Plasmodium falciparum Parasites Are Killed by a Transition State Analogue of Purine Nucleoside Phosphorylase in a Primate Animal Model

María B Cassera,Jemy A Gutierrez,Vern L Schramm,Richard Depinto,Steven C Almo,Yarlagadda S Babu,Gary B Evans,Emilio F Merino,Meng-Chiao Ho,Andrew S Murkin,Nicanor Obaldia,Keith Z Hazleton,Matthew Bogyo

doi:10.1371/journal.pone.0026916

Abstract

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.

Highlights

Plasmodium parasites are purine auxotrophs and require preformed purine bases for synthesis of nucleotides, cofactors, and nucleic acids [1]
Adequate inhibition of purine nucleoside phosphorylase (PNP) requires inhibitors with extraordinary affinity as the enzyme is present at high levels in the host erythrocytes and in P. falciparum [15]
In P. falciparum in vitro cultures, it is reported that the half-maximum inhibitory concentration (IC50) for PNP inhibitors increases with the hematocrit [11]

Summary

Introduction

Plasmodium parasites are purine auxotrophs and require preformed purine bases for synthesis of nucleotides, cofactors, and nucleic acids [1]. Purine salvage in P. falciparum uses hypoxanthine formed in erythrocytes or in parasites by the sequential actions of adenosine deaminase (hADA, PfADA) and/ or purine nucleoside phosphorylase (hPNP, PfPNP) [2] (Figure 1). Hypoxanthine, inosine and adenosine are transported from erythrocytes into parasites by the equilibrative nucleoside transporter (PfNT1) [3,4]. Hypoxanthine and ATP are in dynamic metabolic exchange via ADP, AMP, IMP, inosine and adenosine. Hypoxanthine in the parasite is converted to IMP by hypoxanthine-guanine-xanthine phosphoribosyl-transferase (PfHGXPRT) to provide inosine monophosphate (IMP), a precursor for all required purines. Host and parasite PNPs are essential for the formation of hypoxanthine, making PNPs a target for the purine salvage pathway

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Conclusion