Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC50), assessed by parasite incorporation of [3H]hypoxanthine, were between 0.2 and 1.1 μM. CLT concentrations of 2 μM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (≈48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 μM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.

On the molecular mechanism of chloroquine's antimalarial action.

Chloroquine is thought to exert its antimalarial effect by preventing the polymerization of toxic heme released during proteolysis of hemoglobin in the Plasmodium digestive vacuole. The mechanism of this blockade has not been established. We incubated cultured parasites with subinhibitory doses of [3H]chloroquine and [3H] quinidine. These [3H]quinoline compounds became associated with hemozoin as assessed by electron microscope autoradiography and subcellular fractionation. In vitro, binding of [3H]quinoline inhibitors to the hemozoin chain depended on the addition of heme substrate. These data counter previous conclusions regarding the lack of quinoline association with hemozoin, explain the exaggerated accumulation of quinolines in the plasmodium digestive vacuole, and suggest that a quinoline heme complex incorporates into the growing polymer to terminate chain extension, blocking further sequestration of toxic heme.

The pfmdr1 gene of Plasmodium falciparum confers cellular resistance to antimalarial drugs in yeast cells.

The exact role of the pfmdr1 gene in the emergence of drug resistance in the malarial parasite Plasmodium falciparum remains controversial. pfmdr1 is a member of the ATP binding cassette (ABC) superfamily of transporters that includes the mammalian P-glycoprotein family. We have introduced wild-type and mutant variants of the pfmdr1 gene in the yeast Saccharomyces cerevisiae and have analyzed the effect of pfmdr1 expression on cellular resistance to quinoline-containing antimalarial drugs. Yeast transformants expressing either wild-type or a mutant variant of mouse P-glycoprotein were also analyzed. Dose-response studies showed that expression of wild-type pfmdr1 causes cellular resistance to quinine, quinacrine, mefloquine, and halofantrine in yeast cells. Using quinacrine as substrate, we observed that increased resistance to this drug in pfmdr1 transformants was associated with decreased cellular accumulation and a concomitant increase in drug release from preloaded cells. The introduction of amino acid polymorphisms in TM11 of Pgh-1 (pfmdr1 product) associated with drug resistance in certain field isolates of P. falciparum abolished the capacity of this protein to confer drug resistance. Thus, these findings suggest that Pgh-1 may act as a drug transporter in a manner similar to mammalian P-glycoprotein and that sequence variants associated with drug-resistance pfmdr1 alleles behave as loss of function mutations.

Variations in frequencies of drug resistance in Plasmodium falciparum

Continual exposure of malarial parasite populations to different drugs may have selected not only for resistance to individual drugs but also for genetic traits that favor initiation of resistance to novel unrelated antimalarials. To test this hypothesis, different Plasmodium falciparum clones having varying numbers of preexisting resistance mechanisms were treated with two new antimalarial agents: 5-fluoroorotate and atovaquone. All parasite populations were equally susceptible in small numbers. However, when large populations of these clones were challenged with either of the two compounds, significant variations in frequencies of resistance became apparent. On one extreme, clone D6 from West Africa, which was sensitive to all traditional antimalarial agents, failed to develop resistance under simple nonmutagenic conditions in vitro. In sharp contrast, the Indochina clone W2, which was known to be resistant to all traditional antimalarial drugs, independently acquired resistance to both new compounds as much as a 1,000 times more frequently than D6. Additional clones that were resistant to some (but not all) traditional antimalarial agents acquired resistance to atovaquone at high frequency, but not to 5-fluoroorotate. These findings were unexpected and surprising based on current views of the evolution of drug resistance in P. falciparum populations. Such new phenotypes, named accelerated resistance to multiple drugs (ARMD), raise important questions about the genetic and biochemical mechanisms related to the initiation of drug resistance in malarial parasites. Some potential mechanisms underlying ARMD phenotypes have public health implications that are ominous.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1–42 maturase

The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2’s subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.

Spontaneous rupture of malarial spleen: two case reports and review of literature

Malaria has long been among the most common diseases in the southeast Anatolia region of Turkey. In 1992, 18676 cases were diagnosed in Turkey, and Diyarbakir city had the highest incidence (4168 cases), followed by SanliUrfa city (3578 cases). Malaria was especially common during 1994 and 1995, with 84 345 and 82 094 cases being diagnosed in these years, respectively. Spontaneous rupture of malarial spleen is rare. We saw two cases during 1998, which are reported herein. Both patients were male, and were receiving chloroquine treatment for an acute attack of malaria. One of the patients had developed abdominal pain and palpitations, followed by fainting. The other patient had abdominal pain and fever. Explorative laparotomy revealed an enlarged spleen in both patients. Splenectomy was performed in both patients. We have identified 15 episodes of spontaneous rupture of the spleen in the English language literature published since 1961. Because of increased travel to endemic areas and resistance to antimalarial drugs, malaria is a major medical problem that is becoming increasingly important to surgeons worldwide. Malaria is a particularly important problem in the southeast Anatolia region of Turkey. Prophylactic precautions should be taken by tourists who travel to this region, especially during the summer.

A yeast-like mRNA capping apparatus in Plasmodium falciparum

Analysis of the mRNA capping apparatus of the malaria parasite Plasmodium falciparum illuminates an evolutionary connection to fungi rather than metazoans. We show that P. falciparum encodes separate RNA guanylyltransferase (Pgt1) and RNA triphosphatase (Prt1) enzymes and that the triphosphatase component is a member of the fungal/viral family of metal-dependent phosphohydrolases, which are structurally and mechanistically unrelated to the cysteine-phosphatase-type RNA triphosphatases found in metazoans and plants. These results highlight the potential for discovery of mechanism-based antimalarial drugs designed to specifically block the capping of Plasmodium mRNAs. A simple heuristic scheme of eukaryotic phylogeny is suggested based on the structure and physical linkage of the triphosphatase and guanylyltransferase enzymes that catalyze cap formation.

Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum.

Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance. Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted. The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development. We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A. This represents the first reported crystal structure of a protein from P. falciparum. The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme. The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P. falciparum in culture.