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C J Bacchi

Publications and source records attributed to C J Bacchi.

At least 55 records · Page 3Linked to original sources
PubMed1992

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.

Adenosylmethionine Decarboxylase↗
PubMed1992

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.

Adenosylmethionine Decarboxylase↗
PubMed1992

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.

Amino Acids↗
PubMed1992

Cure of murine Trypanosoma brucei rhodesiense infections with an S-adenosylmethionine decarboxylase inhibitor.

The compound 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL73811), a potent inhibitor of S-adenosylmethionine decarboxylase, was effective in mice against six of eight clinical isolates of Trypanosoma brucei rhodesiense, the causative agent of East African sleeping sickness. In combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO; Ornidyl), MDL73811 acted synergistically to cure seven of eight infections. MDL73811 was effective when given singly at 50 to 100 mg/kg of body weight per day for 7 days (osmotic pumps). In combination with subcurative DFMO levels (0.25 to 1.0% in drinking water for 7 days), the curative MDL73811 dose could be lowered to 25 or 50 mg/kg, depending on the isolate. Oral administration of the MDL73811-DFMO combination was also effective in an acute infection and in a long-term central nervous system model of Trypansoma brucei brucei infection. These data indicate that MDL73811 may be effective therapeutically in drug-refractory and late-stage East African trypanosomiasis.

Adenosylmethionine Decarboxylase↗
PubMed1991

Differential sensitivity of Trypanosoma brucei rhodesiense isolates to in vitro lysis by arsenicals.

Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in murine infections, were examined for resistance in vitro. A rapid lysis assay was developed which was able to predict in vivo sensitivity to melarsoprol (Mel B, Arsobal) and melarsen oxide. The assay was based on the finding that long slender bloodforms of drug-sensitive isolates would lyse in the presence of arsenicals upon incubation in heat-inactivated fetal bovine serum. On the basis of plots of decrease in the absorbance of trypanosome suspensions vs time of incubation with drug, L50 values, reflecting the drug concentration necessary for lysis of 50% of the cells within 30 min. were calculated for five strains. These values ranged from less than 30 microM for arsenical-sensitive strains to greater than 75 microM in proven arsenic refractory isolates. Calcium was essential for lysis, and the presence of the Ca2+ chelator EGTA (10 mM) in serum delayed lysis of sensitive strains. Ca2+ channel antagonists (Verapamil, Diltiazem), however, did not enhance lysis of refractory isolates when used at 20 to 30 microM. Intracellular concentrations of reduced trypanothione, the apparent target of arsenicals, were similar for all isolates, approximately 1.02 +/- 0.28 nmol/10(8) cells, as detected by monobromobimane derivitization and HPLC analysis. Uptake of melarsen oxide was found to be reduced in arsenical refractory strains. Uptake was judged by reduction of free reduced trypanothione as a result of formation of the trypanothione-arsenic complex Mel T. Little change was found in arsenical-resistant strains, but sensitive strains had 50 to 70% reductions in trypanothione levels after incubation with a low (1 microM) level of melarsen oxide.

Animals↗
PubMed1991

Protein methylases in Trypanosoma brucei brucei: activities and response to DL-alpha-difluoromethylornithine.

Protein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-L-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent Km for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent Km for S-AdoMet of 8.4 microM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-L-homocysteine and its analogue sinefungin with apparent Ki values of 12.9 and 1.6 microM, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed.

Adenosine↗
PubMed1991

5'-Alkyl-substituted analogs of 5'-methylthioadenosine as trypanocides.

5'-Deoxy-5'-(methylthio)adenosine (MTA) is a by-product of polyamine metabolism and is phosphoryolytically cleaved to adenine and 5-deoxy-5-(methylthio)ribose-1-phosphate (MTR-1-P) by MTA phosphorylase. In eukaryotes, adenine is subsequently salvaged and converted to nucleotides, while MTR-1-P is converted to methionine. We examined 5'-deoxy-5'-substituted analogs of MTA for trypanocidal activity in vitro and in vivo. 5'-Deoxy-5'-(hydroxyethyl)thioadenosine (HETA) and its 5'-bromo,5'-chloro and 5'-fluoro derivatives were cleaved by extracts of the African trypanosome Trypanosoma brucei brucei (Km for MTA, 11.5 microM; Km for HETA, 13.2 microM) in a phosphate-dependent reaction. HETA and the three halo analogs were 50% inhibitory to growth at 0.5 to 5.0 microM in vitro. Inhibition of growth was reversible by exogenous methionine and 2-keto-4-methylthiobutyric acid, an intermediate in methionine synthesis from MTR-1-P. HETA was selected for further study in vivo. When administered by miniosmotic pump (25 to 150 mg/kg/day for 7 days) to mice infected with T. brucei brucei, HETA effected 70 to 90% cure rates. Results of this study indicate that these analogs of MTA are converted to trypanocidal MTR-1-P analogs and that this approach deserves further consideration in the development of novel chemotherapy of trypanosomiasis.

Adenosine↗
PubMed1990

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.

Animals↗
PubMed1990

Differential susceptibility to DL-alpha-difluoromethylornithine in clinical isolates of Trypanosoma brucei rhodesiense.

DL-alpha-Difluoromethylornithine is an enzyme-activated inhibitor of ornithine decarboxylase and an antagonist of polyamine metabolism that has been successful in clinical trials against West African sleeping sickness caused by Trypanosoma brucei gambiense. Its potential for use against the more virulent East African form of the disease, caused by T. brucei rhodesiense, is not certain. We examined 14 East African clinical isolates from the Kenya Trypanosomiasis Research Institute strain bank plus 2 established isolates for susceptibility to DL-alpha-difluoromethylornithine and to standard trypanocides. Seven of 16 strains were partially or totally refractory to DL-alpha-difluoromethylornithine in our test system. Four strains were also refractory to arsenical drugs, and five were refractory to diamidines. The results indicate that other novel agents or combinations of established agents may be needed for chemotherapy of East African disease.

Animals↗
PubMed1990

Cure of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of S-adenosylmethionine decarboxylase.

A structural analog, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxy adenosine (MDL 73811), of decarboxy S-adenosyl-L-methionine, the product of the reaction catalyzed by S-adenosyl-L-methionine (AdoMet) decarboxylase (DC), was found to inhibit Trypanosoma brucei brucei AdoMet DC. The inhibition was time dependent (tau 50, 0.3 min), exhibited pseudo-first-order kinetics (Ki, 1.5 microM), and was apparently irreversible. The natural substrate of the reaction, AdoMet, protected the enzyme from inactivation, suggesting that MDL 73811 was directed at the enzyme active site and was probably catalytically activated. Administration of MDL 73811 to T. b. brucei-infected rats resulted in rapid inhibition of AdoMet DC activity, a decrease in spermidine, and an increase in putrescine in the trypanosomes isolated from treated rats. Treatment of T. b. brucei-infected mice with MDL 73811 (20 mg/kg of body weight intraperitoneally twice daily for 4 days) resulted in cures of the trypanosome infections. Additionally, drug-resistant T. brucei rhodesiense infections in mice were cured by either a combination of MDL 73811 (50 mg/kg intraperitoneally three times per day for 5 days) and relatively low oral doses of alpha-difluoromethylornithine or MDL 73811 (50 mg/kg per day for 7 days) administered alone in implanted miniosmotic pumps. These data suggest that MDL 73811 and, perhaps, other inhibitors of AdoMet DC have potential for therapeutic use in various forms of African trypanosomiasis.

Adenosylmethionine Decarboxylase↗
PubMed1989

Trypanosoma brucei brucei: regulation of ornithine decarboxylase in procyclic forms and trypomastigotes.

Ornithine decarboxylase (ODC) activity was measured in procyclic forms of Trypanosoma brucei brucei grown in semidefined medium. ODC activity rapidly increased in late log-phase cells which were resuspended in fresh medium. A biphasic induction curve similar to that observed in mammalian cells was observed over an 18-hr period. ODC activity increased 4.5- to 25-fold over control levels measured at zero time. Actinomycin D and cycloheximide inhibited induction by greater than 90%. Polyamines at a level not inhibitory to growth (10 microM) inhibited ODC induction, but only by 30-50%, late in the induction period. Putrescine inhibited the first peak of induction and suppressed activity at 14 hr by 75%. Polyamine analogs such as bis(ethyl)spermidine were not effective suppressors of ODC activity. The half-life of ODC in procyclic forms grown in the presence of cycloheximide was greater than 6 hr, while that of bloodstream trypomastigotes in mice treated with cycloheximide was 5 hr. A single dose of the ODC inhibitor DL-alpha-difluoromethylornithine given to infected rats or mice suppressed trypanosome ODC activity greater than 90% for more than 7 hr. These studies indicate that although trypanosome ODC increases rapidly under log growth conditions, it is less susceptible to fluctuation and external control than the enzyme from mammalian sources. The latter may be a factor in the clinical efficacy of ODC inhibitors.

Animals↗
PubMed1988

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.

Adenosine↗
PubMed1988

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.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1988

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)

Adenosine↗
PubMed1987

Polyamines antagonize both the antileukemic activity and the reverse transcriptase stimulatory activity of 4,4'-diacetyldiphenylurea bis(guanylhydrazone) (DDUG).

(Diacetyldiphenylurea)bis(guanylhydrazone) (DDUG) functions as a cationic trypanocide antagonized in vivo by exogenous concomitant addition of the biologically active polyamine, spermine. It also inhibits the DNA polymerases of L1210 murine leukemia cells. We have found that DDUG stimulates Rauscher murine leukemia virus DNA polymerase activity in a manner similar to polyamines. Such stimulation does not occur if DNA synthesis is carried out on spermine + activated DNA complexes. We also show that the in vivo antileukemic activity of DDUG in the L1210 ascites mouse model is antagonized by biologically active polyamines. These studies suggest a new intracellular target for the antileukemic activity of DDUG: interference with polyamine function.

Animals↗