Polyamine metabolism: a potential therapeutic target in trypanosomes.

alpha-Difluoromethylornithine (RMI 71,782), a specific irreversible inhibitor of the first step in polyamine biosynthesis, that is, the formation of putrescine from ornithine by ornithine decarboxylase, cures mice infected with a virulent, rodent-passaged strain of Trypanosoma brucei brucei. This parasite is closely related to the trypanosomes that cause human sleeping sickness. The drug, which is remarkably nontoxic, was effective when administered in drinking water or by intubation. The ability of the compound to inhibit ornithine decarboxylase in vitro was demonstrated by the reduced amounts of putrescine synthesized from tritiated ornithine in Trypanosoma brucei suspensions. These observations direct attention to polyamine metabolism as a target for chemotherapy of parasitic diseases.

Stimulation of Rauscher leukemia virus DNA polymerase DNA-directed DNA synthesis by cationic trypanocides and polyamines.

Activated DNA-directed DNA synthesis catalyzed by Rauscher leukemia virus (RLV) and other type C mammalian retroviral DNA polymerases is uniquely stimulated by biologically active polyamines. Cationic trypanocides may act as antagonists of polyamine function. As described here, several cationic trypanocides stimulate RLV polymerase-catalyzed DNA-directed DNA synthesis at concentrations significantly inhibiting eukaryotic DNA polymerases. Such stimulation is negated by polyamines. Kinetic analysis of the stimulation of RLV DNA polymerase by three structurally dissimilar cationic trypanocides (Antrycide, Burroughs-Wellcome Compound 64A, and Bayer Compound 1694) suggests that such stimulation is, in part, due to a drug:DNA structural interaction resembling the polyamine:DNA structural complex recognized by the RLV DNA polymerase.

S-adenosylmethionine synthetase in bloodstream Trypanosoma brucei.

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10-250 microM the Km for methionine was 20 microM, and this changed to 200 microM for the range 0.5-5.0 mM. In the range 10-250 microM the Km for ATP was 53 microM, and this changed to 1.75 mM for the range 0.5-5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [14C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (Ki 240 microM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-alpha-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration approximately 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.

Effect of DL-alpha-difluoromethylornithine on methionine cycle intermediates in Trypanosoma brucei brucei.

Activities of enzymes involved in transmethylation reactions were determined in bloodstream trypomastigotes of Trypanosoma brucei brucei infection in rats. S-Adenosyl-L-methionine synthetase (EC 2.5.1.6), S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1), cystathionine synthase (EC 4.2.1.21), as well as several transmethylases were detected and localized in cytosolic rather than particulate fractions. High performance liquid chromatography analysis of methionine cycle intermediates in cells from untreated rats and from rats treated with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO) indicated that the inhibitor causes pronounced changes in concentrations of these intermediates and dramatically alters the methylation index of the cell. These findings demonstrate another in the wide range of metabolite disturbances attributable to DFMO and reflect the belief that multiple biochemical events are a sequel of its action on trypanosomes.

Effect of DL-alpha-difluoromethylornithine on polyamine synthesis and interconversion in Trichomonas vaginalis grown in a semi-defined medium.

Growth of Trichomonas vaginalis in a semi-defined medium was inhibited by 5 mM DL-alpha-difluoromethylornithine (DFMO). Using high pressure liquid chromatography (HPLC) analysis, putrescine and cadaverine levels were found to be 90 and 100% reduced, respectively after 120 h exposure, whilst spermidine and spermine levels were unchanged. Putrescine (40 microM) and cadaverine (6 microM) were detected in the spent media from control cultures. Neither of these diamines was detected in spent media from 72 h DFMO-treated cultures. Changes in intracellular levels of amine precursors were also determined by HPLC. There was a transient increase in ornithine to 39 nmol (mg protein)-1 at 48 h in the DFMO-treated cells while it remained undetectable in control cells throughout the experiment. Arginine and citrulline levels remained high, decreasing to control levels only after 72 h. Only spermine (1 mM) rescued DFMO-treated cells, and this is discussed with respect to the presence of a putative spermine-specific oxidase designated by its sensitivity to aminoguanidine. Aerobic incubation of growing (normal) cells with [14C]spermine resulted in the production of an unknown metabolite (19% of total label), whose content was reduced to 5% under anaerobic conditions. Decarboxylated S-adenosylmethionine remained undetectable in DFMO-treated cells, and the methylation index (ratio of S-adenosylmethionine to S-adenosylhomocysteine) did not change from the control value of 9.3. Ornithine decarboxylase, S-adenosylmethionine synthetase, S-adenosylmethionine:L-homocysteine methyltransferase, and S-adenosylhomocysteine hydrolase enzyme activities were detected. However, S-adenosylmethionine decarboxylase, spermidine synthase or spermine synthase were not detected. These findings are discussed with reference to the arginine dihydrolase pathway whose end products are putrescine and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of ornithine decarboxylase in Leptomonas seymouri.

Ornithine decarboxylase, the initial enzyme of polyamine biosynthesis, was induced in vitro in Leptomonas seymouri, a parasite of Diptera, by resuspending stationary phase cells with fresh medium. Induction was biphasic with peaks at 2 and 8 h. Activity increased about 20-fold over 22 h under control conditions. Induction was completely blocked by cycloheximide and was suppressed by actinomycin D, alpha-amanitin, putrescine, spermidine and spermine. The enzyme half-life was 45 min in cells treated with cycloheximide 24 h post induction. These observations suggest the presence of a highly sensitive mechanism for regulation of ornithine decarboxylase as found in mammalian and other eukaryotic cells.

In vivo effects of difluoromethylornithine on trypanothione and polyamine levels in bloodstream forms of Trypanosoma brucei.

The effect of D,L-alpha-difluoromethylornithine (DFMO) on thiol and polyamine levels in Trypanosoma brucei was investigated by isolating trypanosomes from infected rats treated with DFMO for 12-48 h. Concentrations of thiols, polyamines and other amino-compounds were measured by an automated high-performance liquid chromatography method. The levels of DFMO in rat plasma (0.02-1.34 mM) is similar to that found in the parasites (0.27-0.99 mM), concentrations which exceed the Ki of DFMO for T. brucei ornithine decarboxylase. Treatment with DFMO increases intracellular levels of ornithine, S-adenosylmethionine and decarboxylated S-adenosylmethionine and decreases putrescine and spermidine. Putrescine is undetectable after 12 h treatment with DFMO and after 48 h spermidine is decreased by 76%. By 48 h, the spermidine-glutathione conjugates glutathionylspermidine and dihydrotrypanothione (bis(glutathionyl)spermidine) are also decreased by 41 and 66%, respectively. In contrast, levels of glutathione show a slight increase. These changes in metabolite levels are consistent with the biosynthetic pathway proposed for Crithidia fasciculata, where trypanothione is synthesized from spermidine and glutathione via the intermediates N1- and N8-glutathionyl-spermidine. Trypanothione is thought to have two important roles in trypanosomatid metabolism: the maintenance of intracellular thiols in the correct redox state and in the removal of hydrogen peroxide and other hydroperoxides. Thus, it is proposed that depletion of this metabolite may be an important contributory factor to the selective toxic effect of DFMO, particularly in its synergistic effect with other trypanocidal drugs.

Effect of putrescine on the respiration of Trypanosoma brucei brucei.

When bloodstream forms of Trypanosoma brucei brucei were exposed to exogenous putrescine for 24 h during in vitro culture, the rate of O2 consumption increased significantly in a concentration-related and time-dependent manner. Trypanosomes cultured with 100 microM DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, were depleted of intracellular putrescine, and the rate of O2 consumption decreased by more than 50%. This effect could be abrogated if 100 microM putrescine was also present. A similar pattern was observed in trypanosomes harvested from rats after 36 h of DFMO treatment. If such trypanosomes were placed in culture for 2 h with 100 microM putrescine, the rate of O2 consumption returned to that of controls. When an intraperitoneal injection of putrescine was given to infected rats 18 h after commencement of DFMO treatment, rates of O2 consumption in the trypanosomes were found to return to control values. The addition of putrescine, spermidine or Mg2+ did not affect rate of O2 consumption in enriched mitochondrial preparations. However, when putrescine was present throughout the preparation of mitochondrial fractions, there was an increase of 23% in O2 uptake, which was 23% higher than in the controls. Putrescine may modulate trypanosomal respiration by stabilizing mitochondrial membranes.

Effects of exogenous polyamine and trypanocides on the DNA polymerase activities from Trypanosoma brucei brucei, mouse thymus and murine leukemia virus.

The effects of exogenously added spermine on activated (gapped) DNA-directed and poly(dC) . (dG)12-18-directed DNA synthesis were tested on the chromatographically separated DNA polymerase activities of Trypanosoma brucei brucei. Activated DNA-directed DNA synthesis by the Peak I (eluting from DNA-agarose at 0.15 M KCl) and Peak II (eluting at 0.3 M KCl) polymerase was consistently inhibited or stimulated, respectively, by exogenous spermine. Kinetic analysis revealed that inhibition of the Peak I enzyme with respect to template DNA occurred by a mixed mechanism, while a major factor in the stimulation of the Peak II enzyme by spermine appeared to be the polyamine-mediated reversal of "substrate inhibition' by DNA at concentrations above 10 micrograms/ml. The apparent Km values of Peak I and Peak II DNA polymerase for activated DNA were determined to be 5 and 0.5 microgram/ml, respectively. In contrast to the results observed with activated DNA, activation of Peak II-enzyme-catalyzed poly(dC)-directed DNA synthesis was similar at all template-primer concentrations. Peak I enzyme-catalyzed poly(dG) synthesis was either inhibited or slightly stimulated by spermine, depending upon the presence or absence of heteropolymeric DNA, respectively. Dose-dependent inhibition of DNA-directed DNA synthesis catalyzed by T. b. brucei DNA polymerases, murine thymus DNA polymerase alpha, and Rauscher murine leukemia virus reverse transcriptase by trypanocides was examined to determine a possible mechanism of selective toxicity by such agents. The drugs Antrycide (quinapyramine), pentamidine, imidocarb, Berenil (diminazene aceturate), WR-199-385-[2,5-bis(4-guanylphenyl)furan . 2HCl] and isometamidium inhibited DNA polymerases of the eucaryotic cells at approximately the same degree, and at similar concentrations. The presence of spermine in reaction mixtures did not spare any drug inhibition. Stimulation of reverse transcriptase activity was observed in the presence of Antrycide and imidocarb, however, this could be negated by stimulatory amounts of spermine present in the reaction mixture. The results, obtained using an activated DNA-directed assay system, suggest that trypanosomal DNA polymerases are not the selective target of trypanocidal drugs currently available.

Substrate specificities of 5'-deoxy-5'-methylthioadenosine phosphorylase from Trypanosoma brucei brucei and mammalian cells.

The separation by chromatofocusing of two distinct purine nucleoside cleaving activities from crude extracts of Trypanosoma brucei brucei is described. One catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MeSAdo) and adenosine (Ado) and was designated an MeSAdo/Ado phosphorylase, while the other catalyzes the hydrolysis of adenosine, inosine, and guanosine but not MeSAdo. The substrate specificity of trypanosomal MeSAdo/Ado phosphorylase differed from that of a mammalian MeSAdo phosphorylase (derived from murine Sarcoma 180 cells) in that it was able to phosphorolyze 2'-deoxyadenosine, 3'-deoxyadenosine and 2',3'-dideoxyadenosine. In addition, the trypanosomal phosphorylase was able to utilize the nucleoside analog, 6-methylpurine 2'-deoxyribonucleoside, as an alternative substrate, whereas the mammalian enzyme could not. Because of these differences, cytotoxic analogs of MeSAdo may be designed that are selectively activated by the trypanosomal MeSAdo/Ado phosphorylase.

In vivo effects of alpha-DL-difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei.

The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor alpha-DL-difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72-80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels (less than 1 nmol mg-1 protein) after 36 h treatment. alpha-DL-Difluoromethylornithine-treated cells were unable to synthesize putrescine from [3H]ornithine but were able to convert [3H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S-adenosylmethionine was also altered in treated parasites: decarboxylated S-adenosylmethionine increased more than 1000-fold over untreated cells while S-adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50-100%, while protein synthesis increased up to 4-fold in 36-h treated cells. alpha-DL-Difluoromethylornithine treatment progressively limits the parasites' ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender leads to short stumpy transformation.

(+)-7-Deaza-5'-noraristeromycin as an anti-trypanosomal agent.

The (+)-enantiomer of 7-deaza-5'-noraristeromycin (4) has been found to show IC50 values ranging from 0.16 to 5.3 microM against four strains of African trypanosomes, one Trypanosoma brucei brucei isolate, and several clinical isolates of Trypanosoma brucei rhodesiense (agent of east African sleeping sickness), including a multidrug resistant clone of one isolate. While this compound was originally designed to inhibit S-adenosyl-L-homocysteine hydrolase, it has been found to have no effect on this enzyme.

Synthesis and anti-trypanosomal activity of various 8-aza-7-deaza-5'noraristeromycin derivatives.

A recent observation that (+)-7-deaza-5'-noraristeromycin (1), as an L-like analogue of aristeromycin, possessed meaningful anti-trypanosomal properties has prompted a search of other 7-deazapurines with similar or improved anti-trypanosomal responses. In that direction a series of pyrazolo[3,4-d]pyrimidines (that is, 8-aza-7-deaza-5'-noraristeromycin derivatives, 2-11) related to 1 have been prepared. These derivatives were evaluated against bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense grown in vitro. Of these compounds, the parent L-like derivative 2 was less potent (IC50 40-70 microM) than 1 (IC50 0.165-5.3 microM) whereas the D-like analogue 3 was inactive, which is the same trend observed previously with 7-deaza-5'-noraristeromycin. Interestingly, some moderate activity (IC50 12.2-16.8 microM) was seen in the D-like 4'-methyl derivative 7 while its L-like partner was inactive.

S-(5'-deoxy-5'-adenosyl)-1-aminoxy-4-(methylsulfonio)-2-cyclopentene (AdoMao): an irreversible inhibitor of S-adenosylmethionine decarboxylase with potent in vitro antitrypanosomal activity.

The S-adenosylmethionine (AdoMet) analogue S-(5'-deoxy-5'-adenosyl)-1-aminoxy-4-(methylsulfonio)-2-cycl opentene (AdoMao) was synthesized in two of its four possible diastereomeric forms using a facile chemoenzymatic route. The trans-1R,4R- and trans-1S,4S-diastereomers of AdoMao, as well as the corresponding diastereomers of the unmethylated precursor molecule nor-AdoMao, were then evaluated as inhibitors of S-adenosylmethionine decarboxylase (AdoMet-DC) from both bacterial and human sources. All four of the analogues acted as time-dependent, irreversible inhibitors of AdoMet-DC from Escherichia coli, exhibiting remarkably constant Ki values ranging between 20.6 and 23.7 microM. These analogues also inhibited the human form of AdoMet-DC, although this form of the enzyme was able to discriminate between AdoMao (Ki values of 21.2 microM for the trans-1R,4R form and 19.6 microM for the trans-1S,4S form) and nor AdoMao (Ki values of 95.2 microM for the trans-1R,4R form and 30.9 microM for the trans-1S,4S form). The trans diastereomers of AdoMao and nor-AdoMao were next evaluated for their ability to inhibit trypanosomal growth in vitro against cultured Trypanosoma brucei brucei bloodforms. All four of these analogues were effective growth inhibitors, with IC50 values ranging between 0.9 and 10.1 microM. The two most effective analogues, trans-1S,4S-AdoMao (IC50 0.9 microM) and trans-1S,4S-AdoMao (IC50 3.0 microM) were also effective against two clinical isolates of the pathogenic organism Trypanosoma brucei rhodesiense, KETRI 243 and KETRI 269. The most promising analogue in all respects was trans-1S,4S-AdoMao, which was subsequently found to have minimal effects on cell growth, AdoMet-DC activity, and intracellular polyamine levels in the sensitive human promyelocytic leukemia cell line HL60. Thus, the S-adenosylmethionine analogue trans-1S,4S-AdoMao acts as an effective inhibitor of AdoMet-DC and appears to serve as a parasite-specific trypanocidal agent in vitro.

Synthesis, structure, and antiparasitic activity of sulfamoyl derivatives of ribavirin.

The triazole nucleoside derivatives 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl) [1,2,4]triazole-3-carboxamide (2), 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl) [1,2,4]triazole-3-thiocarboxamide (3), and 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl)-[1,2,4]triazole-3- carbonitrile (4) were synthesized. Suitably protected triazole nucleosides were converted to their corresponding 5'-sulfamoyl derivatives, which on subsequent deprotection gave the desired compounds in good yields. The structures of compounds 2-4 were confirmed by X-ray crystallographic analysis. All three compounds showed significant antiparasitic activity in vitro, while 2 showed significant activity in vivo against Leishmania donovani and Trypanosoma brucei.

Inhibition of Trichomonas vaginalis ornithine decarboxylase by amino acid analogs.

Ornithine decarboxylase (ODC) from Trichomonas vaginalis was inhibited irreversibly by several substrate analogs. Of these, DL-alpha-monofluoromethyldehydroornithine (MFMDO) and DL-alpha-monofluoromethylornithine (MFMO) were the most potent. The enzyme was unaffected by putrescine analogs suggesting that differences exist between the regulation of the trichomonad enzyme and that in other eukaryotes. In culture the ornithine analogs strongly inhibited putrescine synthesis and increased the generation time after 24 hr of exposure. In a semi-defined growth medium MFMDO methyl and ethyl esters increased the generation time from 4.5 hr to 9.0 and 8.2 hr, respectively. In standard undefined growth medium the trichomonad ODC was fully induced only after 15 hr (late log) and had an extended half-life of greater than 8 hr.

Catalytic irreversible inhibition of Trypanosoma brucei brucei ornithine decarboxylase by substrate and product analogs and their effects on murine trypanosomiasis.

Ornithine decarboxylase from Trypanosoma brucei brucei was inhibited by several substrate (ornithine) and product (putrescine) analogs both in vitro and in vivo. Since alpha-difluoromethylornithine is effective for the treatment of experimental and clinical African trypanosomiasis, it was possible that the more potent ornithine and putrescine analogs might be more active in treating the disease. However, only alpha-monofluoromethyldehydroornithine methyl ester was more potent than alpha-difluromethylornithine against mouse trypanosomiasis and warrants further study in model infections.

Irreversible inhibition of S-adenosylmethionine decarboxylase of Trypanosoma brucei brucei by S-adenosylmethionine analogues.

S-Adenosylmethionine analogues designed as active-site directed inhibitors were tested in vitro for their effects on S-adenosylmethionine decarboxylase (AdoMetDC) of Trypanosoma brucei brucei. These analogues contained a tertiary nitrogen atom in place of the sulfonium and had a side chain of variable length ending in a reactive group (hydrazino-, aminooxy-, hydrazido- or a methylnitrosourea). The hydrazino- derivatives were the most potent inhibitors with IC50 values in the range of 40-100 nM. The most active compound (IC50 of 0.04 microM) was 5'-deoxy-5'-[(2-hydrazinoethyl)-methylamino]adenosine (MHZEA). Addition of MHZEA produced a time-dependent inactivation with an apparent Ki of 0.4 microM, and the enzyme half-life at a saturating concentration of MHZEA was 0.4 min. Increasing the length of the side chain or changing the methyl group attached to the nitrogen to an ethyl group reduced the potency. Replacement of the hydrazino moiety with an aminooxy group resulted in about a 30- to 35-fold decrease in inhibition potency. However, the relative order of activities of these aminooxy analogues was similar to that found in the hydrazino series with 5'-deoxy-5'-[(2-aminooxyethyl)methylamino]adenosine (MAOEA), which had an IC50 of 1.3 microM, being the most active. The hydrazido analogs were even less effective with 5'-deoxy-5'-[(3-hydrazino-3-oxopropyl)-methylamino]adenosine, the best inhibitor, having an IC50 value of 8.7 microM. The methylnitrosourea derivatives were inactive. The inactivation of trypanosomal AdoMetDC with MHZEA or MAOEA was irreversible and was greatly stimulated by putrescine, a known activator of the enzyme, indicating that the compounds bind to the active site and form a covalent bond with the enzyme. These inhibitors may have considerable potential as chemotherapeutic agents against trypanosomiasis and other protozoal infections and may also be useful in studying the role of AdoMetDC in the regulation of polyamine levels in these organisms.

Inhibitors of histamine metabolism in vitro and in vivo. Correlations with antitrypanosomal activity.

The effects of antimalarial and antitrypanosomal drugs on the activity of histamine N-methyl transferase and diamine oxidase in vitro, as well as diamine oxidation and histamine levels in vivo, were examined. Diamidine antitrypanosomal drugs which interfere with polyamine metabolism were found to be potent inhibitors both in vitro and in vivo. Antrycide ( quinapyramine ) and isometamidium were the best inhibitors of both enzymes. Ki values for histamine N-methyl transferase were 3 X 10(-8) M for both compounds, and the inhibition was competitive for histamine. Antrycide and isometamidium were both non-competitive inhibitors of diamine oxidase, having Ki values of 6 X 10(-9) M and 3 X 10(-9) M respectively. Isometamidium elevated histamine levels in rat kidney 2-fold and produced a long-term inhibition of putrescine oxidation in vivo. Among the compounds examined, only known active antitrypanosomal agents inhibited both histamine N-methyl transferase and diamine oxidase in vitro as well as putrescine oxidation in vivo. These observations suggest that the enzymes acting on histamine and putrescine as substrates can be used to select compounds which interfere with polyamine metabolism and that persistence of such compounds in vivo, as indicated by inhibition of putrescine oxidation, correlates with favorable chemotherapeutic properties as antitrypanosomal agents.

Resistance to DL-alpha-difluoromethylornithine by clinical isolates of Trypanosoma brucei rhodesiense. Role of S-adenosylmethionine.

The ornithine decarboxylase (ODC) inhibitor DL-alpha-difluoromethylornithine (DFMO) has emerged as a new treatment for West African sleeping sickness but is less effective against East African sleeping sickness. We examined uncloned clinical isolates of Trypanosoma brucei rhodesiense, agent of the disease in East Africa, which were refractory to DFMO in laboratory infections, for characteristics that would explain their resistance. None of the isolates were from patients treated with DFMO. Two isolates took up [3H]DFMO at 50-70% lower rates than drug-sensitive strains but ODC activities, Ki values for DFMO, spermidine and spermine uptake rates, polyamine content and inhibition of polyamine metabolism by DFMO were statistically (P < 0.05) similar between sensitive and refractory isolates. One cloned strain, continuously passaged in vivo under DFMO pressure and included for comparison, had > 85% lower ODC activity and up to 14-fold higher putrescine uptake rates than sensitive controls. A statistically important trend was the metabolism of S-adenosylmethionine (AdoMet): activities of AdoMet synthetase and AdoMet decarboxylase were 2- to 5-fold and 3- to 40-fold lower in resistant strains, respectively, while intracellular AdoMet pools (AdoMet + decarboxylated AdoMet) that were > 60-fold elevated in sensitive strains during DFMO treatment, increased only 9-fold in refractory isolates. The extreme elevation of the AdoMet pool in sensitive isolates from 0.7 to 44 nmol/mg protein and an intracellular pool concentration of approximately 5 mM may lead to an imbalance in methylation of proteins or other cell constituents as a consequence of DFMO action. These studies indicate that the metabolism of AdoMet is altered significantly in DFMO refractory isolates and suggest that differences in AdoMet metabolism may be responsible for increased tolerance to DFMO.