The flagellum and flagellar pocket of trypanosomatids.

The flagellum and flagellar pocket are distinctive organelles present among all of the trypanosomatid protozoa. Currently, recognized functions for these organelles include generation of motility for the flagellum and dedicated secretory and endocytic activities for the flagellar pocket. The flagellar and flagellar pocket membranes have long been recognized as morphologically separate domains that are component parts of the plasma membrane that surrounds the entire cell. The structural and functional specialization of these two membranes has now been underscored by the identification of multiple proteins that are targeted selectively to each of these domains, and non-membrane proteins have also been identified that are targeted to the internal lumina of these organelles. Investigations on the functions of these organelle-specific proteins should continue to shed light on the unique biological activities of the flagellum and flagellar pocket. In addition, work has begun on identifying signals or modifications of these proteins that direct their targeting to the correct subcellular location. Future endeavors should further refine our knowledge of targeting signals and begin to dissect the molecular machinery involved in transporting and retaining each polypeptide at its designated cellular address.

Xanthones as antimalarial agents: stage specificity.

The erythrocytic development of Plasmodium falciparum is divided into the ring, trophozoite, and schizont stages based on morphologic assessment. Using highly synchronous ring and trophozoite cultures of P. falciparum, we observed considerable differences in their sensitivity to hydroxyxanthones: trophozoites were much more sensitive to the drugs than ring-stage parasites. Trophozoites treated with a prototypic xanthone, the 2,3,4,5,6-pentahydroxy derivative (X5), were arrested in their development and became degenerate in appearance within 24 hr of drug exposure. These morphologic changes appeared to reflect the cytotoxic nature of the action of the drug against the parasite, since daughter ring-stage forms were not observed following addition of the drug. That X5 was more active against parasites in the later stages of intraerythrocytic development is consistent with the proposed mode of action, inhibition of heme polymerization. Knowledge of the structure-activity relationships for xanthones as antimalarial agents has also been expanded. Xanthones with a hydroxyl group in the peri-position exhibited decreased antimalarial activity, possibly due to intramolecular hydrogen bonding with the carbonyl and consequent reduced affinity for heme. Paired hydroxyls attached to the lower half of the xanthone greatly enhanced drug potency.

Potentiation of an antimalarial oxidant drug.

In a previous report we described the synergistic antimalarial interaction between two structurally similar compounds, rufigallol and exifone. To explain this phenomenon, we proposed that exifone is transformed inside the parasitized erythrocyte into a xanthone with potent antimalarial properties. We speculated that the transformation process was induced by the prooxidant activity of rufigallol. On the basis of this model we hypothesized that exifone would act synergistically with other oxidant drugs. In the present study we have found a similar synergistic interaction between exifone and ascorbic acid (vitamin C) against both chloroquine-susceptible and multidrug-resistant strains of Plasmodium falciparum. The prooxidant activity of ascorbic acid against Plasmodium-infected erythrocytes is believed to result from an intraerythrocytic Fenton reaction occurring in the acidic food vacuole of the parasite. The hydroxyl radicals produced during this process are believed to attack exifone, which undergoes cyclodehydration to become 2,3,4,5,6-pentahydroxyxanthone (X5). Evidence presented to support this "xanthone hypothesis" includes the demonstration that the exifone ==> X5 transformation occurs readily in vitro under mildly acidic conditions in the presence of iron, ascorbic acid, and oxygen.

Xanthones as antimalarial agents; studies of a possible mode of action.

We recently demonstrated that 2,3,4,5,6-pentahydroxyxanthone (X5) inhibits the in vitro growth of both chloroquine-sensitive and multidrug-resistant strains of P. falciparum. To study the molecular basis of its antimalarial action, we tested X5 and selected hydroxyxanthone analogs as inhibitors of in vitro heme polymerization in a low ionic strength phosphate solution at mildly acidic pH. We found that addition of 1 Eq. of X5 resulted in complete inhibition of polymerization in this system whereas addition of up to 40 Eqs. of standard antimalarial compounds (chloroquine, primaquine, quinacrine, artemisinin and methylene blue) had no such effect although these compounds did co-precipitate with heme. The antimalarial potency of the hydroxyxanthones correlated well with their ability to inhibit in vitro heme polymerization in our assay, suggesting that these compounds exert their antimalarial action by preventing hemozoin formation. Based on the observed structure-activity relationships, we propose a model displaying possible interactions between hydroxyxanthones and heme.

Potentiation of the antimalarial agent rufigallol.

We have discovered a remarkable synergistic antimalarial interaction between rufigallol and the structurally similar compound exifone. The synergistic effects were produced in chloroquine-susceptible and chloroquine-resistant clones of Plasmodium falciparum. The degree of potentiation as estimated by standard isobolar analysis was approximately 60-fold for experiments initiated with asynchronous parasites. The most pronounced synergism was observed in experiments with synchronized trophozoite-infected erythrocytes, in which the degree of synergy was at least 300-fold. While the mechanism underlying this drug potentiation remains unresolved, it is hypothesized that rufigallol acts in pro-oxidant fashion to produce oxygen radicals inside parasitized erythrocytes. These radicals would attack exifone, thereby initiating its transformation into a more potent compound, a xanthone.