Inhibition of multidrug-resistant HIV-1 by interference with cellular S-adenosylmethionine decarboxylase activity.

S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, can be specifically inhibited by the experimental drug SAM486A. The pharmaceutical interference with SAMDC activity results in the depletion of the intracellular pool of spermidine and spermine. In particular, low spermidine levels compromise hypusine modification and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), which is a cellular cofactor of the essential human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev. In the present study, we show that SAM486A efficiently suppresses HIV-1 replication, including the replication of viruses that are resistant to multiple reverse transcriptase and protease inhibitors. At drug concentrations that efficiently inhibit the formation of progeny viruses, no toxic effects of SAM486A on cellular metabolism are observed. It is demonstrated that the antiretroviral effect of SAM486A is based on the fact that Rev activity is severely compromised in drug-treated cells. Thus, inhibition of cellular SAMDC activity may provide a novel strategy to achieve suppression of otherwise drug-resistant viruses.

The putrescine analogue 1-aminooxy-3-aminopropane perturbs polyamine metabolism in the phytopathogenic fungus Sclerotinia sclerotiorum.

The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40-50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.

Effect of retinoic acid on proliferation and polyamine metabolism in cultured bovine retinal pigment epithelial cells.

Reports regarding the effect of all-trans-retinoic acid (RA) on the cell growth of retinal pigment epithelial cells (RPE) have been contradictory. The aims of this study are to clarify the in vitro effect of RA on RPE cells and to examine polyamine metabolism after RA stimulation. A 4-day incubation of fetal-calf-serum (FCS)-stimulated RPE cells with 10 or 25 microM RA significantly increased both cell number and [3H]thymidine incorporation. RPE cells grown over an extended period for 8 days also increased in number and reached full confluency. However, if the incubation was further extended to 12 days, no further increase in cell number was detected. RA treatment of FCS-stimulated RPE cells shifted the peak of ornithine decarboxylase (ODC) activity from 16 to 4 h. S-adenosylmethionine decarboxylase (SAMDC) activity and spermidine/spermine N1-acetyltransferase (SAT) activity of RA-treated RPE cells were significantly greater until 8 and 16 h after incubation, respectively. The putrescine content was significantly increased in RA-treated RPE cells up until 24 h, while spermidine, spermine and N1-acetylspermidine contents were significantly increased until 16 h. Our findings suggest that RA treatment increases the intracellular polyamine concentration of RPE cells via activation of ODC, SAMDC and SAT and that this results in the promotion of RPE cell growth until the cells reach full confluency.

CGP 48664, a potent and specific S-adenosylmethionine decarboxylase inhibitor: effects on regulation and stability of the enzyme.

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) catalyses a regulatory important step in the biosynthesis of polyamines and is a potential target for therapeutic agents against various parasitic diseases and proliferative disorders. In the present study we examined the effects of a newly synthesized AdoMetDC inhibitor. 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664), on polyamine metabolism in the mouse leukaemia cell line L1210. Treatment of the cells with 2 microM CGP 48664 led to a depletion of cellular spermidine and spermine. The putrescine content, in contrast, was markedly increased. Cells seeded in the presence of the inhibitor showed a significant decrease in growth rate, which was fully reversed by the addition of 2 microM spermidine or 1 microM spermine. The syntheses of ornithine decarboxylase and AdoMetDC were greatly increased in cells treated with CGP 48664. These increases were not correlated with similar changes in the mRNA levels, indicating the involvement of a translational mechanism. CGP 48664 was demonstrated to be a very poor competitor of spermidine uptake in the L1210 cells. L1210 cells deficient in polyamine transport were as sensitive to the antiproliferative effect of the inhibitor as were the parental cells, indicating that CGP 48664 did not enter the cells by the polyamine transport system. In addition to inhibiting AdoMetDC, CGP 48664 stabilized the enzyme against degradation. In the present study we also demonstrated that aminoguanidine (AMG), which is frequently used in cellular systems to inhibit any action of serum polyamine oxidase, apparently inhibits AdoMetDC by an irreversible mechanism that markedly stabilizes the enzyme against proteolytic degradation. CGP 48664 and the parental compound methylglyoxal bis(guanylhydrazone), which is also a potent inhibitor of AdoMetDC, contain one or two AMG-like moieties; the importance of these residues in the inhibition of AdoMetDC is discussed.

In vivo effect of berenil on rat liver polyamine metabolism.

1. Berenil, administered to rats in vivo, promoted a decrease in liver SAMDC activity, but an increase in ODC and SAT activity. 2. Its effect on ODC was completely prevented by cycloheximide, that on SAT only partially. 3. Berenil had no effect on ODC activity in adrenalectomized rats. Adrenergic antagonists counteracted the effect of Berenil on ODC activity. 4. Polyamine content was increased. The maximum modification was observed for putrescine and N1-acetylspermidine.

Polyamine-mediated regulation of S-adenosylmethionine decarboxylase expression in mammalian cells. Studies using 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, a suicide inhibitor of the enzyme.

Cell proliferation is dependent on an adequate supply of the polyamines putrescine, spermidine and spermine. One of the key steps in the polyamine biosynthetic pathway is catalyzed by S-adenosylmethionine decarboxylase (AdoMetDC). In the present study we have used a newly synthesized enzyme-activated irreversible AdoMetDC inhibitor, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine [(Z)-AbeAdo], to investigate the regulation of this enzyme. Treatment of mouse L1210 leukemia cells with (Z)-AbeAdo resulted in a total inhibition of their AdoMetDC activity followed by depletion of the spermidine and spermine content. The putrescine content, however, was dramatically increased after treatment with (Z)-AbeAdo. In spite of the cellular depletion of spermidine and spermine, only a minor inhibitory effect was obtained on cell growth, indicating that putrescine at a high concentration might partly replace spermidine and spermine in their growth-promoting functions. Cells grown in the presence of (Z)-AbeAdo exhibited an increased synthesis of AdoMetDC, which was counteracted by the addition of either spermidine or spermine. The change in AdoMetDC synthesis could not be fully explained by a change in the level of AdoMetDC mRNA, indicating also a translational control. Mammalian AdoMetDC is synthesized as a larger proenzyme, which is then cleaved into two subunits of different sizes. The conversion of the proenzyme into the subunits is a very rapid process, which is stimulated greatly by putrescine in vitro. However, the processing of the proenzyme in the (Z)-AbeAdo-treated L1210 cells was not affected by their very high putrescine content, indicating that the conversion might be saturated at low levels of putrescine, or that most of the putrescine in the (Z)-AbeAdo-treated L1210 cells might be bound to sites normally occupied by spermidine and spermine.

Putrescine activated S-adenosylmethionine decarboxylase from Trypanosoma brucei brucei.

Trypanosoma brucei brucei contained a S-adenosyl-L-methionine decarboxylase (AdoMetDC) strongly activated by putrescine. The enzyme was also activated to a lesser extent by cadaverine and 1,3-diaminopropane. Spermidine and spermine had no effect on basal activity of the enzyme. However, they interfered with putrescine activation of trypanosomal AdoMetDC. The trypanosomal enzyme could not be precipitated with antiserum against human AdoMetDC. The trypanosomal AdoMetDC enzyme subunit was labeled by reaction with 35S-decarboxylated AdoMet in the presence of NaCNBH4, and found to have a molecular weight of 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit was readily degraded on storage to a form with a molecular weight of 26 kDa. The specificity of labeling of AdoMetDC by this procedure was confirmed by the prevention of 35S-decarboxylated S-adenosylmethionine (AdoMet) binding in the presence of specific AdoMetDC inhibitors [either methylglyoxal bis(guanylhydrazone (MGBG), a reversible inhibitor, or 5'-deoxy-5'-[(2-hydrazinoethyl)methylamino]adenosine (MHZEA), an irreversible inactivator]. As compared to human AdoMetDC, the trypanosomal enzyme showed weaker binding to a column of MGBG-Sepharose and also was significantly less sensitive to inhibition by MGBG and its congener ethylglyoxal bis(guanylhydrazone) (EGBG). Thus, the trypanosomal AdoMetDC differs significantly from its mammalian and bacterial counterparts and may therefore be exploited as a specific target for chemotherapy of trypanosomiasis.

Role of ornithine decarboxylase suppression and polyamine depletion in the antiproliferative activity of polyamine analogs.

Two transfected cell lines, one carrying a mammalian ornithine decarboxylase (ODC) that is suppressed by polyamines and one carrying a trypanosomal ODC that is not, were used to ask whether ODC suppression is necessary for the antiproliferative activities of two polyamine analogs, N1,N8-bis(ethyl)spermidine (BES) and N1,N14-bis(ethyl)homospermine (BE444). Both analogs accumulated within cells and suppressed S-adenosylmethionine decarboxylase, as well as polyamine-sensitive mouse ODC activity. Neither drug was able to suppress the activity of the polyamine-refractory trypanosome ODC. But, whereas BE444 was able to inhibit growth of both cell lines, BES could inhibit only growth of cells carrying the polyamine-sensitive ODC, under conditions that cause prolonged depletion of endogenous polyamines. We conclude from these studies that the antiproliferative activity of BES, a less potent drug, requires the suppression of ODC. The efficacy of BE444 is enhanced by its ability to suppress ODC. However, it can function without ODC suppression, whereas BES cannot.