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J Biomol Screen. 2014 Jul;19(6):966-72. doi: 10.1177/1087057114528738. Epub 2014 Mar 31.

A High-Throughput Assay to Identify Inhibitors of the Apicoplast DNA Polymerase from Plasmodium falciparum.

Author information

1
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.
2
Biochemistry B.S./M.S. Program at Iowa State University, Ames, IA, USA.
3
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA Ames Senior High School, Ames, IA, USA.
4
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA swn@iastate.edu.

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.

KEYWORDS:

Enzyme assays or enzyme kinetics; anti-infective drugs; assays; binding or purification; chemistry; fluorescence methods; labeling; nucleic acid

PMID:
24687154
DOI:
10.1177/1087057114528738
[Indexed for MEDLINE]

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