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Inhibition of Phosphoribosylaminoimidazolecarboxamide Transformylase by Methotrexate and Dihydrofolic

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Inhibition of Phosphoribosylaminoimidazolecarboxamide Transformylase by Methotrexate and Dihydrofolic Proc. Nati. Acad. Sci. USA Vol. 82, pp. 4881-4885, August 1985 Biochemistry Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates (antimetabolites/breast cancer/purine synthesis/folic acid/enzyme kinetics) CARMEN J. ALLEGRA, JAMES C. DRAKE, JACQUES JOLIVET, AND BRUCE A. CHABNER Cliqical Pharmacology Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD 20205 Communicated by DeWitt Stetten, Jr., March 13, 1985 ABSTRACT We report the enhanced inhibitory potency of describes the 2500-fold enhanced capacity of MTX methotrexate (MTX) polyglutamates and dihydrofolate polyglutamate to inhibit 10-formyltetrahydrofolate:5'-phos- pentaglutamate on the catalytic activity of phosphoribosylami- phoribosyl-5-amino-4-imidazolecarboxamide formyl- noimidazolecarboxamide (AICAR) transformylase purified transferase [5-amino-4-imidazolecarboxamide ribotide from MCF-7 human breast cancer cells. In the present work, (AICAR) transformylase, EC 2.1.2.3; AICAR TFase], a MTX (4-amino-10-methylpteroylglutamic acid) and folate-requiring enzyme that catalyzes the reaction: 10- dihydrofolate, both monoglutamates, were found to be weak formyl-tetrahydrofolate (10-formyl-H4PteGlu) + AICAR, competitive inhibitors ofAICAR transformylase with Kis of 143 yielding 5'-phosphoribosyl-5-formamido-4-imidazole-car- and 63 ,uM, respectively, and their inhibitory capacity was boxamide (formyl-AICAR), an intermediate in the de novo largely unaffected by the glutamated state of the folate purine biosynthetic pathway, and tetrahydrofolic acid cosubstrate. In contrast, MTX polyglutamates were found to (H4PteGlu). We also report that H2PteGlu5, which increases be potent competitive inhibitors, with an =10-fold increase in in the cell following inhibition of H2PteGlu reductase (11), inhibitory potency with the addition of each glutamate group potently inhibits AICAR TFase. These findings disclose a up to four (i.e., the pentaglutamate derivative). MTX tetra- new site of action of this commonly used agent. and pentaglutamates were the most potent, with equivalent Kis of 5.6 x 10-8 M or 2500-fold more potent than MTX. MATERIALS AND METHODS Dihydrofolate pentaglutamate was as potent an inhibitor as Chemicals. MTX was obtained from the Drug Synthesis MTX pentaglutamate, with a Ki of 4.3 x 10-8 M. The potent and Chemistry Branch, National Cancer Institute (Bethesda, inhibitory effects demonstrated by the polyglutamate com- MD) and purified by DEAE-cellulose chromatography with pounds when tested against the folate monoglutamate substrate elution along a linear gradient of 0.1-0.4 M NH4HCO3. were sharply curtailed when folate pentaglutamate was used as Purified synthetic MTX polyglutamates (Glu2.5) and purified the substrate. MTX and dihydrofolate pentaglutamates were folate pentaglutamate (PteGlu5), provided by John Montgom- only 7- and 25-fold more potent than their monoglutamate ery (Southern Research Institute, Birmingham, AL) and counterparts under these conditions. A model depicting these C. M. Baugh (Department of Biochemistry, University of complex interactions is postulated. These findings have signif- South Alabama, Mobile, AL), gave single peaks on HPLC icant implications regarding the mechanism of action of MTX. (12). AICAR, 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide, 'y-L-glutamyl-L-glutamic acid (Glu-Glu), and Methotrexate (MTX, 4-amino-10-methylpteroylglutamic ac- H2PteGlu were purchased from Sigma, and H2PteGlu5 was id), a widely used antineoplastic agent and folate reduced from PteGlu5 as described (13, 14) and purified by [pteroylglutamic acid (PteGlu)] analog, is felt to produce recrystallization. Affi-Gel Blue was purchased from Bio-Rad. cytotoxicity through the inhibition of dihydrofolate All other chemicals were of reagent grade and purchased (H2PteGlu) reductase (5,6,7,8-tetrahydrofolate:NADP+ oxi- from Sigma. doreductase, EC 1.5.1.3; often abbreviated DHFR) (1), thus Preparation of Reduced Folates. Pure, biologically active causing a depletion of reduced folate cofactors required for l-L-10-formyl-H4PteGlu and -Glu5 were prepared by enzy- synthesis of purines and thymidylate. MTX, a monoglutam- matic reduction of H2PteGlu or -Glu5 to H4PteGlu or -Glu5 ate, has been shown to undergo intracellular polyglutamation (15). For the enzymatic reduction, H2PteGlu or -Glu5 (50 mg) in a manner similar to the naturally occurring folates (2), and and NADPH (125 mg) in 20 ml 0.05 M Tris HCl buffer (pH the polyglutamated forms of MTX have been detected in a 7.4) were incubated at 370C with partially purified number of neoplastic and, to a lesser extent, normal tissues Lactobacterium casei H2PteGlu reductase (New England following MTX exposure (3-9). While retaining a potent Enzyme Center, Boston). The reaction was followed inhibitory effect on H2PteGlu reductase, the polyglutamated spectrophotometrically at 340 nm until no additional NADPH forms of MTX differ from parent MTX in that they possess was metabolized. The H4PteGlu and -Glu5 thus formed were a more prolonged intracellular half-life resulting from a elution from a slower efflux rate from cells (10). The selective retention of purified by DEAE-cellulose column by using the MTX polyglutamates results in prolonged antimetabolic a linear gradient of ammonium acetate (pH 6.0) from 0.01 to effects after the removal of extracellular drug. 1.5 M in the presence of 1% 2-mercaptoethanol at 40C (15, We have investigated the possibility that polyglutamation 16). Fractions were examined spectrally, and those contain- of MTX may increase its ability to inhibit other folate- ing H4PteGlu, were pooled and lyophilized overnight. 5- dependent enzymes, particularly those that have a higher Formyl-H4PteGlu, was prepared from the purified H4PteGlu, affinity for polyglutamated folate substrates. This report Abbreviations: MTX, methotrexate; H2PteGlu,, series of dihydrofolic acid (H2PteGlu) polyglutamates; H4PteGlu", series of The publication costs of this article were defrayed in part by page charge tetrahydrofolic acid (H4PteGlu) polyglutamates; AICAR, 5-amino- payment. This article must therefore be hereby marked "advertisement" 4-imidazolecarboxamide ribotide: AICAR TFase, AICAR in accordance with 18 U.S.C. §1734 solely to indicate this fact. transformylase; Glu-Glu, 'y-L-glutamyl-L-glutamic acid. 4881 Downloaded by guest on October 1, 2021 4882 Biochemistry: Allegra et al. Proc. Natl. Acad. Sci. USA 82 (1985) by the method of Moran using formic acid in the presence of Gel Electrophoresis. NaDodSO4/gel electrophoresis on the water-soluble carbodiimide bridging reagent 1-ethyl-3-(3- 10% gels was by the methods of Weber et al. (21) using dimethylaminopropyl)carbodiimide (16). The 5-formyl- myosin (heavy chain) (Mr 200,000), phosphorylase b (Mr H4PteGlu, was purified by elution from a DEAE column with 92,500), bovine serum albumin (Mr 68,000), ovalbumin (Mr a linear gradient of ammonium acetate (pH 6.0) from 0.01 to 43,000), and a-chymotrypsinogen (Mr 25,700) as molecular 1.5 M. Fractions were examined spectrally, and those con- weight standards. taining 5-fortnyl-H4PteGlun were pooled, lyophilized, and AICAR TFase Assay. AICAR TFase activity was measured stored at -40'C. spectrophotometrically by using an assay developed by 10-Formyl-H4PteGlun was formed from 5-formyl- Black et al. (22). The reaction cuvettes contained 50 mM H4PteGlun prior to each experiment according to the method Tris HCl (pH 7.4), 25 mM KCl, 50 mM 2-mercaptoethanol, of Rabinowitz (17) by first acidifying the 5-formyl-H4PteGlun purified enzyme, 50 uM AICAR, and various amounts of to pH 1.5 with 0.1 M HCl and allowing the 5-10-methenyl- 10-formyl-H4PteGlu or -Glu5. Reactions were initiated with H4PteGlun to form over 1 hr at room temperature. 10-Formyl- the addition of AICAR. Reactions were carried out at 37'C, H4PteGlun was then formed by normalizing the pH with 0.1 and 10 min were allowed for temperature equilibration and M KOH. During each experiment, the 10-formyl-H4PteGlun blank rate calculation (prior to the addition of AICAR). was kept in 1% 2-mercaptoethanol in the dark at 40C. The Reported rates are reaction rates minus blank rates (consis- concentration of the 10-formyl-H4PteGlun was determined tently <5% of reaction rate). Reaction rates were monitored spectrophotometrically by using an extinction coefficient of with a Beckman Acta III recording spectrophotometer 22,000 cm-l m-1 at 258 nm at pH 7.0 (18). Concentrations of equipped with a constant-temperature, water-jacketed sam- 10-formyl-H4PteGlun were corroborated by spectral mea- ple compartment. Reaction velocities were initially linear surement of the intermediate 5-10-methenyl-H4PteGlun just with respect to time and protein concentration. Reaction prior to its conversion to the final product by using an velocities, AA/min, were converted to ntnol of H4PteGlu, extinction coefficient of 11,800 cm-1lm-l at 350 nm at pH 2.0 formed per min/mg using the extinction coefficient for the (19) and assuming 100% conversion to 10-formyl-H4PteGlul. reaction of 19.7 x 103 cm-1 m-1 at 298 nm (22). Concentrations derived by the two measurements differed by Data Analysis. The data were first analyzed by conven- <5%. Stability of the folate cosubstrates during the experi- tional double-reciprocal plots, and the graphic estimates of mental period was verified by repeated spectral analyses and parameters were used as initial estimates for computerized by kinetic analysis with the folates (i.e., determination of Km)
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