The endoperoxides are a new class of antimalarial agents, ofwhich artemisinin (qinghaosu) is the prototype.We have previously shown that artemisinin is capable of alkylating proteins in model reactions. In the present study, we showed that when Plasmodium falciparum-infected erythrocytes are treated with a radiolabeled antimalarial endoperoxide, either arteether, dihydroartemisinin, or Ro 42-1611 (arteflene), the radioactivity is largely coverted into a form which can be extracted with sodium dodecyl sulfate (SDS). Autoradiograms of SDS-polyacrylamide gels showed that six malarial proteins are radioactively labeled by the three endoperoxides. This labeling occurs at physiological concentrations of drug and is not stage nor strain specific. The labeled proteins were not the most abundant proteins seen on Coomassie-stained gels. No proteins were labeled when uninfected erythrocytes were treated with these drugs, nor when infected erythrocytes were treated with the inactive analog deoxyarteether. Thus, the antimalarial endoperoxides appear to react with specific malarial proteins.

A sensitive fluorometric method for assaying malarial pigment, haemozoin, has been developed and used to determine the haemozoin content of blood and tissue samples. Plasmodium falciparum rings and trophozoites were found to contain 23 and 339 ng haemozoin/10(6) parasitized red blood cells (PRBCs), respectively. Unsynchronized Plasmodium berghei NK65 or ANKA parasites from infected mice contained 27 and 61 ng haemozoin/10(6) PRBCs, respectively. An exponential accumulation of haemozoin within 18 days after infection was demonstrated in liver and spleen tissue, representing up to 0.2% of the tissue by wet weight by day 18. Histology indicated that the accumulation occurred predominantly in the tissue monocytes. In the brain, the levels of haemozoin after 8 days of infection were considerably lower than they were in the liver or spleen, and most of the pigment appeared to be that present inside parasitized red blood cells. CBA/Ca mice infected with P. berghei ANKA (a cerebral malaria model) had significantly higher amounts of haemozoin in the brain than did ICR mice infected with P. berghei NK65. Thus, haemozoin levels in tissue increase with the duration of infection, and its presence may be associated with cerebral pathology.

Malarial hemozoin may play an important role as a target for antimalarial drugs and in disease pathogenesis. A new assay for hemozoin was developed in which the hemozoin was separated from cells by filtration. Trophozoites have substantially more hemozoin than rings, but there are relatively small differences between chloroquine-sensitive and chloroquine-resistant strains. The effects of hemozoin content of chloroquine and artemisinin, two antimalarial drugs, and E64 and Pepstatin A, two protease inhibitors, were measured. At concentrations at which hypoxanthine incorporation was unaffected, the hemozoin content of rings was decreased by E64, but not by the other three compounds. Artemisinin and Pepstatin A also had little effect on the hemozoin content of trophozoites. Chloroquine and E64 inhibited trophozoite hemozoin formation, but inhibited hypoxanthine uptake to a similar or greater extent. When either rings or trophozoites were exposed to several higher concentrations of chloroquine, hemozoin content was diminished, but significantly less than hypoxanthine uptake. Various concentrations of E64, in contrast, inhibited hemozoin production by both rings and trophozoites significantly more than hypoxanthine incorporation, suggesting that hemozoin production may be directly affected by E64.

Dihydroorotate dehydrogenase (DHOD) is a pyrimidine biosynthetic enzyme which is usually directly linked to the mitochondrial respiratory chain. Antimalarial naphthoquinones such as atovaquone (566c80) inhibit malarial DHOD by inhibiting electron transport. Since atovaquone also has therapeutic activity against Pneumocystis carinii, the P. carinii DHOD may also be an important drug target. Organisms were obtained from immunosuppressed rats, incubated for 24 h in a short-term in vitro culture system, and then lysed. P. carinii lysates catalyzed the generation of orotate from dihydroorotate at a rate of 852 pmol/mg of protein per min. Control preparations made from uninfected mice showed much less total enzymatic activity and enzyme specific activity. As expected, P. carinii DHOD activity was susceptible to respiratory inhibitors such as cyanide, antimycin A, and salicylhydroxamic acid (SHAM). Susceptibility to SHAM suggests the presence of an alternative oxidase. In contrast, neither pentamidine nor 5-hydroxy-6-demethylprimaquine (5H6DP), a quinone metabolite of primaquine, inhibited the enzyme. Atovaquone inhibited DHOD by 76.3% at 100 M and 36.5% at 10 M. A similar degree of inhibition was found when the organisms were preincubated with the drug. Atovaquone inhibited P. carinii growth in vitro at a somewhat lower concentration (between 0.3 and 3 M). In contrast, Plasmodium falciparum growth and enzyme activity are susceptible to nanomolar concentrations of atovaquone. Thus, while it is possible that atovaquone acts by inhibiting the P. carinii electron transport chain, the possibility of another drug target cannot be excluded.Dihydroorotate dehydrogenase (DHOD) is a key enzyme in de novo pyrimidine biosynthesis, catalyzing the conversion of dihydroorotate to orotate (5,12,22). DHOD is a particularly important enzyme, because it is the target of several useful chemotherapeutic agents such as the antitumor agent brequinar sodium (6) and the antimalarial agents atovaquone and menoctone (13,17). Several orotate analogs have antimalarial activities (20,25). Since atovaquone is also an effective treatment for Pneumocystis carinii pneumonia (2), it is possible that the P. carinii DHOD might be an important chemotherapeutic target. But little is currently known about the P. carinii DHOD.In all eukaryotic cells that have been studied except trypanosomatids (23), DHOD is bound to mitochondria. Isolated enzymes donate electrons directly to oxygen and to various artificial electron acceptors, although they usually prefer coenzyme Q (ubiquinone) (22). In intact organisms, carefully broken cells, and isolated mitochondria, most DHODs donate electrons directly to the mitochondrial respiratory chain.In a previous study we have examined the effects of various drugs on the mitochondrion-bound DHOD of Plasmodium falciparum (18). We then looked at the effects of these and other drugs on the P. carinii DHOD, which are described here. MATERIALS AND METHODSDrugs. Atovaquone (566c80) was a gift from the Burroughs Wellcome Co., Researc...

Dihydroorotate dehydrogenase (DHOD) is a key enzyme in de novo pyrimidine biosynthesis and the major source of electrons for the mitochondrial electron transport chain of intraerythrocytic malaria parasites. DHOD and the electron transport chain may also be the site of inhibition by certain antimalarial drugs. In order to test this, Plasmodium falciparum-infected erythrocytes were exposed in vitro to artemisinin or various 8-aminoquinolines, such as primaquine, WR 238605, WR 225448, and WR 255956, and then assayed for both enzyme activity and [3H]hypoxanthine incorporation, which is an indicator of viability. Atovaquone inhibits DHOD activity to a much greater extent than hypoxanthine incorporation, which is consistent with previous reports that it targets the parasite respiratory chain. However, artemisinin and the 8-aminoquinolines inhibit DHOD to the same or lesser extent than hypoxanthine incorporation, suggesting that these compounds have different modes of action.