Purine salvage as an antimalarial target

Abstract

The members of the genus Plasmodia, which are the causative agents of malaria, lack the de novo purine synthesis machinery. It has been repeatedly demonstrated that these parasites rely on the salvage of hypoxanthine released from the host erythrocyte ATP pool. Two keys enzymes in this metabolic pathway, purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), act sequentially to produce hypoxanthine from inosine and then incorporate hypoxanthine into the parasite nucleotide pool as inosine-5'-monophosphate.;Our lab has demonstrated that potent inhibition of both host and parasite PNP by the transition-state inhibitor DADMe-ImmucillinG (DADMe-ImmG) is able to kill P. falciparum, the most lethal of the human malarial species, in cultured human erythrocytes and in the Aotus-model of malaria. In both of these models, death of parasites is caused by hypoxanthine depletion and subsequent starvation of purines.;The mouse model of malaria utilizes the rodent species P. berghei and P. yoelii. We show that in mice DADMe-ImmG is orally available and able to completely inhibit blood PNP in less than 30 minutes at doses below 3 mg kg-1. In spite of this powerful inhibition, treatment of P. berghei infected mice with 10 mg kg-1 DADMe-ImmG resulted in higher peak parasitemias and shortened survival as compared to PBS-injected controls.;We employed ex vivo metabolic labeling studies and LC/MS/MS methods to investigate how P. berghei might bypass inhibition of PNP. Ex vivo metabolic labeling studies of infected erythrocytes and erythrocyte-free parasites isolated from infected mice demonstrate that DADMe-ImmG completely blocks the formation of hypoxanthine from inosine and its subsequent incorporation into the parasite nucleotide pool. However, we also observed that parasites are able to directly salvage adenine nucleotides from the host erythrocyte, presenting one possible bypass to PNP inhibition.;Further, LC/MS/MS metabolite analysis of the blood of infected mice treated with DADMe-IminG demonstrated potent PNP inhibition by the marked rise of blood concentrations of inosine and the malaria-specific PNP substrate 5'-methylthioinosine. However, blood levels of hypoxanthine never decreased in concert with the rise in PNP substrates, suggesting formation of hypoxanthine from HGXPRT or residual PNP activity in a compartment not accessible to DADMe-ImmG. These findings and the positive results in Aotus monkeys suggest that mouse malaria is an inadequate model for testing PNP inhibitors as potential anti-malarials.;In conjunction with our development of PNP inhibitors our lab has synthesized a number of HGXPRT inhibitors, most noteably ImmucillinH-5'-phosphate (ImmHP) a 1 nM inhibitor of recombinant PfHGXPRT. Despite this potent inhibition, ImmHP has failed to show biological activity due to lack of cell permeability and chemical stability issues presented by the 5'-phosphate.;To overcome these problems, we synthesized a new class of Immucillins, the acyclic Immucillin phosphonates (AIP). These new HGXPRT inhibitors are able to match the affinity of ImmHP (0.65 to 10.6 nM), show > 500-fold selectivity for PfHGXPRT over the human enzyme, contain fewer chiral centers and replace the labile 5'-phosphate with a chemically stable phosphonate group. Crystal structures of PfHGXPRT bound to pyrophosphate and either ImmHP or S-SerMe-ImmucillinH phosphonate show the decreased complexity of the AIPs allows a more favorable charge interaction between inhibitor and pyrophosphate.;Unmodified AIPs show no biological activity. Prodrug modification to attach an alkyl-propyloxy side chain to the phosphonate creating a mimic of a lysophospholipid drastically enhanced the biological activity of AIPs. Both the 16 and 18 carbon alkyl prodrugs demonstrate a low micromolar IC50 with cultured P. falciparum and inhibit >85% of radiolabeled hypoxanthine uptake in metabolic labeling experiments.;This work presents a detailed analysis of the role of PNP in purine metabolism of mouse malaria and presents a novel family of PfHGXPRT inhibitors and prodrugs.