemoglobin, indicating that the block in parasite development was due to the inhibition of hemoglobin hydrolysis (26).P. falciparum contains three fairly typical papain family cysteine proteases, known as falcipains (28,38,39). Falcipain-2 and falcipain-3 appear to be the principal cysteine protease hemoglobinases (38, 39). Both of these proteases localize to vacuolar parasite fractions and readily hydrolyze hemoglobin under physiological reducing conditions at acidic pHs (37). Falcipain-2 is considerably more active against small peptide substrates, but the specificities of the two proteases are similar; both enzymes display a strong preference for leucine at the P 2 position (38, 39). The role of falcipain-1 in hemoglobin hydrolysis is unknown.In earlier studies, peptidyl vinyl sulfones inhibited falcipain-2 activity and parasite development at nanomolar concentrations and were active in vivo against murine malaria (22,29,30). We have now init...