Two ornithine decarboxylase inhibitors, DL-alpha-difluoromethylornithine (eflornithine; DFMO) and a-monofluoromethyldehydroornithine methyl ester (delta MFMO X CH3) were compared in their ability to ...cure two distinct Trypanosoma brucei brucei central nervous system murine model infections. Both inhibitors cured the TREU 667 and LUMP 1001 isolates if used in combination with a single (20 mg/kg) injection of suramin, a trypanocide in current clinical use. The curative dose of delta MFMO X CH3 in combination with suramin was 1.09 g/kg/day, administered in the drinking water for 14 days; used with suramin, the curative dose of DFMO was 5.3 g/kg/day for 14 days (5 times the delta MFMO X CH3 dose required). In host animals, delta MFMO X CH3 was not toxic and was accumulated by trypanosomes 6-8 times faster than DFMO. Since DFMO by itself has been highly effective against T. b. gambiense infections in humans (12-15 g/day for 6 weeks) the present data suggest that delta MFMO X CH3 might be effective in a shorter regimen and at lower doses.
Adherence of Plasmodium falciparum-infected erythrocytes (IE) to the venular endothelium in brain and other organs is characteristic of cerebral malaria, an often fatal complication in infected ...individuals. It has been shown that cytoadherence may be mediated through interaction of IE with glycoproteins on host target cell surfaces, including CD36 (GPIV), intercellular adhesion molecule-1 (ICAM-1), and thrombospondin. Inhibitors of glycoprotein synthesis and processing were tested for their abilities to decrease IE adherence to C32 human melanoma cells. The alpha-glucosidase inhibitor, castanospermine, was effective in disrupting cytoadherence in vitro when incubated with C32 cells (IC50 = 600-700 microM). Castanospermine-6-butyrate was even more effective than the parent compound (IC50 = 9 microM) in disrupting cytoadherence. The mannosidase inhibitors, swainsonine and deoxymannojirimycin, had no effect on cytoadherence at concentrations up to 2 mM. No effect on cytoadherence was observed when the glucosidase and mannosidase inhibitors were incubated with IE rather than the C32 cell cultures. The level of CD36 on the C32 cell surface was decreased as measured by fluorescence-activated cell sorting (FACS) analysis with the same inhibitors which inhibited cytoadherence. Cells labeled with fluorescein isothiocyanate (FITC) OKM5 monoclonal antibody, which recognizes CD36 and disrupts cytoadherence, showed decreased fluorescence when treated with tunicamycin and castanospermine-6-butyrate but not when treated with swainsonine or deoxymannojirimycin. ICAM-1 levels, as measured by surface labeling of C32 cells with FITC CD54 monoclonal antibody, were decreased in cells treated with tunicamycin. However, incubation of cells with castanospermine-6-butyrate or deoxymannojirimycin decreased cell surface ICAM-1 levels only slightly. These findings suggest that (1) in C32 cells, levels of cell surface CD36, and not ICAM-1, change proportionally to the level of cytoadherence; (2) drugs which can affect the carbohydrate moiety of cellular glycoproteins decrease cytoadherence of IE to C32 cells; and (3) protection against the development of cerebral malaria may be possible with inhibitors of glycoprotein biosynthesis.
Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five ...compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed.
Bacterial growth was measurably slowed by a combination of drugs which inhibit polyamine-biosynthetic enzymes. Addition of DL-alpha-monofluoromethylornithine, which was shown to inactivate ...irreversibly ornithine decarboxylase extracted from Escherichia coli (Ki = 0.36 mM) and Pseudomonas aeruginosa (Ki = 0.30 mM), DL-alpha-difluoromethylarginine and dicyclohexylammonium sulphate to cultures of E. coli or P. aeruginosa resulted in a 40 and 70% increase in generation times (decreased growth rates) respectively, which was completely reversed by the addition of 0.1 mM-putrescine plus 0.1 mM-spermidine to the medium. Decreased intracellular polyamine concentrations correlated with increased generation times; putrescine concentration was decreased by 70% in E. coli and 80% in P. aeruginosa, while spermidine concentration was decreased by 50% in E. coli and 95% in P. aeruginosa. Subsequent investigation of the inactivation of the ornithine decarboxylase by monofluoromethylornithine indicated that it was active-site directed, as the normal substrate ornithine slowed the rate of inhibition. Specific interference with polyamine biosynthesis may be a viable approach to control of some bacterial infections.
Protein synthesis in Trypanosoma brucei brucei was rapidly inhibited during polyamine depletion by DL-alpha-difluoromethylornithine (DFMO) in vitro and in vivo. 3HLeucine incorporation was depressed ...30-40% by 24 h and 80-90% by 48 h of DFMO treatment. Concomitantly there was an apparent decrease in the synthesis of the variant-specific glycoprotein (VSG) in DFMO-treated trypanosomes, as measured by decreased incorporation of 3Hmyristic acid into VSG. The discovery of decreased protein synthesis in T. b. brucei during DFMO treatment is noteworthy, because it was reported previously that protein synthesis was paradoxically stimulated 2-4-fold during DFMO treatment in these organisms. Decreased protein synthesis probably relates to the biochemical mechanism of action of DFMO on trypanosomes.
When tested for ornithine and arginine decarboxylases, pyrrolizidine alkaloid-bearing Senecio riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants exhibited only ...ornithine decarboxylase activity. This contrasts with previous studies of four species of pyrrolizidine alkaloid-bearing Heliotropium (Boraginaceae) in which arginine decarboxylase activity was very high relative to that of ornithine decarboxylase. Unlike Heliotropium angiospermum and Heliotropium indicum, in which endogenous arginine was the only detectable precursor of putrescine channeled into pyrrolizidines, in the species studied here--using difluoromethylornithine and difluoromethylarginine as the enzyme inhibitors--endogenous ornithine was the main if not the only precursor of putrescine converted into the alkaloid aminoalcohol moiety. In S. riddellii and C. retusa at flowering, ornithine decarboxylase activity was present mainly in leaves, especially the young ones. However, other very young organs such as inflorescence and growing roots exhibited much lower or very low activities; the enzyme activity in stems was negligible. There was no correlation between the enzyme activity and polyamine or alkaloid content in either species. In both species only free polyamines were detected except for C. retusa roots and inflorescence--with relatively very high levels of these compounds--in which conjugated putrescine, spermidine, and spermine were also found; agmatine was not identified by HPLC in any plant organ except for C. retusa roots with rhizobial nodules. Organ- or age-dependent differences in the polyamine levels were small or insignificant. The highest alkaloid contents were found in young leaves and inflorescence.
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A 4',5'-unsaturated 5'-fluoroadenosine inhibitor of S-adenosyl-L-homocysteine hydrolase (SAH hydrolase; EC 3.3.1.1), MDL 28842, was found to inhibit markedly the growth of Plasmodium falciparum in ...vitro and Plasmodium berghei in mice. Inhibition of P. berghei growth was associated with a large increase in the concentration of S-adenosyl-L-homocysteine (SAH) in the erythrocytes of the mice treated with MDL 28842. This increase in SAH was due apparently to inhibition of the mouse erythrocyte SAH hydrolase activity, because SAH hydrolase activity was undetectable in either P. berghei or P. falciparum isolated from infected erythrocytes, although enzyme activity was readily detected in mouse erythrocyte extracts. Therefore, MDL 28842 probably inhibits plasmodial growth indirectly by adversely changing the milieu of the host erythrocyte. SAH hydrolase represents a worthwhile target for the future development of potent inhibitors for the chemotherapy of malaria.
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