title: A High-Throughput Assay to Identify Inhibitors of the Apicoplast DNA Polymerase from Plasmodium falciparum.
abstract: 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.
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.
authors.forename: Morgan E
authors.initials: ME
authors.lastname: Miller
authors.affiliation: Biochemistry B.S./M.S. Program at Iowa State University, Ames, IA, USA.
authors.forename: Eric E
authors.initials: EE
authors.lastname: Parrott
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA Ames Senior High School, Ames, IA, USA.
authors.forename: Risham
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA swn@iastate.edu.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Nelson
mesh_terms: D000962:Antimalarials / Q000737:chemistry*; D063546:Apicoplasts / Q000201:enzymology*; D002736:Chloroplasts / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D055808:Drug Discovery; D005092:Exonucleases / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D016778:Malaria, Falciparum / Q000188:drug therapy; D008928:Mitochondria / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry; D019384:Nucleic Acid Synthesis Inhibitors / Q000737:chemistry*; D019151:Peptide Library; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D000962:Antimalarials; D009097:Multienzyme Complexes; D019384:Nucleic Acid Synthesis Inhibitors; D019151:Peptide Library; D015800:Protozoan Proteins; C503371:prex protein, Plasmodium falciparum; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases
keywords: Enzyme assays or enzyme kinetics; anti-infective drugs; assays; binding or purification; chemistry; fluorescence methods; labeling; nucleic acid