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C J Bacchi

Publications and source records attributed to C J Bacchi.

At least 37 records · Page 2Linked to original sources
PubMed1997

A unique transporter of S-adenosylmethionine in African trypanosomes.

African trypanosomes are pathogens of humans and livestock in equatorial Africa. There is a great deal of resistance to present front line drugs for treating African trypanosomiasis such as melarsoprol (Arsobal) and pentamidine. In the search for new and novel drugs against this disease, we have found a unique transporter of S-adenosylmethionine (AdoMet), a metabolite used in transmethylation reactions and polyamine synthesis. This transporter is distinct from those for methionine and adenosine, since AdoMet uptake was not inhibited by trypanocidal drugs, which compete with adenosine for transport. AdoMet analogs competing with [methyl-3H]AdoMet for uptake required a positively charged sulfonium group on the 5' position of the ribose. Since transport of AdoMet does not normally occur to a significant extent in mammalian cells, the parasite transporter provides a selective and novel route to deliver new chemotherapeutic agents against these organisms.

Adenosine↗
PubMed1997

In vivo efficacies of 5'-methylthioadenosine analogs as trypanocides.

5'-Deoxy-5'-(methylthio)adenosine (MTA), a key by-product of polyamine biosynthesis, is cleaved by MTA phosphorylase and is salvaged as adenine and, through conversion of the ribose moiety, methionine. An analog of MTA, 5'-deoxy-5'-(hydroxyethylthio)adenosine (HETA), is a substrate for trypanosome MTA phosphorylase and is active in vitro and in vivo against Trypanosoma brucei brucei, an agent of bovine trypanosomiasis. In this study, HETA and three O-acylated HETA derivatives were examined for their activities against model infections of T. b. brucei and Trypanosoma brucei rhodesiense, the agent of East African sleeping sickness. HETA was curative (>60%) for infections caused by 5 of 11 clinical isolates of T. b. rhodesiense when it was given to mice at 200 mg/kg of body weight for 7 days as a continuous infusion in osmotic pumps. HETA at 150 to 200 mg/kg also extended the life spans of the mice infected with four additional isolates two- to fivefold. Di- and tri-O-acetylated derivatives of HETA also proved curative for the infections, while a tri-O-propionyl derivative, although also curative, was not as effective. This study indicates that substrate analogs of MTA should be given important consideration for development as novel chemotherapies against African trypanosomiasis.

Animals↗
PubMed1997

Synthesis and antitrypanosomal activities of a series of 7-deaza-5'-noraristeromycin derivatives with variations in the cyclopentyl ring substituents.

Previous work in our laboratories has suggested that (+)-5'-nor-7-deazaaristeromycin (compound 1) may represent a prototype structure for a series of compounds with significant antitrypanosomal activities. To test this possibility, a series of derivatives of compound 1 with changes in the cyclopentyl substituents (compounds 3 to 10) have been studied. Although some growth activity was obtained with the L-like compound 5, related compounds 3 and 7 had little or no activity below 100 microM. D-like compounds 4 and 6 showed some activity at or below 100 microM, but the most interesting finding was that both the D- and L-like compounds having a methyl substituent on the 4' position were most active.

Adenosine↗
PubMed1996

Antitrypanosomal activity of purine nucleosides can be enhanced by their conversion to O-acetylated derivatives.

Fifteen purine nucleosides and their O-acetylated ester derivatives were examined for in vitro antitrypanosomal activity against the LAB 110 EATRO isolate of Trypanosoma brucei brucei and two clinical isolates of Trypanosoma brucei rhodesiense. Initial comparisons of activity were made for the LAB 110 EATRO isolate. Three nucleoside analogs exhibited no significant activity (50% inhibitory concentrations [IC50s] of > 100 microM), whether they were O acetylated or unacetylated; three nucleosides showed almost equal activity (IC50s of < 5 microM) for the parent compound and the O-acetylated derivative; nine nucleosides showed significantly improved activity (> or = 3-fold) upon O acetylation; of these nine analogs, six displayed activity at least 10-fold greater than that of their parent nucleosides. The most significant results were those for four apparently inactive compounds which, upon O acetylation, displayed IC50s of < or = 25 microM. When the series of compounds was tested against T. brucei rhodesiense isolates (KETRI 243 and KETRI 269), their antitrypanosomal effects were comparable to those observed for the EATRO 110 strain. Thus, our studies of purine nucleosides have determined that O acetylation consistently improved their in vitro antitrypanosomal activity. This observed phenomenon was independent of their cellular enzyme targets (i.e., S-adenosylmethionine, polyamine, or purine salvage pathways). On the basis of our results, the routine preparation of O-acetylated purine nucleosides for in vitro screening of antitrypanosomal activity is recommended, since O acetylation transformed several inactive nucleosides into compounds with significant activity, presumably by improving uptake characteristics. O-acetylated purine nucleosides may offer in vivo therapeutic advantages compared with their parent nucleosides, and this possibility should be considered in future evaluations of this structural class of trypanocides.

Acetylation↗
PubMed1996

In vitro trypanocidal activities of new S-adenosylmethionine decarboxylase inhibitors.

A series of novel aromatic derivatives based on the structure of methylglyoxal bis(guanylhydrazone) (MGBG) was examined for in vitro antitrypanosomal activities and cytotoxicities for human cells. One-third of the compounds tested showed trypanocidal activity at concentrations below 0.5 microM after an incubation period of 72 h. Structure-activity analysis revealed that bicyclic compounds with homocyclic rings and unmodified termini were the most active compounds. Results obtained in three laboratories employing different methods and trypanosome populations consistently ranked compound CGP 40215A highest. This compound had a 50% inhibitory concentration of 0.0045 microM for Trypanosoma brucei rhodesiense, was also active against other trypanosome species, including a multidrug-resistant Trypanosoma brucei brucei, and was significantly less toxic than other compounds tested for a human adenocarcinoma cell line, with a 50% inhibitory concentration of 1.14 mM. The effect of CGP 40215A was time and dose dependent, and low concentrations of the compound required exposure times of > 2 days to exert trypanocidal activity. Compounds were inactive against Leishmania donovani and Trypanosoma cruzi amastigotes in murine macrophages in vitro.

Adenocarcinoma↗
PubMed1996

In vivo trypanocidal activities of new S-adenosylmethionine decarboxylase inhibitors.

A series of novel aromatic derivatives based on the structure of methylglyoxal bis(guanylhydrazone) (MGBG) was examined for trypanocidal activities in human and veterinary trypanosomes of African origin. One agent, CGP 40215A, a bicyclic analog of MGBG which also resembles the diamidines diminazene (Berenil) and pentamidine, was curative of infections by 19 isolates of Trypanosoma brucei subspecies as well as a Trypanosoma congolense isolate. Several of these isolates were resistant to standard trypanocides. Curative doses were < or = 25 mg/kg of body weight/day for 3 days in these acute laboratory model infections. In addition, CGP 40215A also cured a model central nervous system infection in combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO; Ornidyl, eflornithine). Curative combinations were 14 days of oral 2% DFMO (approximately 5 g/kg/day) plus 5, 10, or 25 mg/kg/day for 3 or 7 days given by intraperitoneal injection or with a miniosmotic pump. Combinations were most effective if CGP 40215A was given in the second half or at the end of the DFMO regimen. MGBG has modest activity as an inhibitor of trypanosome S-adenosylmethionine decarboxylase (50% inhibitory concentration [IC50]. 130 microM), while CGP 40215A was a more active inhibitor (IC50, 20 microM). Preincubation of trypanosomes with CGP 40215A for 1 h caused a reduction in spermidine content (36%) and an increase in putrescine content (20%), indicating that one possible mechanism of its action may be inhibition of polyamine biosynthesis.

Adenosylmethionine Decarboxylase↗
PubMed1995

Fate of soluble methionine in African trypanosomes: effects of metabolic inhibitors.

The metabolism of [35S]methionine in cultured bloodstream forms of African trypanosomes was followed using flow-through radiodetection linked to liquid chromatography separation. The effects of a transmethylase inhibitor, sinefungin, and of the ornithine decarboxylase inhibitor, DL-alpha-difluoromethylornithine (Ornidyl; DFMO), on methionine metabolism were also observed. Trypanosomes rapidly incorporated [35S]methionine into S-adenosylmethionine (AdoMet) and the metabolites methylthioadenosine, S-adenosylhomocysteine, homocysteine, cystathionine cysteine and glutathione. Untreated trypanosomes excreted large quantities of cystathionine and cysteine into the growth medium. DFMO-treated cells formed larger quantities of AdoMet more rapidly than did control cells, as was evident from initial time points (30 min and 1 h). Decarboxylated AdoMet, present in trace quantities in control cells, accumulated in DFMO-treated cells. Sinefungin increased the AdoMet concentrations approximately 20-fold over that of controls after a 6 h incubation with [35S]methionine, while cystathionine and cysteine levels decreased. The half-life (t1/2) and rate of turnover of AdoMet were measured in cells treated with DFMO or sinefungin. DFMO treatment caused a substantial increase in the rate of AdoMet utilization, while sinefungin extended the t1/2 and lowered AdoMet turnover. These studies show that trypanosomes rapidly metabolize methionine through AdoMet to intermediates of the polyamine and transmethylation pathways. Agents inhibiting these pathways rapidly affect the concentration and rate of utilization of AdoMet, significantly changing the concentrations of metabolites.

Adenosine↗
PubMed1995

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.

Adenosylmethionine Decarboxylase↗
PubMed1995

Crithidia luciliae: effect of purine starvation on S-adenosyl-L-methionine uptake and protein methylation.

The utilization of S-adenosyl-L-[methyl-3H]methionine ([3H-methyl]AdoMet) by Crithidia luciliae was assessed under nutrient-replete and purine-starvation conditions. Uptake experiments with intact cells demonstrated that the radiolabel from this molecule was accumulated by purine-starved organisms at a rate approximately 10-fold greater than that observed in those cultivated in nutrient-replete medium. Purine-starved cells also incorporated the radiolabel into trichloroacetic acid insoluble material at an approximately 10-fold faster rate than nutrient-replete cells. No differences, however, were observed in the intracellular levels of AdoMet and its metabolites between organisms cultivated under the two conditions. Results of comparative labeling studies with [3H-methyl]AdoMet, S-adenosyl-L-[carboxyl-14C]methionine, L-[methyl-3H]methionine and L-[35S]methionine in the presence and absence of cycloheximide demonstrated that the incorporation of label from [3H-methyl]AdoMet was due to transmethylation and was independent of protein synthesis. Further, approximately 15 methylated protein bands were identified by SDS-PAGE analysis. Lysates from both purine-starved and nutrient-replete organisms demonstrated similar levels of activity of three protein methyltransferases (PMI, II, III). The differences observed in [3H-methyl]AdoMet utilization between purine-starved and nutrient-replete C. luciliae may reflect the enhanced purine transport capacity which results from purine starvation.

Adenosine↗
PubMed1995

Methionine recycling pathways and antimalarial drug design.

5'-Deoxy-5'-(methylthio)adenosine (MTA) is an S-adenosylmethionine metabolite that is generated as a by-product of polyamine biosynthesis. In mammalian cells, MTA undergoes a phosphorolytic cleavage catalyzed by MTA phosphorylase to produce adenine and 5-deoxy-5-(methylthio)ribose-1-phosphate (MTRP). Adenine is utilized in purine salvage pathways, and MTRP is subsequently recycled to methionine. Whereas some microorganisms metabolize MTA to MTRP via MTA phosphorylase, others metabolize MTA to MTRP in two steps via initial cleavage by MTA nucleosidase to adenine and 5-deoxy-5-(methylthio)ribose (MTR) followed by conversion of MTR to MTRP by MTR kinase. In order to assess the extent to which these pathways may be operative in Plasmodium falciparum, we have examined a series of 5'-alkyl-substituted analogs of MTA and the related MTR analogs and compared their abilities to inhibit in vitro growth of this malarial parasite. The MTR analogs 5-deoxy-5-(ethylthio)ribose and 5-deoxy-5-(hydroxyethylthio)ribose were inactive at concentrations up to 1 mM, and 5-deoxy-5-(monofluoroethylthio)ribose was weakly active (50% inhibitory concentration = 700 microM). In comparison, the MTA analogs, 5'-deoxy-5'-(ethylthio)adenosine,5'-deoxy-5'-(hydroxyethylthio)ade nosine (HETA), and 5'-deoxy-5'-(monofluoroethylthio)adenosine, had 50% inhibitory concentrations of 80, 46, and 61 microM, respectively. Extracts of P. falciparum were found to have substantial MTA phosphorylase activity. Coadministration of MTA with HETA partially protected the parasites against the growth-inhibitory effects of HETA. Results of this study indicate that P. falciparum has an active MTA phosphorylase that can be targeted by analogs of MTA.

Animals↗
PubMed1994

Combination chemotherapy of drug-resistant Trypanosoma brucei rhodesiense infections in mice using DL-alpha-difluoromethylornithine and standard trypanocides.

Combinations of DL-alpha-difluoromethylornithine (DFMO; eflornithine; Ornidyl) with either suramin or melarsen oxide were found to be effective against acute laboratory model infections with Trypanosoma brucei rhodesiense. We used clinical isolates known to be resistant to these drugs when used singly. An infection with a melarsen oxide-refractory isolate was cured by a combination of low-dose DFMO (0.5% in the drinking water) plus low-dose suramin (1 mg/kg of body weight given intraperitoneally). Another strain, moderately resistant to arsenical drugs, was cured with combinations of 4% DFMO with 5 mg of melarsen oxide per kg. Furthermore, a combination of DFMO (2% in the drinking water) and suramin (20 mg/kg) provided a 100% cure rate in a central nervous system model, although the same doses of these drugs used singly were completely ineffective. The synergism of DFMO and suramin against an acute infection was improved when suramin was given at the end of the DFMO administration. No adverse interactions were observed when high doses of DFMO combined with high doses of suramin were administered to uninfected mice. These results suggest that combinations of DFMO and suramin should be examined clinically for activity in arsenical-drug-refractory cases of East African sleeping sickness.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

Trichomonas vaginalis: characterization of ornithine decarboxylase.

Ornithine decarboxylase (ODC), the lead enzyme in polyamine biosynthesis, was partially purified from Trichomonas vaginalis and its kinetic properties were studied. The enzyme appears to be of special significance in this anaerobic parasite, since the arginine dihydrolase pathway generates ATP as well as putrescine from arginine. ODC from T. vaginalis had a broad substrate specificity, decarboxylating ornithine (100%), lysine (1.0%) and arginine (0.1%). The enzyme had a pH optimum of 6.5, a temperature optimum of 37 degrees C and was pyridoxal 5'-phosphate-dependent. Attempts to separate ornithine- from lysine-decarboxylating activity by thermal-stability and pH-optima curves were not successful. Although Km values for ornithine and lysine were 109 and 91 microM respectively, and the Vmax values for these substrates were 1282 and 13 nmol/min per mg of protein respectively, the most important intracellular substrate is ornithine, since intracellular ornithine levels are 3.5 times those of lysine and extracellular putrescine levels are 7.5 times those of cadaverine. Ornithine was also an effective inhibitor of lysine-decarboxylating activity (Ki 150 microM), whereas lysine was relatively ineffective as inhibitor of ornithine-decarboxylating activity (Ki 14.5 mM). Crude ODC activity was localized (86%) in the 43,000 g supernatant and 3303-fold purification was obtained by (NH4)2SO4 salting and DEAE-Sephacel, agarose-gel and hydroxyapatite chromatography steps. The enzyme bound difluoro[3H]methylornithine ([3H]DFMO) with a ratio of drug bound to activity of 2500 fmol/unit, where 1 unit corresponds to 1 nmol of CO2 released from ornithine/min. The enzyme had a native M(r) of 210000 (gel filtration), with a subunit M(r) of 55,000 (by SDS/PAGE), suggesting that the trichomonad enzyme is a tetramer. From the subunit M(r) and binding ratio of DFMO, there is about 137 ng of ODC per mg of T. vaginalis protein (0.013%). The significant amount of ODC protein present supports the view that putrescine synthesis in T. vaginalis plays an important role in the metabolism of the parasite.

Animals↗
PubMed1993

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.

Amines↗
PubMed1993

Effects of antagonists of polyamine metabolism on African trypanosomes.

This review outlines the metabolism of polyamines in African trypanosomes and summarizes evidence to indicate that trypanosome polyamine metabolism differs in several important aspects from that of the mammalian host. These differences relate to the halflife, turnover, substrate specificity and regulation of enzymes within the mainstream of polyamine synthesis and the related pathway of transmethylation. The common denominator for the uniqueness of parasite polyamine metabolism concerns S-adenosylmethionine (AdoMet) whose synthesis is unregulated and, upon accumulating in the cell, appears to result in abnormally high transmethylation activity. Similarly, the catabolism of the AdoMet product of polyamine synthesis, methylthioadenosine, is governed by a phosphorylase having broad substrate specificity, and which, if presented with substrate analogs, can generate cytotoxic metabolites.

Adenosylmethionine Decarboxylase↗
PubMed1993

Resistance to clinical drugs in African trypanosomes.

Drug resistance in African trypanosomes continues to confound clinicians and to stymy development o f equatorial Africa, taking its toll in lives and economic development. Drugs in current, widespread use have been employed continuously for over 60 years in some instances. The recent studies of Fairlamb and colleagues have outlined a defective purine-transport system in drug-resistant trypanosomes, which appears to explain resistance to several established tryponocides and suggests a guide for the development of new drugs. The recently developed agent dl-alpha-di fluoromethylornithine (DFMO) is effective against West African, but not East African, disease and its activity may be the result of the unregulated synthesis of S-odenosylmethionine in tryponosomes. In this report, Cyrus Bacchi outlines recent developments in the elucidation of mechanisms of resistance to established drugs and naturally occurring resistance to DFMO.

Journal Article↗