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 a Z’ 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.

Replication and maintenance of the Plasmodium falciparum apicoplast genome

“…PfPREXpol was reported to yield 4 mg of purified recombinant protein per liter of culture [20], whereas the yield of apPOL is 50 mg of pure protein per liter (although, these constructs were expressed in different E. coli strains) [43]. The total yield of KPom1 was not reported, and the protein was expressed with a maltose-binding-protein (MBP) fusion tag as opposed to the hexahistidine tags used for PfPREXpol and apPOL [24].…”

“…There may be some concern of cross-reactivity between DNA ligase, SSB, and Prex helicase and human cellular functions (as these proteins have distant human homologs), but it is likely that apicoplast-specific features could be exploited. Efforts to identify additional inhibitors of the Prex polymerase are underway [43]. As we gain more biochemical and structural knowledge concerning the proteins involved with replication and maintenance of the apicoplast genome, we begin to elucidate key features that can be employed in our efforts in anti-malarial drug discovery.…”

“…3) [67]. Prex polymerase is able to perform the required strand-displacement synthesis reaction, making it a prime candidate for the long-patch BER pathway [43]. An APN1 homolog (gene ID PF3D7_1332600) appears to be the apurinic/apyrimidinic endonuclease and contains an apicoplast targeting sequence.…”

Crystallization and preliminary X-ray analysis of thePlasmodium falciparumapicoplast DNA polymerase

“…Exonuclease mutation primer previously (Miller et al, 2014). Aliquots of purified apPOL exoÀ were flash-frozen in liquid nitrogen and stored at 193 K. The freezing process does not impact on the polymerase activity or crystallization.…”

“…Isopropyl -d-1-thiogalactopyranoside (IPTG) was added to a concentration of 0.2 mM to induce translation. The induced cultures were grown overnight at 291 K. The cells were harvested by centrifugation for 20 min at 3000g and 277 K, suspended in a minimal volume of 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 5 mM imidazole (buffer 1) and stored at 193 K. The polymerase was purified at 277 K as described by Miller et al (2014). The lysate from homogenized cells (EmulsiFlex-C5) was centrifuged for 1 h at 30 000g and 277 K. The supernatant was loaded onto a 5 ml Ni-agarose column and washed with buffer 1 and then with 20 mM Tris-HCl pH 8.0, 1 M NaCl, 25 mM imidazole.…”