ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1990, p. 1485-1490
0066-4804/90/081485-06$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 34, No. 8
Cure of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense Infections in Mice with an Irreversible Inhibitor of S-Adenosylmethionine Decarboxylase ALAN J. BITONTI,1* TIMOTHY L. BYERS,' TAMMY L. BUSH,' PATRICK J. CASARA,1 CYRUS J. BACCHI,2 ALLEN B. CLARKSON, JR.,3 PETER P. McCANN,' AND ALBERT SJOERDSMA' Merrell Dow Research Institute, Cincinnati, Ohio 452151; Haskins Laboratories of Pace University, New York, New York 100382; and Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, New York 100033 Received 22 February 1990/Accepted 3 June 1990
A structural analog, 5'-{[(Z)4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (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 (T.s, 0.3 min), exhibited pseudo-first-order kinetics (Ki, 1.5 ,uM), 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 a-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.
Trypanosomiasis, or sleeping sickness, remains endemic in many countries in Africa despite vigorous attempts to eradicate the disease (30). Most patients can be treated effectively with the presently available drugs suramin, pentamidine, and melarsoprol, but serious side effects often accompany their use (18) and trypanosome infections resistant to the effects of melarsoprol, the only drug approved for treatment of the late-stage disease with central nervous system involvement, are increasing (14). Recently, eflornithine (a-difluoromethylornithine; Ornidyl) (DFMO), an irreversible inhibitor of ornithine decarboxylase (4, 19), the first enzyme in the biosynthesis of the polyamines putrescine, spermidine, and spermine (22), has been found effective for treatment of both early- and late-stage Gambian sleeping sickness in human patients (2, 25, 28). However, although DFMO is remarkably effective, development of a more potent agent with broader therapeutic and similar nontoxic characteristics is still a worthy goal. S-Adenosyl-L-methionine (AdoMet) decarboxylase (DC) (EC 4.1.1.50) is another obligatory enzyme in the biosynthesis of spermidine and spermine in higher eucaryotes (22) and spermidine in African trypanosomes (1). This enzyme catalyzes the formation of decarboxy AdoMet, which then donates an aminopropyl group to one of the amines of putrescine in a reaction catalyzed by spermidine synthase (Fig. 1). For many years, AdoMet DC has been considered a target for chemotherapy of proliferative disorders, such as cancer, on the basis of the finding that methylglyoxal bis (guanylhydrazone), a cytotoxic drug which inhibits cell proliferation (13), is also a potent competitive inhibitor of *
AdoMet DC (29). In addition to their antitumor effects, methylglyoxal bis(guanylhydrazone) and other bis(guanylhydrazones) were shown to cure murine Trypanosoma brucei brucei infections (10, 26, 27). More recently, it was demonstrated that the trypanocidal drugs diminazene aceturate (Berenil) and pentamidine are inhibitors of trypanosomal AdoMet DC (6). Although the correlation of inhibition of AdoMet DC with the trypanocidal effects of these drugs is not certain, in view of the known effects of DFMO, it seemed logical to consider AdoMet DC as a rational chemotherapeutic target in trypanosomiasis. A number of structural analogs of decarboxy AdoMet have been synthesized as potential inhibitors of AdoMet DC, and several of these have been shown to inhibit the enzyme from rat liver (16, 20) and cultured L1210 cells (21) irreversibly. Two of these compounds were found to inhibit the growth of these cells in culture. The inhibitory effect could be overcome by provision of spermidine or spermine in the culture medium (21), suggesting that growth inhibition was due to specific inhibition of AdoMet DC and depletion of polyamines. More recently, a new analog of decarboxy AdoMet, 5' -{[(Z)-4-amino-2-butenyl]methylamino}-5' -deoxyadenosine (MDL 73811; Fig. 2), has been shown to be an even more potent inhibitor of AdoMet DC in vitro (9). We now report that MDL 73811 is at least 100 times as potent as DFMO against murine T. b. brucei infections and that complete cures of mice infected with a multidrugresistant strain of T. brucei rhodesiense can be obtained when the drug is administered either by injection in combination with low oral doses of DFMO or singly in miniosmotic pumps.
Corresponding author. 1485
1486
ANTIMICROB. AGENTS CHEMOTHER.
BITONTI ET AL. COOH
COOH
H2N
ii
CH
NH2
Onfidhine HO
OH
S-Adenosyl-L-Methionine
(D
CH3
Putresdne H,2N
H HO
OHAo
aminopopyl transfer
0 Spermidine
FIG. 1. Polyamine biosynthesis in African trypanosomes. The following enzymes are involved: 1, ornithine decarboxylase; 2, AdoMet DC; 3, spermidine synthase. Ado, Adenosine. MATERIALS AND METHODS
Chemicals, MDL 73811 and DFMO were synthesized at the Merrell Dow Research Institute (9). S-Adenosyl-L-[carboxyl-"'C]methionine (56.9 mCi/mmol) was purchased from Dupont, NEN Research Products. Trypanosomes. T. b. brucei Lab l11/EATRO and T. b. rhodesiense KETRI 2538 (acquired from the Kenya Trypanosomiasis Research Institute strain bank at Muguga, Kenya) were maintained by syringe passage in male SpraqueDawley rats. Lab 110/EATRO is susceptible to all standard antitrypanosomal drugs, whereas KETRI 2538 is apparently multidrug resistant in mice (C. J. Bacchi and A. B. Clarkson, Jr., submitted for publication). Trypanosomes for biochemical studies were taken from heavily infected male Sprague-Dawley rats as described previously (4) and separated from blood by DEAE-cellulose chromatography (17). Trypanosome extracts and AdoMet DC assays. Purified trypanosomes were suspended in a solution of 50 mM sodium phosphate (ppH 7.4) containing 2 mM dithiothreitol, 3 mM putrescine, and 0.1 mM EDTA and disrupted with three 15-s periods of continuous sonication (setting 3, Branson Sonifier) on ice interrupted by 30-s periods without sonication. Disrupted trypanosomes were then spun at 10,000 x g in a refrigerated (4°C) centrifuge, and the supernatant fraction, containing 7 to 10 mg of protein per ml, was decanted and stored frozen at -20°C for up to 2 weeks without loss of AdoMet DC activity. AdoMet DC activity was measured as described previously (23). The enzyme reaction contained 50 mM sodium NH2
kN
NN HO
NW
OH
5'-{[(Z)-4-Amino-2-butenylJmethylamino)-5'-deoxyadenosine FIG. 2. Structure of AdoMet DC inhibitor MDL 73811.
phosphate (pH 7.4), 2 mM dithiothreitol, 0.25 to 0.5 mM AdoMet, 3 mM putrescine, 0.1 ,uCi of [carboxyl-'4C] AdoMet, and 25 to 30 ,ug of trypanosome protein in a total volume of 0.25 ml. The reactions were run for 15 to 25 min at 37°C in a shaking water bath, terminated by addition of 0.25 ml of 40% trichloroacetic acid, and incubated for an additional 30 min at 37°C, during which time "'CO2 was trapped on a filter paper soaked in methylbenzethonium hydroxide. Trapped 14CO2 was then determined by liquid scintillation counting in Omnifluor (Dupont, NEN) cocktail. Protein concentrations were determined by the method of Bradford (8). Inactivation of AdoMet DC activity. T. b. brucei extract was warmed to 37°C for 45 s and then incubated at 37°C with 0.25 to 1.0 ,uM MDL 73811. At various times, 0.025-ml samples were diluted into ice-cold AdoMet DC assay buffer (described above) containing 2.5 mM unlabeled AdoMet and this mixture was kept on ice, to prevent further inactivation of AdoMet DC activity, until all samples were collected. After the final sample was taken, 0.1 ,uCi of [carboxyl-_4C] AdoMet in assay buffer was added to give a final AdoMet concentration of 0.5 mM in a reaction volume of 0.25 ml and AdoMet DC activity was determined as described above. Kinetic analysis of AdoMet DC inhibition was performed as described previously for acetylcholinesterase (15). In an experiment to test for reversibility of inactivation, T. b. brucei extract was warmed to 25°C and incubated with or without 1 p,M MDL 73811. Samples (0.025 ml) of inactivated and control AdoMet DC were taken at 0 and 5 min and diluted into assay buffer kept on ice. Portions of the extracts (0.3 ml) which had been incubated for 5 min were chromatographed on a 10-ml column of Sephadex G-25 to separate AdoMet DC from unbound MDL 73811. These samples were then added to the assay buffer on ice, 0.1 pCi of [carboxyl"'C]AdoMet (final AdoMet concentration, 0.5 mM) was added to all of the samples, and AdoMet DC activity was determined as described above. This column chromatography procedure was shown previously to reverse inhibition of AdoMet DC by methylglyoxal bis(guanylhydrazone), a competitive inhibitor of the enzyme (6). Measurement of polyamines. Trypanosomes purified as described above were washed twice in 90 mM Tris hydrochloride (pH 7.8) containing 50 mM NaCl and 2% (wt/vol) glucose, sedimented by centrifugation at 15,000 x g for 10 min, and then extracted with small volumes of 10% trichloroacetic acid. Precipitated proteins were removed by centrifugation, and the supernatant fractions were filtered through 0.45-pm-pore-size membranes. The precipitated proteins were dissolved with 1 N NaOH for measurement of protein concentrations. Polyamines were determined by high-pressure liquid chromatography as described previously (24). Drug testing. Male CD-1 mice (20 to 25 g) were infected with T. b. brucei Lab 110/EATRO by intraperitoneal injection of 2.5 x 105 trypomastigotes from frozen samples, and the infection was allowed to develop for 24 h before drug treatments were begun. Untreated control mice usually succumbed 4 to 6 days postinfection. Animals were considered cured if they survived for 30 days beyond the deaths of the untreated controls and were free of trypanosomes at that time. Infection of mice with 105 T. b. rhodesiense KETRI 2538 organisms was also by intraperitoneal injection, but the infection was allowed to develop for 72 h before drug treatment. Untreated mice survived for 14 days, on the average, when infected with T. b. rhodesiense. Mice were considered cured if they survived for 60 days postinfection and were free of trypanosomes at that time.
AdoMet DC INHIBITOR CURES TRYPANOSOME INFECTIONS
VOL. 34, 1990
A
1487
TABLE 1. AdoMet prevents time-dependent inhibition of T. b. brucei AdoMet DC activity by MDL 73811a
1004
Mean + SD AdoMet DC activity
(nmol/h per mg of protein)
Addition (concn)
80
0066-4804/90/081485-06$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 34, No. 8
Cure of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense Infections in Mice with an Irreversible Inhibitor of S-Adenosylmethionine Decarboxylase ALAN J. BITONTI,1* TIMOTHY L. BYERS,' TAMMY L. BUSH,' PATRICK J. CASARA,1 CYRUS J. BACCHI,2 ALLEN B. CLARKSON, JR.,3 PETER P. McCANN,' AND ALBERT SJOERDSMA' Merrell Dow Research Institute, Cincinnati, Ohio 452151; Haskins Laboratories of Pace University, New York, New York 100382; and Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, New York 100033 Received 22 February 1990/Accepted 3 June 1990
A structural analog, 5'-{[(Z)4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (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 (T.s, 0.3 min), exhibited pseudo-first-order kinetics (Ki, 1.5 ,uM), 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 a-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.
Trypanosomiasis, or sleeping sickness, remains endemic in many countries in Africa despite vigorous attempts to eradicate the disease (30). Most patients can be treated effectively with the presently available drugs suramin, pentamidine, and melarsoprol, but serious side effects often accompany their use (18) and trypanosome infections resistant to the effects of melarsoprol, the only drug approved for treatment of the late-stage disease with central nervous system involvement, are increasing (14). Recently, eflornithine (a-difluoromethylornithine; Ornidyl) (DFMO), an irreversible inhibitor of ornithine decarboxylase (4, 19), the first enzyme in the biosynthesis of the polyamines putrescine, spermidine, and spermine (22), has been found effective for treatment of both early- and late-stage Gambian sleeping sickness in human patients (2, 25, 28). However, although DFMO is remarkably effective, development of a more potent agent with broader therapeutic and similar nontoxic characteristics is still a worthy goal. S-Adenosyl-L-methionine (AdoMet) decarboxylase (DC) (EC 4.1.1.50) is another obligatory enzyme in the biosynthesis of spermidine and spermine in higher eucaryotes (22) and spermidine in African trypanosomes (1). This enzyme catalyzes the formation of decarboxy AdoMet, which then donates an aminopropyl group to one of the amines of putrescine in a reaction catalyzed by spermidine synthase (Fig. 1). For many years, AdoMet DC has been considered a target for chemotherapy of proliferative disorders, such as cancer, on the basis of the finding that methylglyoxal bis (guanylhydrazone), a cytotoxic drug which inhibits cell proliferation (13), is also a potent competitive inhibitor of *
AdoMet DC (29). In addition to their antitumor effects, methylglyoxal bis(guanylhydrazone) and other bis(guanylhydrazones) were shown to cure murine Trypanosoma brucei brucei infections (10, 26, 27). More recently, it was demonstrated that the trypanocidal drugs diminazene aceturate (Berenil) and pentamidine are inhibitors of trypanosomal AdoMet DC (6). Although the correlation of inhibition of AdoMet DC with the trypanocidal effects of these drugs is not certain, in view of the known effects of DFMO, it seemed logical to consider AdoMet DC as a rational chemotherapeutic target in trypanosomiasis. A number of structural analogs of decarboxy AdoMet have been synthesized as potential inhibitors of AdoMet DC, and several of these have been shown to inhibit the enzyme from rat liver (16, 20) and cultured L1210 cells (21) irreversibly. Two of these compounds were found to inhibit the growth of these cells in culture. The inhibitory effect could be overcome by provision of spermidine or spermine in the culture medium (21), suggesting that growth inhibition was due to specific inhibition of AdoMet DC and depletion of polyamines. More recently, a new analog of decarboxy AdoMet, 5' -{[(Z)-4-amino-2-butenyl]methylamino}-5' -deoxyadenosine (MDL 73811; Fig. 2), has been shown to be an even more potent inhibitor of AdoMet DC in vitro (9). We now report that MDL 73811 is at least 100 times as potent as DFMO against murine T. b. brucei infections and that complete cures of mice infected with a multidrugresistant strain of T. brucei rhodesiense can be obtained when the drug is administered either by injection in combination with low oral doses of DFMO or singly in miniosmotic pumps.
Corresponding author. 1485
1486
ANTIMICROB. AGENTS CHEMOTHER.
BITONTI ET AL. COOH
COOH
H2N
ii
CH
NH2
Onfidhine HO
OH
S-Adenosyl-L-Methionine
(D
CH3
Putresdne H,2N
H HO
OHAo
aminopopyl transfer
0 Spermidine
FIG. 1. Polyamine biosynthesis in African trypanosomes. The following enzymes are involved: 1, ornithine decarboxylase; 2, AdoMet DC; 3, spermidine synthase. Ado, Adenosine. MATERIALS AND METHODS
Chemicals, MDL 73811 and DFMO were synthesized at the Merrell Dow Research Institute (9). S-Adenosyl-L-[carboxyl-"'C]methionine (56.9 mCi/mmol) was purchased from Dupont, NEN Research Products. Trypanosomes. T. b. brucei Lab l11/EATRO and T. b. rhodesiense KETRI 2538 (acquired from the Kenya Trypanosomiasis Research Institute strain bank at Muguga, Kenya) were maintained by syringe passage in male SpraqueDawley rats. Lab 110/EATRO is susceptible to all standard antitrypanosomal drugs, whereas KETRI 2538 is apparently multidrug resistant in mice (C. J. Bacchi and A. B. Clarkson, Jr., submitted for publication). Trypanosomes for biochemical studies were taken from heavily infected male Sprague-Dawley rats as described previously (4) and separated from blood by DEAE-cellulose chromatography (17). Trypanosome extracts and AdoMet DC assays. Purified trypanosomes were suspended in a solution of 50 mM sodium phosphate (ppH 7.4) containing 2 mM dithiothreitol, 3 mM putrescine, and 0.1 mM EDTA and disrupted with three 15-s periods of continuous sonication (setting 3, Branson Sonifier) on ice interrupted by 30-s periods without sonication. Disrupted trypanosomes were then spun at 10,000 x g in a refrigerated (4°C) centrifuge, and the supernatant fraction, containing 7 to 10 mg of protein per ml, was decanted and stored frozen at -20°C for up to 2 weeks without loss of AdoMet DC activity. AdoMet DC activity was measured as described previously (23). The enzyme reaction contained 50 mM sodium NH2
kN
NN HO
NW
OH
5'-{[(Z)-4-Amino-2-butenylJmethylamino)-5'-deoxyadenosine FIG. 2. Structure of AdoMet DC inhibitor MDL 73811.
phosphate (pH 7.4), 2 mM dithiothreitol, 0.25 to 0.5 mM AdoMet, 3 mM putrescine, 0.1 ,uCi of [carboxyl-'4C] AdoMet, and 25 to 30 ,ug of trypanosome protein in a total volume of 0.25 ml. The reactions were run for 15 to 25 min at 37°C in a shaking water bath, terminated by addition of 0.25 ml of 40% trichloroacetic acid, and incubated for an additional 30 min at 37°C, during which time "'CO2 was trapped on a filter paper soaked in methylbenzethonium hydroxide. Trapped 14CO2 was then determined by liquid scintillation counting in Omnifluor (Dupont, NEN) cocktail. Protein concentrations were determined by the method of Bradford (8). Inactivation of AdoMet DC activity. T. b. brucei extract was warmed to 37°C for 45 s and then incubated at 37°C with 0.25 to 1.0 ,uM MDL 73811. At various times, 0.025-ml samples were diluted into ice-cold AdoMet DC assay buffer (described above) containing 2.5 mM unlabeled AdoMet and this mixture was kept on ice, to prevent further inactivation of AdoMet DC activity, until all samples were collected. After the final sample was taken, 0.1 ,uCi of [carboxyl-_4C] AdoMet in assay buffer was added to give a final AdoMet concentration of 0.5 mM in a reaction volume of 0.25 ml and AdoMet DC activity was determined as described above. Kinetic analysis of AdoMet DC inhibition was performed as described previously for acetylcholinesterase (15). In an experiment to test for reversibility of inactivation, T. b. brucei extract was warmed to 25°C and incubated with or without 1 p,M MDL 73811. Samples (0.025 ml) of inactivated and control AdoMet DC were taken at 0 and 5 min and diluted into assay buffer kept on ice. Portions of the extracts (0.3 ml) which had been incubated for 5 min were chromatographed on a 10-ml column of Sephadex G-25 to separate AdoMet DC from unbound MDL 73811. These samples were then added to the assay buffer on ice, 0.1 pCi of [carboxyl"'C]AdoMet (final AdoMet concentration, 0.5 mM) was added to all of the samples, and AdoMet DC activity was determined as described above. This column chromatography procedure was shown previously to reverse inhibition of AdoMet DC by methylglyoxal bis(guanylhydrazone), a competitive inhibitor of the enzyme (6). Measurement of polyamines. Trypanosomes purified as described above were washed twice in 90 mM Tris hydrochloride (pH 7.8) containing 50 mM NaCl and 2% (wt/vol) glucose, sedimented by centrifugation at 15,000 x g for 10 min, and then extracted with small volumes of 10% trichloroacetic acid. Precipitated proteins were removed by centrifugation, and the supernatant fractions were filtered through 0.45-pm-pore-size membranes. The precipitated proteins were dissolved with 1 N NaOH for measurement of protein concentrations. Polyamines were determined by high-pressure liquid chromatography as described previously (24). Drug testing. Male CD-1 mice (20 to 25 g) were infected with T. b. brucei Lab 110/EATRO by intraperitoneal injection of 2.5 x 105 trypomastigotes from frozen samples, and the infection was allowed to develop for 24 h before drug treatments were begun. Untreated control mice usually succumbed 4 to 6 days postinfection. Animals were considered cured if they survived for 30 days beyond the deaths of the untreated controls and were free of trypanosomes at that time. Infection of mice with 105 T. b. rhodesiense KETRI 2538 organisms was also by intraperitoneal injection, but the infection was allowed to develop for 72 h before drug treatment. Untreated mice survived for 14 days, on the average, when infected with T. b. rhodesiense. Mice were considered cured if they survived for 60 days postinfection and were free of trypanosomes at that time.
AdoMet DC INHIBITOR CURES TRYPANOSOME INFECTIONS
VOL. 34, 1990
A
1487
TABLE 1. AdoMet prevents time-dependent inhibition of T. b. brucei AdoMet DC activity by MDL 73811a
1004
Mean + SD AdoMet DC activity
(nmol/h per mg of protein)
Addition (concn)
80
