Antimalarial drugs have been used for centuries. Early natural products, including the bark of the cinchona tree in South America and extracts of the wormwood plant in China, were among the first effective antimicrobial agents to be used. Cinchona bark was used in Europe beginning in the 17th century, and upon its isolation from bark in 1820, quinine became widely used. In the last 50 years, extensive efforts, including the screening of hundreds of thousands of compounds, have led to the development of a number of effective synthetic antimalarial drugs. The most important of these, chloroquine, has been the mainstay of antimalarial chemotherapy for the last 50 years. The compound eradicates parasites rapidly, has minimal toxicity, is widely available at low cost throughout the world, and needs to be taken only once a week for chemoprophylaxis. However, resistance to chloroquine has been steadily increasing since the drug's initial use in South America and Southeast Asia in the late 1950s. Chloroquine resistance is now widespread in most P. falciparum-endemic areas of the world (3). Thus, the use of chloroquine for presumptive treatment of falciparum malaria or for chemoprophylaxis is usually no longer appropriate (7). Moreover, resistance to chloroquine of P. vivax, the second most lethal human malaria parasite, is increasing in South Asia (8).

No other antimalarial drug (9-12) is as efficacious and safe as chloroquine (Table 1). The best antimalarial drug for treating chloroquine-resistant falciparum malaria remains quinine (or intravenous quinidine), which is fairly toxic; quinine resistance is increasing in Southeast Asia, particularly in the border areas of Thailand (9). Amodiaquine, used to treat chloroquine-resistant malaria in developing countries, is also quite toxic, and resistance to it is also common (13">13). Mefloquine (14) is widely used for chemoprophylaxis against chloroquine-resistant P. falciparum, but its use is limited by toxicity (15) and (in the developing world) high cost. Mefloquine is not approved for treatment of malaria in the United States because of the neurotoxicity of doses required for the treatment. Fansidar, a combination of sulfadoxine and pyrimethamine, is no longer recommended for chemoprophylaxis because of its dermatologic toxicity (15). Fansidar is also not an ideal drug for treatment because it is slow acting, but it is increasingly important in treating chloroquine-resistant malaria in developing countries because economic constraints limit the use of other agents (16). The use of both mefloquine and Fansidar will increasingly be limited by drug resistance, already widespread in parts of Southeast Asia (9,17).

Other antimalarial drugs have specialized uses. Tetracyclines and some other antibiotics (clindamycin, sulfas) are slow acting and generally best used as an adjunct to quinine therapy in treating falciparum malaria (9). Doxycycline is also used for chemoprophylaxis in regions with high levels of drug resistance, especially Southeast Asia (10,17). Other drugs for chemoprophylaxis include proguanil, which remains effective in combination with chloroquine in many areas other than Southeast Asia, and Maloprim, a combination of dapsone and pyrimethamine (10,17). Resistance to these drugs is fairly common, however. Primaquine has a well-defined specific role: eradicating chronic liver stages of P. vivax and P. ovale after treating the acute blood infection with chloroquine.

Relatively few new antimalarial drugs are undergoing clinical testing (Table 2). Halofantrine, identified in the 1940s, was not developed until the 1980s; its use has been limited by variable oral absorption and cardiac toxicity (18). The drug is approved in the United States for treatment of chloroquine-resistant <I>P. falciparum</I> infection, although in most cases quinine (or intravenous quinidine) is preferable. The most effective new drugs are artemisinin and related compounds. Artemisinin was isolated in 1972 from <I>Artemisia annua</I>, a plant used in China for centuries to treat fever (19). Artemisinin derivatives (artesunate, artelinate, artemether, arteether, dihydroartemisinin) have been synthesized and are undergoing extensive clinical testing. These compounds, which are already widely used in some areas, are potent, rapidly acting antimalarials that are effective against chloroquine-resistant P. falciparum (20). Because recrudescences of infection after treatment are common, however, artemisinin and related compounds might best be used in combination with another drug.

Other compounds are under evaluation. Atovaqone (21), which is approved for treating patients with Pneumocystis infections, appears to be effective against malaria in combination with proguanil (22), but its use has been limited by recrudescence after treatment. Pyronaridine, an acridine derivative used to treat malaria in China, has shown efficacy against falciparum malaria (23). The iron chelator desferrioxamine enhances the clearance of parasites in mild malaria (24) and, in conjunction with quinine and Fansidar, hastens recovery from deep coma in severe falciparum malaria (25). Azithromycin, a quinolone antibiotic, appears efficacious in malaria chemoprophylaxis (26).

Figure

Figure. Protease targets in erythrocytic malaria parasites. The Plasmodium falciparum erythrocytic life cycle is shown schematically, and data supporting cysteine (CP), serine (SP), and aspartic (AP) proteases of the different parasite stages...

The limitations of antimalarial chemotherapy underscore the need for new drugs, ideally directed against new targets. Potential targets for chemotherapy include malarial proteases (27). The erythrocytic life cycle, which is responsible for all clinical manifestations of malaria, begins when free merozoites invade erythrocytes. The intraerythrocytic parasites develop from small ring-stage organisms to larger, more metabolically active trophozoites and then to multinucleated schizonts. The erythrocytic cycle is completed when mature schizonts rupture erythrocytes, releasing numerous invasive merozoites. Proteases appear to be required for the rupture and subsequent reinvasion of erythrocytes by merozoite-stage parasites and for the degradation of hemoglobin by intraerythrocytic trophozoites (Figure).

The rupture of erythrocytes by mature schizonts and the subsequent invasion of erythrocytes by free merozoites appear to require malarial protease activity, possibly to breach the erythrocyte cytoskeleton, a complex network of proteins. In addition, a number of malarial proteins are proteolytically processed during the late schizont and merozoite life-cycle stages; for example, merozoite surface protein-1 is processed in a manner inhibited by serine protease inhibitors (28), presumably to facilitate the complex series of events involved in erythrocyte rupture and invasion (29). Although the specific roles of different classes of proteases are not completely clear, inhibitors of cysteine and serine proteases have consistently blocked erythrocyte rupture and invasion (27).

Candidate P. falciparum rupture/invasion proteases have been identified, but none has been fully characterized biochemically or molecularly: 1) a 68 kD cysteine protease was identified in schizonts and merozoites and localized to the merozoite apex, suggesting that it may be released from the rhoptry organelle during invasion (30); 2) a cysteine protease of mature schizonts and a serine protease of merozoites were identified in highly synchronized parasites (31); 3) a serine protease was shown to be bound to the schizont/merozoite membrane by a glycosyl-phosphatidylinositol anchor, to be activated by phosphatidylinositol-specific phospholipase C during the merozoite stage, and to be capable of cleaving the erythrocyte cytoskeletal protein band 3 (32,33); 4) another protease, inhibited by both cysteine and serine protease inhibitors, hydrolyzed the erythrocyte cytoskeletal proteins spectrin and band 4.1 (34); and 5) the serine repeat antigen (35,36) and the related protein SERP H (37), both expressed in mature schizonts, have important similarities in their sequences with cysteine proteases. Further research should identify the specific biologic roles of the proteases mentioned and better characterize these enzymes, thus fostering the development of specific inhibitors.

Host proteases may also play a role in erythrocyte rupture by P. falciparum. In recent studies, host urokinase was shown to bind to the surface of P. falciparum-infected erythrocytes, and the depletion of urokinase from parasite culture medium inhibited erythrocyte rupture by mature schizonts (38). This inhibition was reversed by exogenous urokinase.

Extensive evidence suggests that the degradation of hemoglobin is necessary for the growth of erythrocytic malaria parasites, apparently to provide free amino acids for parasite protein synthesis (27,50). In P. falciparum, hemoglobin degradation occurs predominantly in trophozoites and early schizonts, the stages at which the parasites are most metabolically active. Trophozoites ingest erythrocyte cytoplasm and transport it to a large central food vacuole. In the food vacuole, hemoglobin is broken down into heme, a major component of malarial pigment (51), and globin, which is hydrolyzed to its constituent amino acids. The food vacuole is an acidic organelle analogous to lysosomes. Several lysosomal proteases are well characterized, including cysteine (cathepsins B, H, and L) and aspartic (cathepsin D) proteases (52), and malaria parasites contain analogous food vacuole proteases that degrade hemoglobin. At least two aspartic proteases and one cysteine protease have been isolated from purified P. falciparum food vacuoles (53).

Malarial aspartic protease activities have been identified (54-60). Two recently characterized aspartic proteases (plasmepsin I and plasmepsin II) are located in the food vacuole, have acid pH optima, and share sequence homology with other aspartic proteases (41,53,61,62). Furthermore, the aspartic proteases can cleave hemoglobin. One of the enzymes, plasmepsin I, cleaves native hemoglobin (53,59). Plasmepsin II appears to prefer denatured globin as a substrate (53). On the basis of these data, plasmepsin I is thought to be responsible for initial cleavages of hemoglobin after the molecule is transported to the food vacuole (53).

Incubation of cultured P. falciparum parasites with the protease inhibitor leupeptin caused trophozoite food vacuoles to fill with apparently undegraded erythrocyte cytoplasm (63-65). Analysis of the leupeptin-treated parasites showed that they contained large quantities of undegraded globin, while minimal globin was detectable in control parasites (64,66). Leupeptin inhibits both cysteine and some serine proteases, but the highly specific cysteine protease inhibitor E-64 also caused undegraded globin to accumulate. After parasites were incubated with inhibitors of other classes of proteases including the aspartic protease inhibitor pepstatin (63-67), globin did not accumulate. More recent studies that used nondenaturing electrophoretic methods demonstrated that cysteine protease inhibitors not only blocked malarial globin hydrolysis, but also inhibited earlier steps in hemoglobin degradation, including denaturation of the hemoglobin tetramer and the release of heme from globin (68). Another study showed that E-64, but not pepstatin, inhibited the production of hemozoin (the malarial end product of heme) by cultured parasites (69). These results suggest that a cysteine protease is required for initial steps in hemoglobin degradation by P. falciparum.

A P. falciparum trophozoite cysteine protease with biochemical features expected for a food vacuole hemoglobinase has been identified (31) and biochemically (70-72) and molecularly (73) characterized. This protease, called falcipain, degraded denatured and native hemoglobin in vitro; its acid pH optimum, substrate specificity, and inhibitor sensitivity indicated that it was a papain family cysteine protease (64,70,71). Specific inhibitors of falcipain blocked hemoglobin degradation and prevented parasite development. The degree of inhibition of falcipain by fluoromethyl ketones (44) and vinyl sulfones (46) correlated with their inhibition of hemoglobin degradation and parasite development, supporting the hypothesis that falcipain is the cysteine protease required for hemoglobin degradation.

The specific mechanism for hemoglobin degradation in the malarial food vacuole remains unclear. As noted above, both the aspartic protease plasmepsin I and the cysteine protease falcipain have been identified in parasite food vacuoles and shown to cleave denatured and native hemoglobin in vitro (53,71). Results showing that only cysteine protease inhibitors block hemoglobin processing and globin hydrolysis in cultured parasites suggest that falcipain is required for initial steps of hemoglobin degradation (66-68,74). However, other studies have shown that native hemoglobin is cleaved by plasmepsin I, but not falcipain, in nonreducing conditions that may be present in the food vacuole (53,59,72). In any event, regardless of the exact sequence of hemoglobin processing, multiple enzymes, including at least the three proteases already identified, appear to participate in the degradation of hemoglobin. These proteases are thus logical targets for antimalarial drug development.

Aminopeptidase activity has also been described in malaria parasites (75-77). This activity, with a neutral pH optimum, was not found in food vacuole lysates (77). When these lysates were incubated with hemoglobin, discrete peptide fragments, but not free amino acids, were identified (77). These results suggest that hemoglobin is degraded to small peptides in the food vacuole, that these peptides are transported to the parasite cytosol, and that additional processing of hemoglobin peptides is mediated by cytosolic aminopeptidase activity (77).