GATEWAY™ vectors for Plasmodium falciparum transfection Skinner-Adams, Tina S.; Hawthorne, Paula L.; Trenholme, Katharine R. ...
Trends in parasitology,
2003, 2003-Jan, 2003-1-00, 20030101, Letnik:
19, Številka:
1
Journal Article
Recenzirano
Techniques that allow the genetic manipulation of
Plasmodium falciparum are important for improving our knowledge of the biology of this human pathogen. Although recent advances in transfection ...technology have significantly increased the number of genetic manipulations that are now possible, the process is still slow and tedious. Poor transfection efficiencies are largely responsible for this problem, and the cloning of nucleotides, particularly AT-rich sequences, into
P. falciparum transfection vectors can be time-consuming. We have used the GATEWAY™ conversion technology to provide us with a fast and convenient method of cloning such sequences into transfection vectors.
Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable ...environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs.
The sensitivities
in vitro of
Plasmodium falciparum to the benzimidazoles, albendazole, thiabendazole, mebendazole, omeprazole and 2 albendazole metabolites, albendazole sulphone and albendazole ...sulphoxide, were investigated and compared to those of the commonly used antimalarial drugs chloroquine and quinine. Quinine and chloroquine were the most potent drugs tested (EC
50 values of 8 × 10
−9–6 × 10
−8 mol/L and 5–7 × 10
−9 mol/L, respectively). Thiabendazole, mebendazole, albendazole sulphone and albendazole sulphoxide reached maximum growth inhibitions of 13–36% at the highest concentration tested (1 × 10
−4 mol/L). Albendazole (EC
50 range: not achieved-2 × 10
−6 mol/L) and omeprazole (EC
50 range: 2–4 × 10
−5 mol/L) were the most effective benzimidazoles. The activity of albendazole was pH dependent, as was that of chloroquine, and variable. Albendazole has its primary mode of action on trophozoites, suggesting that the drug may target parasite tubulin polymerization. Omeprazole, although also primarily effective against trophozoites, had additional activity against schizonts and ring forms, suggesting a distinct or additional parasitic target. Given the variable activity of albendazole and its rapid metabolism
in vivo into compounds with even less antimalarial activity, it appears unlikely that this benzimidazole will be useful in the treatment of malaria. The rapid activity and different stage-specific profile of the more soluble benzimidazole omeprazole warrants further investigation.
Novel targets for new drug development are urgently required to combat malaria, a disease that puts half of the world's population at risk. One group of enzymes identified within the genome of the ...most lethal of the causative agents of malaria, Plasmodium falciparum, that may have the potential to become new targets for antimalarial drug development are the aminopeptidases. These enzymes catalyse the cleavage of the N-terminal amino acids from proteins and peptides. P. falciparum appears to encode for at least nine aminopeptidases, two neutral aminopeptidases, one aspartyl aminopeptidase, one aminopeptidase P, one prolyl aminopeptidase and four methionine aminopeptidases. Recent advances in our understanding of these genes and their protein products are outlined in this review, including their potential for antimalarial drug development.
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