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The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and ⌬arg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The ⌬arg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the ⌬arg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the ⌬arg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.

Trypanosomatids depend on spermidine for growth and survival. Consequently, enzymes involved in spermidine synthesis and utilization, i.e. arginase, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase, trypanothione synthetase (TryS), and trypanothione reductase (TryR), are promising targets for drug development. The ODC inhibitor alpha-difluoromethylornithine (DFMO) is about to become a first-line drug against human late-stage gambiense sleeping sickness. Another ODC inhibitor, 3-aminooxy-1-aminopropane (APA), is considerably more effective than DFMO against Leishmania promastigotes and amastigotes multiplying in macrophages. AdoMetDC inhibitors can cure animals infected with isolates from patients with rhodesiense sleeping sickness and leishmaniasis, but have not been tested on humans. The antiparasitic effects of inhibitors of polyamine and trypanothione formation, reviewed here, emphasize the relevance of these enzymes as drug targets. By taking advantage of the differences in enzyme structure between parasite and host, it should be possible to design new drugs that can selectively kill the parasites.

When ornithine decarboxylase, the initial and highly regulated enzyme in polyamine biosynthesis, is irreversibly inactivated by ␣-difluoromethylornithine, F9 teratocarcinoma stem cells are depleted of putrescine and spermidine and as a result differentiate into a cell type which phenotypically resembles the parietal endoderm cells of the early mouse embryo. Simultaneously the level of decarboxylated S-adenosylmethionine (dcAdoMet), the aminopropyl group donor in spermidine and spermine synthesis, increases dramatically, as the aminopropyl group acceptor molecules (putrescine and spermidine) become limiting. When this excessive accumulation of dcAdoMet is prevented by specific inhibition of the AdoMet decarboxylase activity, the differentiative effect is counteracted, despite the fact that the extent of polyamine depletion remains almost identical. Therefore, it may be concluded that dcAdoMet plays an important role in the induction of differentiation. Moreover, this key metabolite acts as a competitive inhibitor of DNA methyltransferase and is therefore capable of interfering with the maintenance methylation of newly replicated DNA. During the course of F9 cell differentiation, the highly methylated genome is gradually demethylated, and its pattern of gene expression is changed. Our present findings, that the DNA remains highly methylated and that the differentiative process is counteracted when the build-up of dcAdoMet is prevented, provide strong evidence for a causative relation between the level of dcAdoMet and the state of DNA methylation as well as cell differentiation.Cells require optimal levels of the polyamines, putrescine, spermidine, and spermine, for their growth and differentiation and are therefore equipped with many intricate mechanisms for the control of these levels (1, 2). The polyamine biosynthetic pathway consists of two highly regulated enzymes, ornithine decarboxylase (ODC) 1 and S-adenosylmethionine decarboxylase (AdoMetDC), and two constitutively expressed enzymes, spermidine synthase and spermine synthase (Fig. 1). Any cell that becomes polyamine-deficient is severely limited in its ability to grow and proliferate. There is also evidence suggesting that polyamines play a role in apoptotic cell death (3, 4). More specifically, the polyamines have been shown to regulate ion channel gating (5-7) and polyamine derivatives are constituents of venoms produced by many invertebrates (e.g. funnel-web spiders), where they act as potent neurotoxins (8). Polyamines also regulate programmed translational frameshifting, which is essential for the expression of ODC antizyme, a protein involved in the regulation of polyamine levels both in yeast (9) and in mammalian (10) cells.Of particular interest, with regard to growth regulation, is the finding that when ODC genes are transfected into cells and overexpressed, the cells go through malignant transformation (11). The fact that inhibition of the ODC activity counteracts malignant transformation suggests that the ODC gene is a proto-oncogene. In this co...