Transition state inhibitors of purine nucleoside phosphorylase: Mechanism and biological effects

Abstract

Cells that engage in rapid proliferation synthesize RNA and DNA with nucleic acid precursors obtained through purine salvage pathways. We have developed powerful transition-state inhibitors of a key enzyme in purine salvage, purine nucleoside phosphorylase (PNP), to test the hypothesis that disrupting purine salvage will prevent growth of specific cell types. Human T-cells and the malarial parasite, Plasmodium falciparum, were chosen as experimental models because a genetic deficiency in PNP causes a T-cell immunodeficiency and P. falciparum is a purine auxotroph.;In vitro T-cell proliferation studies revealed that as little as 5 nM of the PNP inhibitor, immucillin-H (Imm-H), completely prevented proliferation of the poorly differentiated human T-cell leukemia lines, CCRF-CEM and MOLT-4 in the presence of 2'-deoxyguanosine. The inhibitor had no effect on non T-cell lines, even at high concentrations. Normal human peripheral T-cells are sensitive to Imm-H when supra-stimulated, but unstimulated T-cells are resistant to the inhibitor. Metabolic studies correlate deoxycytidine kinase activity with the cellular accumulation of dGTP to cause cell susceptibility to Imm-H.;In the SCID mouse model for T-cell leukemia, immunodeficient mice were injected (I.P.) with human T-cell leukemia cells. Oral administration of Imm-H caused significant reduction in the growth of abdominal tumors in one experiment. When repeated on a large scale with additional conditions, no effect of immucillin-H was seen.;Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP), which is present at high levels in human erythrocytes and in P. falciparum. We characterized the Plasmodium falciparum PNP (PfPNP) and identified inhibitors that can be used to induce a purine-less death of the parasites in culture.;This work is supported by Research Grant GM41916 from the NIH and MSTP Training Grant T32GM07288. (Abstract shortened by UMI.).