Abstract
Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.
Highlights
Each year, Plasmodium species infect 300 to 500 million people and cause nearly two million deaths, mostly in children under the age of five in sub-Saharan Africa [1]
Prior studies have shown that PfPNP and PfADA have an additional specificity for 5Ј-methylthiopurines, and that salvage of 5Ј-methylthioadenosine (MTA), a dead-end molecule of polyamine synthesis, is through the malarial purine salvage enzymes [5]
Most immucillins tested in malaria cultures, such as ImmH, 4 The abbreviations used are: PNP, purine nucleoside phosphorylase; ADA, adenosine deaminase; HGPRT, hypoxanthine-guanine phosphoribosyl transferase; MTA, methylthioadenosine; WT, wild type
Summary
P. falciparum Culture—Human erythrocytes were collected from local volunteers under protocol CCI 99 –240 of the Albert Einstein College of Medicine. Vector Construction and Parasite Transfection—The pfpnp allelic exchange fragment was PCR-amplified from 3D7 genomic DNA, using the primer combination p10/p11 (see supplementary materials). The IC50 values Ϯ the mean Ϯ S.E. were calculated from the IC50 values from 3 to 12 independent experiments, and for each drug tested a oneway analysis of variance analysis in combination with a Bonferroni post hoc test was performed to determine the p values of the various group pairs using KaleidaGraph version 4.03 (Synergy Software, Reading, PA). Mean Ϯ S.D. was determined, and twotailed unpaired t tests were carried out to determine the statistical significance for each disrupted line as compared with the wild-type control line using KaleidaGraph version 4.03 (Synergy Software) These results were confirmed using the lactate dehydrogenase assay, as described in Ref. 23.
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