Reductase 10-Deazaaminopterin, and Dihydrofolate

Interaction of Polyglutamyl Derivatives of Methotrexate, 10-Deazaaminopterin, and Dihydrofolate with Dihydrofolate Reductase Piyush Kumar, Roy L. Kisliuk, Yvette Gaumont, et al. Cancer Res 1986;46:5020-5023. Published online October 1, 1986.

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[CANCER RESEARCH 46, 5020-5023, October 1986]

Interaction of Polyglutamyl Derivatives of Methotrexate, Dihydrofolate with Dihydrofolate ReducÃ-ase1

10-Deazaaminopterin,

and

Piyush Kumar, Roy L. Kisliuk,2 Yvette Gaumont, Madhavan G. Nair, Charles M. Baugh, and Bernard T. Kaufman Department of Biochemistry and Pharmacology, Tufts University Health Science Campus, Boston, Massachusetts 02111 fP.K., R.L.K., Y.GJ; Department of Biochemistry, University of South Alabama, Mobile, Alabama 36688 [M. G. N., C. M. B.J; and Laboratory of Nutrition and Endocrinology, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland 2020S [B. T. K.I

ABSTRACT Polyglutamyl derivatives of methotrexate (MIX) and 10-deazaaminopterin (10-DAM) containing a total of one through six glutamate residues (Glu residues) were tested as inhibitors of dihydrofolate reductase (DHFR) derived from sheep, chicken, and beef Over. The ability of dihydropteroylpentaglutamate to antagonize the inhibitory activity of these analogues was also studied. The most striking effects were seen with sheep liver DHFR, where polyglutamylation of MIX causes stepwise decreases in the concentration required for 50% inhibition (IQo) with each additional Glu residue until MIX with a total of six Glu residues has an 1C* value '/>that of MIX With 10-DAM the pattern is more complex. The 1CÂ»values increase with addition of Glu residues until a maximum is reached with 10-DAM having a total of three Glu residues which has a value twice that of 10-DAM. 10-DAM with a total of four Glu residues and 10-DAM with a total of five GiÃ¹residues have progressively lower IQo values, the latter being equipment with 10DAM. With dihydropteroylpentaglutamate as substrate instead of dihy drofolate, the ICjo values are increased 2- to 5-fold for both MIX and 10-DAM derivatives. The results obtained with chicken liver and beef liver DHFR are generally similar to those described for the sheep liver enzyme, but the effects of polyglutamylation are less pronounced. The addition of 0.2 M KC1 to the assay system reduces the differences in inhibitory potency of the polyglutamyl derivatives with all three enzymes tested. We conclude that polyglutamylation can alter the interaction of folate analogues and dihydrofolate with DHFR.

INTRODUCTION Both MTX3 and 10-DAM are potent inhibitors of dihydro folate reducÃ-ase(1, 2). The detection of polyglutamate deriva tives of these antifolates in animal tissues and neoplastic cells raises the possibility of a role for polyglutamate forms in cytotoxicity (3, 4), since they have a prolonged intracellular half-life (5) and have an increased affinity for folate-requiring enzymes such as thymidylate synthase and aminoimidazolecarboxamide ribonucleotide transformylase (6). Folate substrates also exist intracellularly as polyglutamate forms, and many folate-requiring enzymes show a higher affinity for polyglutamates than monoglutamates (6). While binding studies indicate that antifolyl polyglutamates have at least as high an affinity for mammalian DHFR as does MTX (7-9), the effect of antifolyl polyglutamates on mamma lian DHFR activity has not been studied with the exception of LI210 DHFR (10) where little difference was detected between the inhibitory potency of MTX and MTX +d. Received 1/10/86; revised 5/20/86; accepted 6/20/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by Grants CA 10914 (R. L. K.) and CA 32687 and CA 27101 (M. G. N.) from the National Cancer Institute. 2To whom requests for reprints should be addressed. 3The abbreviations used are: MTX, methotrexate (4-amino-4-deoxy-JV10-methylpteroylglutamate); DHFR, dihydrofolate reducÃ-ase;10-DAM, 10-deazaaminopterin (4-amino-4-deoxy-10-deazapteroylglutamate); +Gâ€žthe number (n) of glutamyl residues added to the folate analogue; KM,, the concentration of drug inhibiting enzyme activity 50%; H2PteGlu5, dihydropteroylpentaglutamate; HPLC, high-performance liquid chromatography; PteGlu5, pteroylpentaglutamate.

We report here on the inhibitory potency of polyglutamates of MTX and 10-DAM for DHFR derived from sheep, chicken, and beef liver using either dihydrofolate or H2PteGlu5 as sub strate. We show that the activity of mammalian DHFR can be influenced by the polyglutamate status of inhibitors and sub strates. MATERIALS

AND METHODS

NADPH was purchased from Sigma Chemical Co., St. Louis, MO. Tetrabutyl ammonium phosphate for HPLC was purchased from East man Kodak Co., Rochester, NY. Methanol and ammonium phosphate were obtained from Fisher Scientific Co., Fair Lawn, NJ. Dihydrofolate and H2PteGlu5 were prepared from folate and PteGlu5, respectively, by dithionite reduction (11) and stored in 1.0 M 2-mercaptoethanol at â€”20Â°C. Their authenticity and concentration were verified by UV spectroscopy in 0.2 M 2-mercaptoethanol, pH 7.5. The absorbance at 282 nni was used to calculate concentration using a molar absorbance coefficient of 19,000 (12). The dihydrofolate reductases were purified by affinity chromatogra phy and isoelectric focusing as described by Kaufman et al. (13, 14). All three enzymes were greater than 95% pure. Electrophoresis of 20Â¿tgsamples of each preparation on polyacrylamide gels containing sodium dodecyl sulfate (15) showed no contaminant bands when stained with Coomassie blue. Polyglutamyl derivatives of 10-DAM, MTX, and folate were synthe sized from the corresponding pteroate derivatives by the solid phase method (16) and purified by DEAE-celluIose chromatography. Their authenticity and purity were verified by UV spectroscopy in 0.1 N KOH, the absorbance at 256 nm being used to calculate concentrations using molar absorbance coefficients of 30,000 (2) and 23,000 (12) for 10DAM and MTX derivatives, respectively. HPLC was performed on dihydrofolate, H2PteGlu5, and on polyglu tamates of 10-DAM and MTX using an Altex Model 330 isocratic liquid Chromatograph with a 5-mm x 256- mm Altex Trasphere column. A solvent system containing 45% methanol, 0.018 M ammonium phos phate, and 0.005 M tetrabutyl ammonium phosphate was used for 10DAM and MTX derivatives. For dihydrofolate and H2PteGlu5, the methanol concentration was reduced to 40%. Fig. 1 shows the results of HPLC of 10-DAM and MTX derivatives. None of the compounds showed contamination. Due to differences between the sheep, chicken, and beef liver DHFRs it was necessary to determine assay conditions by preliminary experi ments which yield initial linear reaction rates which are maintained over the 5-min assay period. These conditions, which are different for each enzyme, are summarized in Table 1. Similar rates were obtained with either dihydrofolate or 11..Pti-( Â¡ Uu as substrates in all three systems (Table 2). The reactions were monitored in a Gilford Model 250 spectrophotometer by utilizing the decrease in absorbance at 340 nm that occurs when NADPH and dihydrofolate are converted to NADP+ and tetrahydrofolate. The reactions were initiated by addition of an amount of enzyme sufficient to obtain a change in absorbance of 0.027/ min at 30Â°C.The pH was verified for each reaction system at the end of the incubation period. Although the specific activities of the enzymes used in this study (Table 2) were lower than when freshly isolated, their activity remained constant within 15% during the course of this study. Reaction velocities obtained at various inhibitor concentrations were plotted to determine the IC50 values as illustrated for 10-DAM +G2 and sheep liver DHFR in Fig. 2. Protein was determined by the method of Lowry et al. (17).

5020




POLYGLUTAMATE

INTERACTION WITH DHFR

H2PteGlu5 as substrate, polyglutamylation of MTX results in a stepwise decrease in IC50 values. In most instances the decrease in ICso values with progressive polyglutamylation of MTX is greater with H2PteGlu5 as substrate. The enhanced activity of DHFR caused by increased ionic strength is presumed to be due to conformational changes which alter the rates of ligand binding and dissociation (22). In the present study we show that, in most instances, 0.2 M KC1 diminishes the differences in inhibitory potency between differ ent polyglutamate inhibitors. Thus the interaction of the polyglutamate tail with the enzyme is less influential in the presence of0.2MKCl. We conclude that polyglutamylation of MTX makes it a better inhibitor of sheep, chicken, and beef liver dihydrofolate reductases, whereas polyglutamylation of 10-DAM generally results in reduced inhibition. In addition, replacement of dihy drofolate with H2PteGlu5 generally results in reduced inhibition with MTX and 10-DAM and their polyglutamate forms. Thus the state of polyglutamylation of inhibitors and of the substrate alters DHFR inhibition and could be important in determining the level of DHFR inhibition in vivo. REFERENCES 1. Sirotnak, F. M., DeGraw, J. I., Chello, P. L., Moccio, D. M., and Dorick, D. M. Biochemical and pharmacologie properties of a new folate analog, 10deazaaminopterin, in mice. Cancer Treat. Rep., 66:351-358, 1982. 2. DeGraw, J. I., Kisliuk, R. L., Gaumont, Y., Baugh, C. M., and Nair, M. G. Synthesis and antifolate activity of 10-deazaaminopterin. J. Med. Chem., 17: 552-553, 1974. 3. Baugh, C. M., Krumdieck, C. L., and Nair, M. G. Polyglutamyl metabolites of methotrexate. Biochem. Biophys. Res. Commun., 52: 27-34, 1973. 4. Samuels, L. L., Moccio, D. M., and Sirotnak, F. M. Similar differential for total polyglutamylation and cytotoxicity among various folate analogues in human and murine tumor cells in vitro. Cancer Res., 45:1488-1495, 1985. 5. Galivan, J. H. Transport and metabolism of methotrexate in normal and resistant cultured rat hepatoma cells. Cancer Res., 39:735-743, 1979. 6. Kisliuk, R. L. The biochemistry of folates. In: F. M. Sirotnak, J. J. Burchall, W. B. Ensminger, and J. A. Montgomery (eds.), Folate Antagonists as Therapeutic Agents, Vol. 1, pp. 1-68. New York: Academic Press, Inc., 1984.

7. Whitehead, V. M. Synthesis of methotrexate polyglutamates in LI210 mu rine leukemia cells. Cancer Res., 37: 408-412, 1977. 8. Galivan, J. H. Evidence for the cytotoxic activity of polyglutamate derivatives of methotrexate. Mol. Pharmacol., 17: 105-110, 1980. 9. Matherly, L. H., Voss, M. K., Anderson, L. A., Fry, D. W., and Goldman, I. D. Enhanced polyglutamylation of aminopterin relative to methotrexate in the Ehrlich ascites tumor cell in vitro. Cancer Res., 45: 1073-1078, 1985. 10. Jacobs, S. A., AdamsÂ«m,R. H., Chabner, B. A., Derr, C. J., and Johns, D. G. Stoichiometric inhibition of mammalian dihydrofolate reducÃ-aseby the gamma-glutamyl metabolite of methotrexate. Biochem. Biophys. Res. Commun., 63:692-698, 1975. 11. Fried kin, M., Crawford, E. J., and Misra, D. Reduction of folate derivatives with dithionite in mercaptoethanol. Fed. Proc., 21:176, 1962. 12. Blakley, R. L. The Biochemistry of Folie Acid and Related Pteridines, p. 93. Amsterdam: North Holland, 1969. 13. Kaufman, B. T., and Kemmerer, V. F. Characterization of chicken liver dihydrofolate reducÃ-aseafter purification by affinity chromatography and isoelectric focusing. Arch. Biochem. Biophys., 779:420-431, 1977. 14. Kaufman, B. T., and Kemmerer, V. F. Purification and characterization of beef liver dihydrofolate reductase. Arch. Biochem. Biophys. /"_'.â€¢ 289r300, 1976. 15. Weber, K., and Osbom, M. J. Proteins and sodium dodecyl sulfate: molecular weight determination on polyacrylamide gels and related procedures. In: H. Neurath and R. L. Hill (eds.), The Proteins, Ed. 3, pp. 179-223. New York: Academic Press, 1975. 16. Nair, M. G., and Baugh, C. M. Synthesis and biological evaluation of polyglutamyl derivatives of methotrexate. Biochemistry, 12: 3293-3927, 1973. 17. Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol.Chem., Â¡93:265-275, 1951. 18. Kumar, A. A., Blankenship, D. T., Kaufman, B. T., and Freisheim, J. H. Primary structure of chicken liver dihydrofolate reductase. Biochemistry, 19: 667-678, 1980. 19. Peterson, D. L., Gleisner, J. M., and Blakley, R. L. Bovine liver dihydrofolate reductase: purification and properties of the enzyme. Biochemistry, 14:52615267, 1975. 20. Kisliuk, R. L., Gaumont, Y., Kumar, P., Courts, M., Nair, M. G., Nanavati, M. T., and Kaiman, T. I. The effect of polyglutamylation on the inhibitory activity of folate analogs. In: I. D. Goldman (ed.), Proceedings of the Second Workshop on Folyl and Antifolyl Polyglutamates, pp. 319-328. New York: Praeger, 1985. 21. Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., Kaufman, B. T., Beddell, C. R., Champness, J. N., Stammers, D. K., and Kraut, J. Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim. J. Biol. Chem., 260: 381-389, 1985. 22. Subramanian, S., Shindo, H., and Kaufman, B. T. Kinetic and chlorine-35 nuclear magnetic resonance studies of the effect of chloride on the properties of chicken liver dihydrofolate reductase. Biochemistry, 20:3226-3230,1981.

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