zaprinast 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

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, which has limited potential for the prophylaxis, transmission blocking, and eradication efforts that will be needed in the future. To address these unmet needs, a high-throughput phenotypic liver-stage Plasmodium parasite screen was developed to systematically identify molecules with liver-stage efficacy. The screen recapitulates liver-stage infection by isolating luciferase-expressing Plasmodium berghei parasites directly from the salivary glands of infected mosquitoes, adding them to confluent human liver cells in 384-well plates, and measuring luciferase activity after a suitable incubation period. Screening 5,375 known bioactive compounds identified 37 liver-stage malaria inhibitors with diverse modes of action, as shown by inhibition time course experiments. Further analysis of the hits in the Food and Drug Administration-approved drug subset revealed compounds that seem to act specifically on the liver stage of infection, suggesting that this phase of the parasite's life cycle presents a promising area for new drug discovery. Notably, many active compounds in this screen have molecular structures and putative targets distinctly different from those of known antimalarial agents.

Topics: Animals; Anopheles; Antimalarials; Drug Evaluation, Preclinical; Hep G2 Cells; Humans; Inhibitory Concentration 50; Insect Vectors; Life Cycle Stages; Liver; Malaria; Malaria, Falciparum; Male; Mice; Mice, Inbred C57BL; Plasmodium berghei; Plasmodium falciparum; Treatment Outcome