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A High-Throughput Assay to Identify Inhibitors of the Apicoplast DNA Polymerase from Plasmodium falciparum
Morgan E Miller1, Eric E Parrott2, Risham Singh3
1Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.
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.
Insights
Researchers developed a new assay to find drugs targeting the malaria parasite Plasmodium falciparum. This assay screens for inhibitors of the apicoplast DNA polymerase POM1, a potential antimalarial drug target.
Area of Science:
- Biochemistry
- Parasitology
- Drug Discovery
Background:
- Malaria, caused by Plasmodium falciparum, is a significant global health threat.
- The parasite possesses a unique apicoplast organelle with its own genome, essential for survival.
- The apicoplast DNA polymerase (POM1) lacks mammalian homologs, making it a promising antimalarial drug target.
Purpose of the Study:
- To develop and validate a high-throughput assay for screening potential inhibitors of Plasmodium falciparum POM1.
- To identify novel compounds that can inhibit POM1 activity as a strategy for antimalarial drug discovery.
Main Methods:
- A fluorescent DNA polymerase assay utilizing a DNA hairpin substrate with a Cy3 dye and quencher.
- Strand-displacement synthesis by POM1 separates the dye and quencher, generating a measurable signal.
- Assay validation using 384-well plates, achieving a signal window of 7.90 and a Z' factor of 0.71.
Main Results:
- A pilot screen of a 2880-compound library was conducted.
- 62 compounds were identified as potential inhibitors, demonstrating >50% inhibition of POM1 activity.
- The assay demonstrated simplicity and statistical robustness suitable for large-scale screening.
Conclusions:
- The developed fluorescent assay is effective for identifying inhibitors of the Plasmodium falciparum apicoplast DNA polymerase POM1.
- This assay provides a robust platform for discovering novel antimalarial lead compounds targeting POM1.
- The identified inhibitors warrant further investigation as potential antimalarial drug candidates.
