Polyamine Metabolism in Disease-causing Microorganisms and Viruses

A. Protozoa. The polyamine metabolism of disease-causing protozoa has been of particular interest because of the pronounced sensitivity of several species to the effects of several polyamine inhibitors, primarily DFMO.

The original observations of the effects of DFMO on protozoa were made in African trypanosomes when the inhibitor was found to cure acute, lethal infections of Trypanosoma brucei brucei in mice. These initial results led to the rapid clinical use of DFMO in Africa against what would have been fatal cases of late-stage, arsenic-resistant West African sleeping sickness caused by Trypanosoma brucei gambiense.

Numerous experiments have been conducted concerning the role of polyamines in trypanosomal growth and differentiation. The African trypanosome appears to be unusually sensitive to DFMO, due in part to both its rapid doubling time and lack of spermine. ODC in the trypanosome, however, appears to be kinetically similar to the mammalian enzyme. Again, analogous to mammalian cells, DFMO enters the trypanosome by passive diffusion rather than facilitated transport.

An intact host immune system is necessary for cures of trypanosome infections, and other evidence indicates that polyamines are required for the shift of antigenic determinants on the parasite membrane. Furthermore, DFMO and consequent polyamine depletion induces morphological and biochemical shifts of entire trypanosome populations from parasitic bloodstream forms to “short, stumpy” forms, which do not replicate. This differentiation event is independent of an earlier actual block of bloodstream form replication.

Trypanosomes also contain a novel spermidine-containing cofactor, which is necessary for glutathione reductase activity. This cofactor, trypanothione, uniquely present in trypanosomatids, is greatly reduced in T. b. brucei after DFMO treatment and is probably yet another target for polyamine depletion in these protozoa.

Recent experiments on molecular aspects of ODC from T. b. brucei have demonstrated that the trypanosomal ODC has a much longer half-life than the mammalian enzyme. Although the trypanosomal enzyme is very homologous to the mammalian enzyme, a likely explanation for this is that ODC from T. b. brucei lacks a specific 36-amino-acid carboxyterminal peptide containing a “PEST” (prolineglutamate - aspartate - serine-threonine - containing) region, which promotes rapid protein degradation.

The other polyamine biosynthetic enzymes have been less studied in trypanosomes and, although the AdoMetDC of T. b. brucei is inhibited by drugs such as Berenil®, pentamidine, and MGBG, the effects of such inhibition contribute little or nothing to the antiprotozoal effects of these agents. As is the case with the mammalian enzyme, spermidine synthase from T. b. brucei is quite sensitive to both cyclohexylamine and AdoDato. Neither one has any in vivo effect on trypanosome infections as might be predicted, because neither one actually alters trypanosome polyamine levels.

DFMO will also inhibit the growth of promasti- gotes of the trypanosomatid Leishmania donovani but has no effect on its intracellular-infective cousin Trypanosoma cruzi, the cause of American trypanosomiasis (Chagas’ disease). Unexpectedly, a-difluoro- methylarginine, an irreversible inhibitor of arginine decarboxylase (an enzyme not present in mammalian cells), significantly inhibited T. cruzi infection of macrophages as well as actual replication of the parasite. These findings indicate the apparent presence of arginine decarboxylase in T. cruzi and a possible unique target for this now incurable disease.

A number of the sporozoea class of protozoa are quite sensitive to the effects of DFMO, including Eimeria spp. and Plasmodia spp., the latter causing various forms of malaria, the most common parasitic disease worldwide. DFMO inhibits the in vitro replication of Plasmodium falciparum and will cure exoerythrocytic infections of P. berghei in mice and also block the sporogonous cycle of P. berghei in mosquitoes. Although not curative of erythrocytic infection of P. berghei, DFMO will significantly lower parasitemia and, in combination with a bis(benzyl)polyamine analogue, will actually cure P. berghei-infected mice. [See Malaria.]

Pneumocystis carinii, the cause of the lethal opportunistic pneumonia in acquired immunodeficiency syndrome patients, has been thought to be related to the protozoal class Sporozoea, subclass Coccidia, although recently it has been shown to have ribosomal RNA more homologous to certain fungi. In any case, DFMO has been successfully used in numerous clinical instances as therapy for P. carinii pneumonia. Experiments in cell culture and immunosuppressed rats have shown that P. carinii is inhibited by DFMO, and its growth-inhibitory effects can be reversed by putrescine.

B. Bacteria. In bacteria and in plants, an alternate pathway exists for the synthesis of putrescine via the decarboxylation of arginine to form agmatine, which then yields putrescine (see Fig. 1). As bacteria have both ODC and arginine decarboxylase, inhibition of either enzyme alone induces the other to increase and thus maintain a specific intracellular concentration of putrescine.

Although bacteria, in general, contain only putrescine and spermidine and not spermine, it has been difficult to deplete intracellular polyamines and thus have specific effects on cell growth and replication.

Remarkably, several bacteria such as Escherichia coli and Klebsiella pneumoniae have ODC enzymes that are wholly unreactive to DFMO. Other analogues, however, such as a-difluoromethylputrescine and α-monofluoromethylornithine, do completely and irreversibly inhibit the enzymes from these organisms. α-Difluoromethylarginine, as well as even other more potent arginine and agmatine analogues, was found to irreversibly inhibit the arginine decarboxylases found in both bacteria and plants.

AdoMetDC from E. coli is inhibited by MGBG in a cell-free system, but this drug is not effective on intact cells. Some analogues of AdoMet as well as pentamidine and Berenil® are also in vitro inhibitors of cell-free bacterial AdoMetDC. With regard to spermidine synthase, both cyclohexylamine and AdoDato are potent inhibitors of the E. coli and Pseudomonas aeruginosa cell-free enzymes. However, cyclohexylamine is taken up by both organisms and will inhibit growth specifically due to depletion of spermidine, unlike AdoDato, which is not taken up.

The most significant growth inhibition of bacteria (e.g., E. coli, P. aeruginosa, and Serratia marcescens) was demonstrated with combinations of a-monoflu-oromethylornithine, a-difluoromethylarginine, and cyclohexylamine, wherein all three polyamine biosynthetic enzymes (ODC, arginine decarboxylase, and spermidine synthase) were simultaneously inhibited. Although appreciable growth inhibitory effects were noted, they were not as profound as in a number of protozoa and fungi. Therefore, the aforementioned inhibitors of polyamine biosynthesis will unlikely be useful as therapy for any bacteriall caused infections.

C. Fungi. Filamentous fungi and most yeast synthesize putrescine solely via ODC, unlike bacteria. Although only limited experimental work has been done with zoo-philic and disease-causing fungi and yeast, the ODC in Saccharomyces cervisiae and S. uvarum is sensitive to DFMO. Furthermore, polyamines are depleted by DFMO in several human pathogenic Candida spp., whereas C. tropicalis growth was also significantly inhibited by DFMO as well. α-Monofluoromethyl- dehydroornithine methylester was shown to be a potent inhibitor of two species of dermatophyte fungi, Microsporum and Trichophyton. In fact, it was 25 times more effective than DFMO itself, which also significantly arrested the growth of these organisms.

D. Viruses. Viruses as intracellular parasites depend completely on their metabolically active host cells for replication. However, in some cases, viral enzymes necessary for replication are encoded in the viral DNA or RNA. The poxviruses such as vaccinia apparently encode both ODC and AdoMetDC and have been shown to respond to polyamine inhibitors such as α-methylor-nithine, DFMO, and MGBG. DNA viruses in the herpes family, such as herpes simplex virus and human cytomegalovirus, have been shown to be sensitive to the effects of DFMO and MGBG as well.

A correlation was also made between the ability of herpes simplex virus to induce the synthesis of polyamines and the antiviral effects of the inhibitors. It was found that depletion of polyamines was necessary in the host cells prior to infection with cytomegalovirus to have an effect on viral replication by DFMO. Overall, although polyamine biosynthesis is required for viral replication, more experimental work is needed to effectively determine any possible utility of inhibitors for viral diseases.