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The toxicity of antifolates in Babesia bovis
lnrernarional Printed
Journalfor
in Grenl
Porasifology
Vol. 23. No. 3, pp. 399-402,
1993
Britain 0
002&7519/93 %6.00 + 0.00 Pergamon Press Lfd Somryfor Parasirology
1993 Australian
RESEARCH NOTE THE TOXICITY OF ANTIFOLATES IN BABESIA BO VIS SUSAN E. NOTT
and ALDO S. BAGNARA*
School of Biochemistry and Molecular Genetics, University of New South Wales, P.O. Box I, Kensington, New South Wales 2033, Australia (Received 6 January 1993; accepted 8 February 1993)
S. E. and BAGNARAA. S. 1993. The toxicity of antifolates in Babesia bovis. International Parasitology 23: 399402. A variety of anti-folate compounds have been tested for their ability to inhibit the growth of Babesia bovis as measured by the incorporation of [‘Hlhypoxanthine into the Abstract-Norm
Journalfor
parasite’s nucleic acids. Inhibitors of folate synthesis (including 7-methylguanosine and several sulpha drugs) were without effect but several structural analogues of folate were toxic. The most potent folate analogues were the lipophilic compounds piritrexim and trimetrexate, each causing 50% inhibition of [‘Hlhypoxanthine incorporation (IC,,) at a concentration of 2.9 nM; other classical anti-folates such as pyrimethamine, methotrexate and trimethoprim were at least IOO-fold less effective with IC,, values of 1.2, 0.29 and 0.50 ,UM, respectively. From these results we conclude that B. bovis does not synthesize folate de nova under cell culture conditions. However, the toxic effects of piritrexim and trimetrexate suggest that dihydrofolate reductase (DHFR) activity is essential for the parasite, most probably because of the role of this enzyme in the synthesis
of thymidine
nucleotides
via thymidylate
synthase.
INDEX KEY WORDS: Babesia bovis; protozoan parasites; folic acid metabolism; folic acid analogues; methotrexate; pyrimethamine; dihydrofolate reductase; thymidylate synthase.
BECAUSEof the involvement of the folate derivatives in the synthesis of the precursors for proteins and nucleic acids, perturbation of folate metabolism has long been a target for antimicrobial and anticancer chemotherapy (Schweitzer, Dicker & Bertino, 1990). Organisms which synthesize folate de now are usually sensitive to drugs which inhibit the synthesis of the pteridine ring or to the sulphonamide family of drugs which inhibit the incorporation of p-aminobenzoate into the folate structure (Anand, 1983), while most organisms are sensitive to drugs which inhibit the reduction of folate to its functional tetrahydrofolate form. The metabolism of folate and its derivatives has not been studied in detail in protozoan parasites. In the malarial parasite Plasmodium falciparum, the metabolism of folate includes the salvage of preformed folate (provided by the host), the salvage of other folate precursors (e.g. pterin aldehyde and p-aminobenzoylglutamate) complete bioand synthesis de nova from GTP, p-aminobenzoate and glutamate (Krungkrai, Yuthavong & Webster, 1985; Krungkrai, Webster & Yuthavong, 1989; Fairlamb, 1989; Hyde, 1990; Krungkrai, Webster & Yuthavong,
* To whom all correspondence
1990). The origin of folate in other protozoans is less clear. Indirect evidence suggests that Leishmania species are dependent on an exogenous supply of preformed folate (Peixoto & Beverley, 1987; Scott, Coombs & Sanderson, 1987; Beck & Ullman, 1990) while other genera such as Entamoeba, Giardia and the trichomonads appear to lack folate metabolism altogether and utilise S-adenosylmethionine for their one-carbon transfer reactions (Hyde, 1990). To our knowledge there are no published reports on folate metabolism in any species of Babesia. The toxic effects reported here of a variety of folate analogues and other folate antagonists on B. bovis in cell culture give insight into the metabolism of folate in this parasite and suggest that dihydrofolate reductase (DHFR) activity is a sensitive target for chemotherapy in this parasite. The cultivation of B. bovis and the design of the experiments to determine the toxicity of drugs have been described elsewhere (Nott, O’Sullivan, Gero & Bagnara, 1990). The toxicity assay is based on the ability of a drug to inhibit the incorporation of isotopically-labelled hypoxanthine into the nucleic acids of the parasite over a fixed incubation period. The concentration of drug that inhibits this incorporation by 50% (IC,,) can be determined graphically (Nott et al., 1990). Using these methods,
should be addressed. 399
400
S. E. NOTT and A. S. BAGNARA
TABLE 1. ANTIFOLA~E DRUGS AND THEIR EFFECTS ON GROWTHOF Babesia bovis Drug* Pyrimethamine Methotrexate Trimethoprim Trimetrexate Piritrexim PDDF DDATHF DACTHF Sulphafurazole (sulphisoxazole)t Sulphadimidine (sulphamethazine)? Sulphamethoxazolet Sulphadoxinet 7-Methylguanosinet
THE
1% (IJM) 1.2 0.29 0.50 0.0029 0.0029 0.40 >2.5 >2.5 > 100 1100 ZlOO >I00 >lOO
*Most of the folate analogues including pyrimethamine, methotrexate, trimethoprim, trimetrexate, piritrexim, IOpropargyl-5,8-dideazafolate (PDDF), 5deaza-acyclo-5,6,7,8tetrahydrofolate (DACTHF) and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) were kindly supplied by Dr Richard Christopherson (University of Sydney). The sulpha drugs were obtained as follows: sulphadoxine (Dr Karl Rieckmann, Australian Army Malarial Research Unit, Ingleburn, New South Wales); sulphamethoxazole, sulphafurazole and sulphadimidine (Sigma); 7-methylguanosine was also obtained from Sigma. The drugs that were insoluble in the culture medium or phosphate-buffered saline were dissolved in dimethyl sulphoxide; the stocks were then sterilized by filtration. Control experiments were performed with a range of concentrations of dimethyl sulphoxide and the results showed that this solvent alone was not inhibitory in the toxicity assay at the dilutions used in these experiments. tNo inhibition of babesial growth was observed up to final
concentrations of 100 PM. the anti-babesial properties of a variety of antifolate drugs have been determined (Table 1). 7-Methylguanosine, an inhibitor of GTP cyclohydrolase activity in P. falciparum (Krungkrai et al., 1985) and a selection of sulpha drugs were not inhibitory (Table 1). These results should be interpreted cautiously because of the possibility that pre-formed folate and p-aminobenzoate (respectively 2.3 and 7.3 jJM in RPM1 1640) from the culture medium (RPM1 1640 supplemented with 40% bovine serum; see Nott et al., 1990) could by-pass the specific metabolic blocks caused by 7-methylguanosine and the sulpha drugs as previously found in similar studies conducted with P. falciparum (Milhous, Weatherly, Bowdre & Desjardins, 1985; Tan-Ariya, Brockelman & Menabandhu, 1987). However, despite the presence of folate in the culture medium, several of the folate analogues were potent inhibitors of babesial growth (Table I), the most potent being piritrexim and trimetrexate while PDDF, methotrexate and
Plritrexim
Pyrlmethamlne H
2&+&H
*CH 3
FIG. 1. The structure of dihydrofolate and some of the structural analogues of folate used in this study.
trimethoprim were about two orders of magnitude less effective. Pyrimethamine also was moderately toxic but its IC,, (Table 1) was approximately lOOOfold higher than that found for pyrimethaminesensitive isolates of P. falciparum (Milhous et al., 1985; Cowman, Morry, Biggs, Cross & Foote, 1988; Thaithong, Chan, Songsomboon, Wilairat, Seesod, Sueblinwong, Goman, Ridley & Beale, 1992). These results indicate that B. bovis does not synthesize folates de nova under cell culture conditions and the parasite therefore must rely entirely on the salvage of pre-formed folates provided from the growth medium or from the host bovine red cell. Of the antifolate drugs tested, the two most potent inhibitors of babesial growth, piritrexim and trimetrexate, contain the structures analogous to the pteridine and p-aminobenzoate rings of the natural folates but they lack the (poly)glutamate residues (Fig. 1; see also Christopherson & Lyons, 1990). These inhibitors are most likely to exert their effects by inhibiting the DHFR activity in B. bovis, an enzyme which is essential for thymidylate synthesis because the parasite appears to lack the ability to
401
Research Note salvage either thymine or thymidine (Matias, Nott, Bagnara, O’Sullivan & Gero, 1990). It is not known why piritrexim and trimetrexate are the most effective inhibitors of those tested but the following suggestions are put forward. First, these two drugs are more lipophilic than the glutamate-containing analogues such as methotrexate and PDDF and they may therefore have greater access to the babesial DHFR through their ability to penetrate the hostcell and parasite membranes. However, a second alternative is that our results simply reflect that piritrexim and trimetrexate are specific and potent inhibitors of the babesial DHFR. We note that the two drugs share a similar 5-deaza-5-methyl function in the moiety analogous to the pteridine ring (see Fig. 1) which might be important in causing inhibition of the DHFR activity. These hypotheses might be tested with cell-free extracts of B. bovis or with purified enzyme preparations but we do not have access to the amount of parasite material required to perform this work. For these reasons we plan to produce sufficient quantities of DHFR by isolating its gene and cloning it into a suitable expression vector; a similar approach has been used successfully to obtain the enzyme from Leishmania major (Grumont, Sirawaraporn & Santi, 1988). However, regardless of the actual mechanism of action of piritrexim and trimetrexate against the growth of B. bovis, these two antifolate drugs are as inhibitory against this parasite as pyrimethamine is against sensitive isolates of P. falciparum. Further study of DHFR and/or thymidylate synthase activities as potential targets for chemotherapy is therefore warranted, especially if these two activities are present on a single, bifunctional protein as has been found in other protozoan parasites (Garrett, Coderre, Meek, Garvey, Claman, Beverley & Santi, 1984). Finally, our data give some insight into folate metabolism in B. bovis. We conclude that the parasite is able to utilise folate provided from exogenous sources and convert it by reduction to the dihydroand tetrahydro-forms. In addition, although our data do not unequivocally rule out the possibility that B. bovis can synthesize folates de novo, the fact that all of the sulpha drugs and 7-methylguanosine were completely without effect up to concentrations of 100 PM (Table 1) argues strongly against this possibility. We suggest, therefore, that the metabolism of folate in B. bovis is limited to its uptake from exogenous sources, its conversion to the dihydroand tetrahydro-forms and its primary use as a onecarbon donor (via 5,10-methylenetetrahydrofolate) for thymidylate synthesis. This metabolic scheme for folate, though simple, plays a vital role in providing
the only source of thymidine nucleotides for DNA synthesis and repair in B. bovis and it therefore defines an extremely sensitive target for chemotherapy. Acknowledgements-This
work was supported by the Australian Research Council and a Special Research Grant from the University of New South Wales. We thank Drs Richard Christopherson and Karl Rieckmann for the supply of drugs. REFERENCES ANAND N. 1983. Sulfonamides: structure-activity relationships and mechanism of action. In: Handbook of Experimental Pharmacology, Vol. 64 (Edited by HITCHINGSG. H.), pp. 25-54. Springer, New York. BECK J. T. & ULLMAN B. 1990. Nutritional requirements of wild-type and folate transport-deficient Leishmaniu donovani for pterins and folates. Molecular and Biochemical Parasitology 43: 221-230. CHRISTOPHERSONR. I. & LYONSS. D. 1990. Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents. Medicinal Research Reviews 10: 50%
548. COWMANA. F., MORRY M. J., BIGGS B. A., CROSS G. A. M. & F~XXE S. J. 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductasethymidylate synthase gene of Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 85: 9109-9113. FAIRLAMB A. H. 1989. Novel biochemical pathways in parasitic protozoa. Parasitology 99: S93-S112. GARRETS C. E., CODERRE J. A., MEEK T. D., GARVEY T. P., CLAMA~ D. M., BEVERLEYS. M. & SANX D. V. 1984. A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa. Molecular and Biochemical Parasitology 11:257-265. GRUMONT R., SIRAWARAPORNW. & SAN~I D. V. 1988. Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major. Biochemistry 21: 3776-3784. HYDE J. E. 1990. Molecular Parasitology, pp. 181-228. Open University Press, Buckingham. KRUNGKRAI J., YU~HAVONG Y. & WEBSTER H. K. 1985. Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species. Molecular and Biochemical Parasitology 17: 265-276. KRUNGKRAI J., WEBSTERH. K. and YU~HAVONGY. 1989. De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum. Molecular and Biochemical Parasitology 32: 25-37. KRUNGKRAI J., WEBSTER H. K. and YUTHAVONGY. 1990. Folate and cobalamin metabolism in Plasmodium falciparum. Parasitology Today 6: 388-39 1. MATIAS C., NOTT S. E., BAGNARAA. S., O’SULLIVANW. J. & GERO A. M. 1990. Purine salvage and metabolism in Babesia bovis. Parasitology Research 16: 207-213. MILHOUS W. K., WEA~HERLY N. F., BOWDRE J. H. & DESIARDINS R. E. 1985. In vitro activities of and
402
S. E. NOTT and A. S. BAGNARA
mechanisms of resistance to antifol antimalarial drugs. Antimicrobial Agents and Chemotherapy 27: 525-530. NOI-T S. E., O’SULLIVAN W. J., GERO A. M. & BAGNARAA. S. 1990. Routine screening for potential babesicides using cultures of Babesia bovis. International Journal for Parasitology 20: 797-802. PEIXOTOM. P. & BEVERLEYS. M. 1987. In vitro activity of sulfonamides and sulfones against Leishmania major promastigotes. Antimicrobial Agents and Chemotherapy 31: 1575-1578. SCHWEITZERB. I., DICKER A. P. & BERTINO J. R. 1990. Dihydrofolate reductase as a therapeutic target. The FASEB Journal 4: 244-2452. SCOTTD. A., CRUMBSG. H. & SANDERSONB. E. 1987. Folate
utilisation by Leishmania species and the identification of intracellular derivatives and folate-metabolising enzymes. Molecular and Biochemical Parasitology 23: 139-149. TAN-ARIYA P., BROCKELMANC. R. & MENABANDHUC. 1987. Optimal concentrations of p-aminobenzoic acid and folic acid in the in vitro assay of antifolates against Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 37: 4248. THAITHONG S., CHAN S-W., SONCSOMBOONS., WILAIRAT P., SEESOD N., SUEBLINWONGT., GOMAN M., RIDLEY R. & BEALE G. 1992. Pyrimethamine resistant mutations in Plasmodium falciparum. Molecular and Biochemical Parasitology 52: 149-158.
Journalfor
in Grenl
Porasifology
Vol. 23. No. 3, pp. 399-402,
1993
Britain 0
002&7519/93 %6.00 + 0.00 Pergamon Press Lfd Somryfor Parasirology
1993 Australian
RESEARCH NOTE THE TOXICITY OF ANTIFOLATES IN BABESIA BO VIS SUSAN E. NOTT
and ALDO S. BAGNARA*
School of Biochemistry and Molecular Genetics, University of New South Wales, P.O. Box I, Kensington, New South Wales 2033, Australia (Received 6 January 1993; accepted 8 February 1993)
S. E. and BAGNARAA. S. 1993. The toxicity of antifolates in Babesia bovis. International Parasitology 23: 399402. A variety of anti-folate compounds have been tested for their ability to inhibit the growth of Babesia bovis as measured by the incorporation of [‘Hlhypoxanthine into the Abstract-Norm
Journalfor
parasite’s nucleic acids. Inhibitors of folate synthesis (including 7-methylguanosine and several sulpha drugs) were without effect but several structural analogues of folate were toxic. The most potent folate analogues were the lipophilic compounds piritrexim and trimetrexate, each causing 50% inhibition of [‘Hlhypoxanthine incorporation (IC,,) at a concentration of 2.9 nM; other classical anti-folates such as pyrimethamine, methotrexate and trimethoprim were at least IOO-fold less effective with IC,, values of 1.2, 0.29 and 0.50 ,UM, respectively. From these results we conclude that B. bovis does not synthesize folate de nova under cell culture conditions. However, the toxic effects of piritrexim and trimetrexate suggest that dihydrofolate reductase (DHFR) activity is essential for the parasite, most probably because of the role of this enzyme in the synthesis
of thymidine
nucleotides
via thymidylate
synthase.
INDEX KEY WORDS: Babesia bovis; protozoan parasites; folic acid metabolism; folic acid analogues; methotrexate; pyrimethamine; dihydrofolate reductase; thymidylate synthase.
BECAUSEof the involvement of the folate derivatives in the synthesis of the precursors for proteins and nucleic acids, perturbation of folate metabolism has long been a target for antimicrobial and anticancer chemotherapy (Schweitzer, Dicker & Bertino, 1990). Organisms which synthesize folate de now are usually sensitive to drugs which inhibit the synthesis of the pteridine ring or to the sulphonamide family of drugs which inhibit the incorporation of p-aminobenzoate into the folate structure (Anand, 1983), while most organisms are sensitive to drugs which inhibit the reduction of folate to its functional tetrahydrofolate form. The metabolism of folate and its derivatives has not been studied in detail in protozoan parasites. In the malarial parasite Plasmodium falciparum, the metabolism of folate includes the salvage of preformed folate (provided by the host), the salvage of other folate precursors (e.g. pterin aldehyde and p-aminobenzoylglutamate) complete bioand synthesis de nova from GTP, p-aminobenzoate and glutamate (Krungkrai, Yuthavong & Webster, 1985; Krungkrai, Webster & Yuthavong, 1989; Fairlamb, 1989; Hyde, 1990; Krungkrai, Webster & Yuthavong,
* To whom all correspondence
1990). The origin of folate in other protozoans is less clear. Indirect evidence suggests that Leishmania species are dependent on an exogenous supply of preformed folate (Peixoto & Beverley, 1987; Scott, Coombs & Sanderson, 1987; Beck & Ullman, 1990) while other genera such as Entamoeba, Giardia and the trichomonads appear to lack folate metabolism altogether and utilise S-adenosylmethionine for their one-carbon transfer reactions (Hyde, 1990). To our knowledge there are no published reports on folate metabolism in any species of Babesia. The toxic effects reported here of a variety of folate analogues and other folate antagonists on B. bovis in cell culture give insight into the metabolism of folate in this parasite and suggest that dihydrofolate reductase (DHFR) activity is a sensitive target for chemotherapy in this parasite. The cultivation of B. bovis and the design of the experiments to determine the toxicity of drugs have been described elsewhere (Nott, O’Sullivan, Gero & Bagnara, 1990). The toxicity assay is based on the ability of a drug to inhibit the incorporation of isotopically-labelled hypoxanthine into the nucleic acids of the parasite over a fixed incubation period. The concentration of drug that inhibits this incorporation by 50% (IC,,) can be determined graphically (Nott et al., 1990). Using these methods,
should be addressed. 399
400
S. E. NOTT and A. S. BAGNARA
TABLE 1. ANTIFOLA~E DRUGS AND THEIR EFFECTS ON GROWTHOF Babesia bovis Drug* Pyrimethamine Methotrexate Trimethoprim Trimetrexate Piritrexim PDDF DDATHF DACTHF Sulphafurazole (sulphisoxazole)t Sulphadimidine (sulphamethazine)? Sulphamethoxazolet Sulphadoxinet 7-Methylguanosinet
THE
1% (IJM) 1.2 0.29 0.50 0.0029 0.0029 0.40 >2.5 >2.5 > 100 1100 ZlOO >I00 >lOO
*Most of the folate analogues including pyrimethamine, methotrexate, trimethoprim, trimetrexate, piritrexim, IOpropargyl-5,8-dideazafolate (PDDF), 5deaza-acyclo-5,6,7,8tetrahydrofolate (DACTHF) and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) were kindly supplied by Dr Richard Christopherson (University of Sydney). The sulpha drugs were obtained as follows: sulphadoxine (Dr Karl Rieckmann, Australian Army Malarial Research Unit, Ingleburn, New South Wales); sulphamethoxazole, sulphafurazole and sulphadimidine (Sigma); 7-methylguanosine was also obtained from Sigma. The drugs that were insoluble in the culture medium or phosphate-buffered saline were dissolved in dimethyl sulphoxide; the stocks were then sterilized by filtration. Control experiments were performed with a range of concentrations of dimethyl sulphoxide and the results showed that this solvent alone was not inhibitory in the toxicity assay at the dilutions used in these experiments. tNo inhibition of babesial growth was observed up to final
concentrations of 100 PM. the anti-babesial properties of a variety of antifolate drugs have been determined (Table 1). 7-Methylguanosine, an inhibitor of GTP cyclohydrolase activity in P. falciparum (Krungkrai et al., 1985) and a selection of sulpha drugs were not inhibitory (Table 1). These results should be interpreted cautiously because of the possibility that pre-formed folate and p-aminobenzoate (respectively 2.3 and 7.3 jJM in RPM1 1640) from the culture medium (RPM1 1640 supplemented with 40% bovine serum; see Nott et al., 1990) could by-pass the specific metabolic blocks caused by 7-methylguanosine and the sulpha drugs as previously found in similar studies conducted with P. falciparum (Milhous, Weatherly, Bowdre & Desjardins, 1985; Tan-Ariya, Brockelman & Menabandhu, 1987). However, despite the presence of folate in the culture medium, several of the folate analogues were potent inhibitors of babesial growth (Table I), the most potent being piritrexim and trimetrexate while PDDF, methotrexate and
Plritrexim
Pyrlmethamlne H
2&+&H
*CH 3
FIG. 1. The structure of dihydrofolate and some of the structural analogues of folate used in this study.
trimethoprim were about two orders of magnitude less effective. Pyrimethamine also was moderately toxic but its IC,, (Table 1) was approximately lOOOfold higher than that found for pyrimethaminesensitive isolates of P. falciparum (Milhous et al., 1985; Cowman, Morry, Biggs, Cross & Foote, 1988; Thaithong, Chan, Songsomboon, Wilairat, Seesod, Sueblinwong, Goman, Ridley & Beale, 1992). These results indicate that B. bovis does not synthesize folates de nova under cell culture conditions and the parasite therefore must rely entirely on the salvage of pre-formed folates provided from the growth medium or from the host bovine red cell. Of the antifolate drugs tested, the two most potent inhibitors of babesial growth, piritrexim and trimetrexate, contain the structures analogous to the pteridine and p-aminobenzoate rings of the natural folates but they lack the (poly)glutamate residues (Fig. 1; see also Christopherson & Lyons, 1990). These inhibitors are most likely to exert their effects by inhibiting the DHFR activity in B. bovis, an enzyme which is essential for thymidylate synthesis because the parasite appears to lack the ability to
401
Research Note salvage either thymine or thymidine (Matias, Nott, Bagnara, O’Sullivan & Gero, 1990). It is not known why piritrexim and trimetrexate are the most effective inhibitors of those tested but the following suggestions are put forward. First, these two drugs are more lipophilic than the glutamate-containing analogues such as methotrexate and PDDF and they may therefore have greater access to the babesial DHFR through their ability to penetrate the hostcell and parasite membranes. However, a second alternative is that our results simply reflect that piritrexim and trimetrexate are specific and potent inhibitors of the babesial DHFR. We note that the two drugs share a similar 5-deaza-5-methyl function in the moiety analogous to the pteridine ring (see Fig. 1) which might be important in causing inhibition of the DHFR activity. These hypotheses might be tested with cell-free extracts of B. bovis or with purified enzyme preparations but we do not have access to the amount of parasite material required to perform this work. For these reasons we plan to produce sufficient quantities of DHFR by isolating its gene and cloning it into a suitable expression vector; a similar approach has been used successfully to obtain the enzyme from Leishmania major (Grumont, Sirawaraporn & Santi, 1988). However, regardless of the actual mechanism of action of piritrexim and trimetrexate against the growth of B. bovis, these two antifolate drugs are as inhibitory against this parasite as pyrimethamine is against sensitive isolates of P. falciparum. Further study of DHFR and/or thymidylate synthase activities as potential targets for chemotherapy is therefore warranted, especially if these two activities are present on a single, bifunctional protein as has been found in other protozoan parasites (Garrett, Coderre, Meek, Garvey, Claman, Beverley & Santi, 1984). Finally, our data give some insight into folate metabolism in B. bovis. We conclude that the parasite is able to utilise folate provided from exogenous sources and convert it by reduction to the dihydroand tetrahydro-forms. In addition, although our data do not unequivocally rule out the possibility that B. bovis can synthesize folates de novo, the fact that all of the sulpha drugs and 7-methylguanosine were completely without effect up to concentrations of 100 PM (Table 1) argues strongly against this possibility. We suggest, therefore, that the metabolism of folate in B. bovis is limited to its uptake from exogenous sources, its conversion to the dihydroand tetrahydro-forms and its primary use as a onecarbon donor (via 5,10-methylenetetrahydrofolate) for thymidylate synthesis. This metabolic scheme for folate, though simple, plays a vital role in providing
the only source of thymidine nucleotides for DNA synthesis and repair in B. bovis and it therefore defines an extremely sensitive target for chemotherapy. Acknowledgements-This
work was supported by the Australian Research Council and a Special Research Grant from the University of New South Wales. We thank Drs Richard Christopherson and Karl Rieckmann for the supply of drugs. REFERENCES ANAND N. 1983. Sulfonamides: structure-activity relationships and mechanism of action. In: Handbook of Experimental Pharmacology, Vol. 64 (Edited by HITCHINGSG. H.), pp. 25-54. Springer, New York. BECK J. T. & ULLMAN B. 1990. Nutritional requirements of wild-type and folate transport-deficient Leishmaniu donovani for pterins and folates. Molecular and Biochemical Parasitology 43: 221-230. CHRISTOPHERSONR. I. & LYONSS. D. 1990. Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents. Medicinal Research Reviews 10: 50%
548. COWMANA. F., MORRY M. J., BIGGS B. A., CROSS G. A. M. & F~XXE S. J. 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductasethymidylate synthase gene of Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 85: 9109-9113. FAIRLAMB A. H. 1989. Novel biochemical pathways in parasitic protozoa. Parasitology 99: S93-S112. GARRETS C. E., CODERRE J. A., MEEK T. D., GARVEY T. P., CLAMA~ D. M., BEVERLEYS. M. & SANX D. V. 1984. A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa. Molecular and Biochemical Parasitology 11:257-265. GRUMONT R., SIRAWARAPORNW. & SAN~I D. V. 1988. Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major. Biochemistry 21: 3776-3784. HYDE J. E. 1990. Molecular Parasitology, pp. 181-228. Open University Press, Buckingham. KRUNGKRAI J., YU~HAVONG Y. & WEBSTER H. K. 1985. Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species. Molecular and Biochemical Parasitology 17: 265-276. KRUNGKRAI J., WEBSTERH. K. and YU~HAVONGY. 1989. De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum. Molecular and Biochemical Parasitology 32: 25-37. KRUNGKRAI J., WEBSTER H. K. and YUTHAVONGY. 1990. Folate and cobalamin metabolism in Plasmodium falciparum. Parasitology Today 6: 388-39 1. MATIAS C., NOTT S. E., BAGNARAA. S., O’SULLIVANW. J. & GERO A. M. 1990. Purine salvage and metabolism in Babesia bovis. Parasitology Research 16: 207-213. MILHOUS W. K., WEA~HERLY N. F., BOWDRE J. H. & DESIARDINS R. E. 1985. In vitro activities of and
402
S. E. NOTT and A. S. BAGNARA
mechanisms of resistance to antifol antimalarial drugs. Antimicrobial Agents and Chemotherapy 27: 525-530. NOI-T S. E., O’SULLIVAN W. J., GERO A. M. & BAGNARAA. S. 1990. Routine screening for potential babesicides using cultures of Babesia bovis. International Journal for Parasitology 20: 797-802. PEIXOTOM. P. & BEVERLEYS. M. 1987. In vitro activity of sulfonamides and sulfones against Leishmania major promastigotes. Antimicrobial Agents and Chemotherapy 31: 1575-1578. SCHWEITZERB. I., DICKER A. P. & BERTINO J. R. 1990. Dihydrofolate reductase as a therapeutic target. The FASEB Journal 4: 244-2452. SCOTTD. A., CRUMBSG. H. & SANDERSONB. E. 1987. Folate
utilisation by Leishmania species and the identification of intracellular derivatives and folate-metabolising enzymes. Molecular and Biochemical Parasitology 23: 139-149. TAN-ARIYA P., BROCKELMANC. R. & MENABANDHUC. 1987. Optimal concentrations of p-aminobenzoic acid and folic acid in the in vitro assay of antifolates against Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 37: 4248. THAITHONG S., CHAN S-W., SONCSOMBOONS., WILAIRAT P., SEESOD N., SUEBLINWONGT., GOMAN M., RIDLEY R. & BEALE G. 1992. Pyrimethamine resistant mutations in Plasmodium falciparum. Molecular and Biochemical Parasitology 52: 149-158.