Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax.

Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.

Cysteine proteases of malaria parasites: targets for chemotherapy.

New drugs to treat malaria are urgently needed. Cysteine proteases of malaria parasites offer potential new chemotherapeutic targets. Cysteine protease inhibitors block parasite hemoglobin hydrolysis and development, indicating that cysteine proteases play a key role in hemoglobin degradation, a necessary function of erythrocytic trophozoites. These inhibitors also block the rupture of erythrocytes by mature parasites, suggesting an additional role for cysteine proteases in the hydrolysis of erythrocyte cytoskeletal proteins. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites is larger than was previously realized. Plasmodium falciparum, the most virulent human malaria parasite, expresses three papain-family cysteine proteases, known as falcipains. All three proteases are expressed by trophozoites and hydrolyze hemoglobin at acidic pH, suggesting roles in this process. Falcipain-2 also hydrolyzes ankyrin at neutral pH, suggesting additional activity against erythrocyte cytoskeletal targets. Multiple orthologs of the falcipains have been identified in other plasmodial species. Analysis of orthologs from animal model rodent parasites identified similar features, but some noteworthy biochemical differences between the cysteine proteases. These differences must be taken into account in interpreting in vivo experiments. A number of small molecule cysteine protease inhibitors blocked parasite hemoglobin hydrolysis and development, and inhibitory effects against parasites generally correlated with inhibition of falcipain-2. Some compounds also cured mice infected with otherwise lethal malaria infections. Current research priorities are to better characterize the biological roles and biochemical features of the falcipains. In addition, efforts to identify optimal falcipain inhibitors as antimalarials are underway.

Tetracyclines specifically target the apicoplast of the malaria parasite Plasmodium falciparum.

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.

Stage-specific antimalarial activity of cysteine protease inhibitors.

Cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are promising targets for antimalarial chemotherapy. We evaluated cultured parasites for the stage-specific expression of cysteine proteases and sensitivity to cysteine protease inhibitors. Protease activity and inhibitor sensitivity varied markedly over time. Cysteine protease activity was greatest in early trophozoites, while sensitivity to cysteine protease inhibitors was greatest in mature trophozoites. Our results indicate the importance of considering the stage-specific effects of antimalarials and are consistent with the conclusion that the principal antimalarial activity of cysteine protease inhibitors is due to a block in hemoglobin hydrolysis.

Folding of the Plasmodium falciparum cysteine protease falcipain-2 is mediated by a chaperone-like peptide and not the prodomain.

Papain-family cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are hemoglobinases and potential drug targets. Available data suggest that papain-family proteases require prodomains for correct folding into functional conformations. However, in prior studies of falcipain-2, an Escherichia coli-expressed construct containing only a small portion of the prodomain refolded efficiently, suggesting that this enzyme differs in this regard from other papain-family enzymes. To better characterize the determinants of folding for falcipain-2, we expressed multiple pro- and mature constructs of the enzyme in E. coli and assessed their abilities to refold. Mature falcipain-2 refolded into active protease with very similar properties to those of proteins resulting from the refolding of proenzyme constructs. Deletion of a 17-amino acid amino-terminal segment of the mature protease yielded a construct incapable of correct folding, but inclusion of this segment in trans allowed folding to active falcipain-2. The prodomain was a potent, competitive, and reversible inhibitor of mature falcipain-2 (K(i) 10(-10) m). Our results identify a chaperone-like function of an amino-terminal segment of mature falcipain-2 and suggest that protease inhibition, but not the mediation of folding, is a principal function of the falcipain-2 prodomain.

Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites.

Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity ( k cat/ K m increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.

Structure-activity relationships for inhibition of cysteine protease activity and development of Plasmodium falciparum by peptidyl vinyl sulfones.

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.