tretinoin and Malaria--Falciparum

Infection by Plasmodium falciparum is the leading cause of malaria in humans. The parasite contains a unique and essential plastid-like organelle called the apicoplast that, similar to the mitochondria and chloroplast, houses its own genome that must undergo replication and repair. The putative apicoplast replicative DNA polymerase, POM1, has no direct orthologs in mammals, making the P. falciparum POM1 an attractive antimalarial drug target. Here, we report on a fluorescent high-throughput DNA polymerase assay that relies on the ability of POM1 to perform strand-displacement synthesis through the stem of a DNA hairpin substrate, thereby separating a Cy3 dye from a quencher. Assay-validation experiments were performed using 384-well plates and resulted in a signal window of 7.90 and aZ' factor of 0.71. A pilot screen of a 2880-compound library identified 62 possible inhibitors that cause more than 50% inhibition of polymerase activity. The simplicity and statistical robustness of the assay suggest it is well suited for the screening of novel apicoplast polymerase inhibitors that may serve as lead compounds in antimalarial drug-discovery efforts.

Topics: Antimalarials; Apicoplasts; Chloroplasts; DNA; DNA-Directed DNA Polymerase; Drug Discovery; Exonucleases; Humans; Kinetics; Malaria, Falciparum; Mitochondria; Multienzyme Complexes; Nucleic Acid Synthesis Inhibitors; Peptide Library; Plasmodium falciparum; Protozoan Proteins; Spectrometry, Fluorescence

With 3·3 billion people at risk of infection, malaria remains one of the world's most significant health problems. Increasing resistance of the main causative parasite to currently available drugs has created an urgent need to elucidate the pathogenesis of the disease in order to develop new treatments. A possible clue to such an understanding is that the malaria parasite Plasmodium falciparum selectively absorbs vitamin A from the host and appears to use it for its metabolism; serum vitamin A levels are also reduced in children with malaria. Although vitamin A is essential in low concentration for numerous biological functions, higher concentrations are cytotoxic and pro-oxidant, and potentially toxic quantities of the vitamin are stored in the liver. During their life cycle in the host the parasites remain in the liver for several days before invading the red blood cells (RBCs). The hypothesis proposed is that the parasites emerge from the liver packed with vitamin A and use retinoic acid (RA), the main biologically active metabolite of vitamin A, as a cell membrane destabilizer to invade the RBCs throughout the body. The characteristic hemolysis and anemia of malaria and other symptoms of the disease may thus be manifestations of an endogenous form of vitamin A intoxication associated with high concentrations of RA but low concentrations of retinol (ROL). Retinoic acid released from the parasites may also affect the fetus and cause preterm birth and fetal growth restriction (FGR) as a function of the membranolytic and growth inhibitory effects of these compounds, respectively. Subject to testing, the hypothesis suggests that parasite vitamin A metabolism could become a new target for the treatment and prevention of malaria.

Topics: Anemia; Endocytosis; Erythrocytes; Hemolysis; Humans; Liver; Malaria, Falciparum; Plasmodium falciparum; Tretinoin; Vitamin A

Retinol (vitamin A alcohol) may have a beneficial role in the host response to malaria in humans and previously published data have suggested that it has a direct inhibitory effect on the growth of Plasmodium falciparum in vitro. To further investigate the role of retinoids as potential antimalarial agents, we assessed the effect of all-trans-retinoic acid (RA), 9-cis-RA and 13-cis-RA, as well as retinol itself and its ester, retinyl palmitate, on 3H-hypoxanthine uptake by the laboratory-adapted strains of P. falciparum 3D7 and K1. In addition, we examined the influence of three specific RA receptor antagonists, ER 27191, Ro 415253 and AGN 194301, on retinoid-induced growth inhibition of 3D7. All-trans-RA, 9-cis-RA and 13-cis-RA in concentrations ranging from 1 x 10(-4) to 5 x 10(-10) M each had antimalarial activity, but at IC50 values (5.9 x 10(-5) to 7.9 x 10(-5) M) that were less than those of retinol (2.5 x 10(-5) to 3.2 x 10(-5) M). Retinyl palmitate had minimal effect on 3H-hypoxanthine uptake. Each of the three specific antagonists inhibited growth of 3D7 (IC50 range 1.2 x 10(-5) to 3.0 x 10(-5) M) but, in isobolographic analysis, were antagonistic to retinol (dose factor potentiation, DFP 0.46-0.79) and, in the case of Ro 415253, to all-trans-RA (DFP=0.39). Although we did not attempt to quantify losses of retinoids from the system, these data suggest that retinol has greater antimalarial activity than its RA metabolites and especially retinyl palmitate. The specific RA receptor antagonists showed paradoxical antimalarial activity but consistently antagonised the effect of retinol and all-trans-RA in isobolographic experiments. We conclude that RA metabolites may be less suitable than retinol per se as antimalarial agents and that P. falciparum might possess or acquire a RA receptor-like moiety.

Topics: Animals; Anthracenes; Antimalarials; Benzoates; Chromans; Drug Interactions; Hypoxanthine; Inhibitory Concentration 50; Malaria, Falciparum; Plasmodium falciparum; Pyrroles; Receptors, Retinoic Acid; Retinoids; Tretinoin; Vitamin A

Supplementation with vitamin A potentiates host resistance to malaria, however, the underlying mechanism is unknown. We tested the effects of 9-cis-retinoic acid, a metabolite of vitamin A, on CD36 expression, non-opsonic phagocytic clearance of parasitised erythrocytes, and TNFalpha production in human monocytes and macrophages. We found reduced secretion of TNFalpha, upregulated CD36 expression, and increased phagocytosis of Plasmodium falciparum-parasitised erythrocytes. Increased parasite clearance and reduced proinflammatory cytokine responses to infection might partly explain the beneficial effects of supplementation with vitamin A in malaria.

Topics: Alitretinoin; Animals; Antiprotozoal Agents; CD36 Antigens; Child; Erythrocytes; Humans; Malaria, Falciparum; Phagocytosis; Plasmodium falciparum; Tretinoin; Tumor Necrosis Factor-alpha; Up-Regulation; Vitamin A