Changes in the plasmodial surface anion channel reduce leupeptin uptake and can confer drug resistance in Plasmodium falciparum-infected erythrocytes.

Cysteine protease inhibitors kill malaria parasites and are being pursued for development as antimalarial agents. Because they have multiple targets within bloodstream-stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. In the present study, we used in vitro selection to generate a parasite resistant to growth inhibition by leupeptin, a broad-profile cysteine and serine protease inhibitor. Resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in the plasmodial surface anion channel (PSAC), an ion channel on infected erythrocytes that functions in nutrient and bulky organic solute uptake. Osmotic fragility measurements, electrophysiological recordings, and leupeptin uptake studies revealed selective reductions in organic solute permeability via PSAC, altered single-channel gating, and reduced inhibitor affinity. These changes yielded significantly reduced leupeptin uptake and could fully account for the acquired resistance. PSAC represents a novel route for the uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance development though the selection of PSAC mutants.

A role for falcilysin in transit peptide degradation in the Plasmodium falciparum apicoplast.

Falcilysin (FLN) is a zinc metalloprotease thought to degrade globin peptides in the acidic vacuole of the human malaria parasite Plasmodium falciparum. The enzyme has been found to have acidic or neutral pH optima on different peptides and to have additional distribution outside the food vacuole. These data suggested that FLN has an additional function in the parasite. To further probe the functions of FLN, we created a transgenic parasite clone expressing a chromosomally encoded FLN-GFP fusion. Unexpectedly, FLN was found in the apicoplast, an essential chloroplast-like organelle. Nuclear encoded apicoplast proteins are targeted to the organelle by a bipartite N-terminal sequence comprised of a signal sequence followed by a positively charged transit peptide domain. Free transit peptides are thought to be toxic to the plastid and need to be rapidly degraded after proteolytic release from proproteins. We hypothesized that FLN may participate in transit peptide degradation in the apicoplast based on its preference for basic residues at neutral pH and on phylogenetic comparison with other M16 family metalloproteases. In vitro cleavage by FLN of the transit peptide from the apicoplast-resident acyl carrier protein supports this idea. The importance of FLN for parasite development is suggested by our inability to truncate the chromosomal FLN open reading frame. Our work indicates that FLN is an attractive target for antimalarial development.

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

Degradation of host hemoglobin by the human malaria parasite Plasmodium falciparum is a massive metabolic process. What role this degradation plays and whether it is essential for parasite survival have not been established, nor have the roles of the various degradative enzymes been clearly defined. We report that P. falciparum can grow in medium containing a single amino acid (isoleucine, the only amino acid missing from human hemoglobin). In this medium, growth of hemoglobin-degrading enzyme gene knockout lines (missing falcipain-2 and plasmepsins alone or in combination) is impaired. Blockade of plasmepsins with the potent inhibitor pepstatin A has a minimal effect on WT parasite growth but kills falcipain-2 knockout parasites at low concentrations and is even more potent on falcipain-2, plasmepsin I and IV triple knockout parasites. We conclude that: (i) hemoglobin degradation is necessary for parasite survival; (ii) hemoglobin degradation is sufficient to supply most of the parasite's amino acid requirements; (iii) external amino acid acquisition and hemoglobin digestion are partially redundant nutrient pathways; (iv) hemoglobin degradation uses dual protease families with overlapping function; and (v) hemoglobin-degrading plasmepsins are not promising drug targets.

The role of Plasmodium falciparum food vacuole plasmepsins.

Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions.

Novel and potent anti-malarial agents.

Readily accessible, novel, and potent anti-malarial compounds have been developed. Optimization of the initial lead structure resulted in derivatives with IC50 values from 7 to 35 nM against chloroquine-sensitive and 70-350 nM against chloroquine-resistant strains of Plasmodium falciparum.

Interference with heme binding to histidine-rich protein-2 as an antimalarial strategy.

The erythrocytic growth stage of Plasmodium falciparum involves hemoglobin proteolysis as the primary nutrient source with the concomitant release of free heme. The liberated heme is processed by the parasite into hemozoin, a polymeric porphyrin dimer. Histidine-rich protein binds heme and mediates the formation of hemozoin, which is inhibited by the antimalarial drug chloroquine. Interference with heme binding was determined using a microtiterplate assay. Combinatorial libraries were screened and tested against parasite growth, revealing a good correlation between heme binding interference and the inhibition of parasite growth. Several of these compounds retain their potency against a chloroquine-resistant strain of Plasmodium falciparum. The most potent compounds have IC(50) values less than or equal to 50 nM against chloroquine-resistant and chloroquine-sensitive parasites.