Impact of Field Isolate Identified Nonsynonymous Single Nucleotide Polymorphisms on Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitor Efficacy

Abstract

Plasmodium falciparum causes the most severe form of malaria and causes approximately 500 000 deaths per year. P. falciparum parasites resistant to current antimalarial treatments are spreading. Therefore, it is imperative to develop new antimalarial drugs. Malaria parasites are purine auxotrophic. They rely on purine import from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Recently, inhibitors of the P. falciparum ENT1 (PfENT1) that inhibit proliferation of malaria parasites in culture have been identified as promising starting points for antimalarial drug development. Genome sequencing of P. falciparum field isolates has identified nonsynonymous single nucleotide polymorphisms (SNPs) in the gene encoding PfENT1. Here we evaluate the impact of these PfENT1 SNPs on purine substrate affinity and inhibitor efficacy. We expressed each PfENT1-SNP in Saccharomyces cerevisiae. Using PfENT1-SNP-expressing yeast, we characterized the PfENT1 purine substrate affinity using radiolabeled substrate uptake inhibition experiments. Four of the 13 SNPs altered affinity for one or more purines by up to 7-fold. Three of the SNPs reduced the potency of a subset of the inhibitors by up to 7-fold. One SNP, Q284E, reduced the potency of all six inhibitor chemotypes. We tested drug efficacy in available parasite strains containing PfENT1 SNPs. While PfENT1-SNP-expressing yeast had decreased sensitivity to PfENT1 inhibitors, parasite strains containing SNPs showed similar or more potent inhibition of proliferation with all PfENT1 inhibitors. Thus, parasite strains bearing PfENT1 SNPs are not resistant to these PfENT1 inhibitors. This supports PfENT1 as a promising target for further development of novel antimalarial drugs.

Inhibition and Mechanism of Plasmodium falciparum Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferase

“…37 A new downfield resonance signal corresponding to 16 O in the bridging position would be observed by NMR. 38 Taking into consideration the low forward commitment factor and the S N 1-like mechanism from transition state analysis, 23 the lack of an 16 O− 18 O PIX demonstrates constrained torsional rotation of the pyrophosphate. Strong coordination of the pyrophosphate leaving group to the divalent magnesium ions or a transition state lifetime shorter than phosphoryl rotation could prevent phosphoryl rotation to prevent isotope scrambling.…”

“…PIX isotopic scrambling is expected if a reversible riboxocarbenium intermediate is formed during the reaction (Figure 3A), the Michaelis complex releases the substrate faster than is converted to products, and there is free torsional rotation of the pyrophosphate α-phosphoryl in the active site. 37 A new downfield resonance signal corresponding to 16 O in the bridging position would be observed by NMR. 38 Taking into consideration the low forward commitment factor and the S N 1-like mechanism from transition state analysis, 23 the lack of an 16 O− 18 O PIX demonstrates constrained torsional rotation of the pyrophosphate.…”

“…PIX was monitored by measuring 16 O/ 18 O isotope scrambling at C-1 of [1-13 C, 1- were acquired at 25 °C using a Bruker AVIII 600 MHz spectrometer running TopSpin 3.6 and equipped with a 5 mm H/F-TCI cryogenic probe. The 1-13 C of PRPP was monitored in a high-resolution 2D 1 H− 13 C HSQC to resolve the cross peak belonging to the 1-13 C bonded to 18 O from that bonded to 16 O expected to appear if PIX occurred during the enzymatic reaction. Each HSQC timepoint was run for 13 min with eight scans acquired for each increase using a spectral width of 14 ppm and 1 ppm for 1 H and 13 C, respectively, and a recycle delay of 1.1 s. The data sets were collected using 4096 and 32 complex points for 1 H and 13 C, respectively, and the time-domain NMR data were linear predicted in the indirect dimension and multiplied with a shifted sine-bell function (SSB = 3) in each dimension prior to Fourier transformation.…”

“…This occurs if a stabilized ribocation has a lifetime sufficient to permit rotation of the α-phosphoryl group, reformation of [1-13 C, bridge- 16 O]PRPP, and release to the substrate PRPP pool. No isotope scrambling in PRPP was observed by monitoring the 13 C chemical shift as influenced by the adjacent bridge-18 O (Figure 3B).…”

“…6-Oxopurine nucleosides are in equilibrium with 6-oxopurine bases by the action of purine nucleoside phosphorylases (PNP) present both in erythrocytes and in P. falciparum. − We and others have previously reported Pf HGXPRT to be a target for the development of novel antimalarials and demonstrated that transition state analogue (TSA) inhibitors and their lipophilic prodrugs of Pf HGXPRT block incorporation of extracellular purines and inhibit the growth of P. falciparum parasites in culture. ,, Other reports have demonstrated the validity of the Pf HGXPRT target by inhibition with nucleoside phosphonates. , Targeting PNP with transition state analogues in cells and Aotus primates blocks hypoxanthine formation and results in antiparasitic activity .…”