agents, is believed to exert its antitumor effects by virtue of its tight-binding inhibition of dihydrofolate reductase (EC. 1.5.1.3) (2). Although the parent compound ...
THEJOURNAL
OF
BIOLOGICAL CHEMISTRY
Vol. 260, No. 17, Issue of August 15, pp. 9720-9726, 1985 Printed in U.S.A.
Enhanced Inhibitionof Thymidylate Synthase by Methotrexate Polyglutamates* (Received for publication, August 3, 1984)
Carmen J. AllegraSj, Bruce A. ChabnerS, James C. Drake$, Rudolf Lutzll, David Rodbardll, and Jacques Jolivet 11 From the $Clinical Pharmacology Branch, Division of Cancer Treatment, National Cancer Institute and the Wuboratoryof Theoretical and Physical Biology, National Institute of Child Health and Human Development, Bethesda, Maryland 20205 and IIInstitut d u Cancer de Montreal and Hospital Notre-Dame, Montreal H2L 4M1, Canada
We have studied the effects of methotrexate (MTXMethotrexate (MTX-Glul’; 4-NHz-10-CH3pteroyl glutaGlul) and the polyglutamate derivatives of methotrex- mate), one of the most widely usedand effective antineoplastic ate (MTXPGs) with 2,3,4, and5 glutamyl residues on agents, is believed to exert its antitumor effects by virtue of the catalytic activityof thymidylate synthase purified its tight-binding inhibition of dihydrofolate reductase (EC from MCF-7 human breast cancer cells and on the 1.5.1.3) (2). Although the parentcompound has potent inhibkinetics of the ternarycomplex formation by 5-fluoro- itory activity in its own right, itis now appreciated that 2’-deoxyuridine5’-monophosphate,folatecofactor, MTX-Glul may undergo metabolism to polyglutamate derivandthymidylatesynthase. MTX-Glul exhibitedunatives (MTXPGs) in a manner similar to thepolyglutamation competitive inhibition of thymidylate synthase when of physiologic folates. These MTXPGs, whichbecome the reaction kinetics were analyzed by either double reciprocalplots or a computerizedmathematical model predominant form of intracellular drug in some malignant Ki cells and to a lesser extent in selected normal tissues, are as based on nonlinear least-squares curve fitting. The for MTX-Glu, inhibition was 13 PM and theIS0was 22 potent as MTX-Glulin inhibiting dihydrofolate reductase but p ~ irrespective , of the degree of polyglutamation of have other properties not found in the parent compound (3the folate. In contrast, the polyglutamated derivatives 17). The MTXPGs, particularly those with three or more glutamyl groups, have a much slower effluxrate from cells, as ofMTX all acted as noncompetitive inhibitors. The MTXPGs had 75-300-fold greater potency than MTX- compared to the efflux of MTX-Glul,andare selectively retained by cells following the removal of extracellular drug, Glul as inhibitors of thymidylate synthase catalytic activity, with Ki values from 0.17 to 0.047 p~ for thereby prolonging the duration of inhibition of DNA syntheMTX-Glu2 to MTX-Glu6, respectively. Neither MTX- sis (17). In addition, in studies with intact tumor cells, the Glu, nor MTXPGs promoted the formation of a char- MTXPGs appeared to have slower rates of dissociation from coal-stable ternary complex with thymidylate synthasedihydrofolate reductase than does MTX-Glul, the half-life for and 5-fluro-Zf-deoxyuridine 5‘-monophosphate. CH2- MTX-Glu5dissociation being 120min as compared to 16 min H4PteGlu6 (where PteGlu represents pteroylglutamic for MTX-Glul (18). acid) was found to be 40-fold more potent than CH2In the present work, we have investigated an additional H4PteGlul in participating in the formation of a ter- aspect of MTXPG action-the possibility that these derivanary complex, and 10 p~ MTX-Glus significantly in- tives have sites of inhibition in addition to dihydrofolate hibited the formationof a ternary complex containing reductase. This possibility is suggested by evidence that polthis folate as cofactor. The inhibition was determined yglutamation of physiologic folates greatly enhances their to be due toa reduction in the k,,,,.The potency of this affinity for a number of folate-requiring enzymes (19). For inhibition was markedly greater in the presence of example, pentaglutamated folate substrates have 30-fold inCH2-H4PteGlul as compared to CH2-H4PteGlu6. This finding suggests that the degree of interference with creased affinity for methylenetetrahydrofolate reductase and complex formation in intact cells would depend on the a 250-fold enhanced affinity for AICAR transformylase, as state of polyglutamation of available folate cofactor. compared to thecorresponding monoglutamate substrates (20, Ternary complex formation withH2PteGlu5 as the fo- 21). Thymidylate synthase from human leukemic cells has a late cofactor was also investigated, anda 50%reduc- 15-foldincreased affinity for the triglutamated substrate5,lOcompared to tion in complex formation was found in the presence methylene tetrahydrofolate (CHz-H4PteGlu3) as of a 2 p~ concentration of MTX-Glu6. These findings its affinity for the monoglutamate (21). While MTX-Glul is a weak inhibitor of thymidylate synthase, with a Kiof 3 X have significant implications regarding mechanism the M, preliminary observations suggest that MTX-Glu3 and of action of MTX-Glul and contribute to an understanding of the complex interactions of MTX-Glul and MTX-Glu7 have at least 200-fold greater affinity for this enzyme (23). In the present study, we have examined the 5-fluorouracil. parent compound and its polyglutamate derivatives as inhib-
* 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. § T o whom correspondence should be addressed Building 10, Room 12N226,National Cancer Institute, Bethesda, MD20205.
The abbreviations used are: MTX, methotrexate (as defined at the first Symposium on Folyl and Antifolyl Polyglutamates (1);the total number of glutamyl moieties appended to the pteroyl ring of MTX is denoted by the suffix -Glu,); MTXPG, polyglutamate derivatives of MTX;FdUMP, 5-fluoro-2’-deoxyuridine5’-monophosphate; 5-FU, 5-fluorouracil; PteGlu, pteroylglutamic acid; AICAR, 5-amino-l-(5-O-phosphoro-~-~-ribofuranosyl)-lH-imidazole-4-carboxamide.
9720
Inhibition Enhanced
of Thymidylate Synthase
itors of the catalytic activityof this enzyme, and as inhibitors of the formation of a ternary complex between thymidylate synthase, CHz-H,PteGlu, and 5-FdUMP (the inhibitory nucleotide product of 5-fluorouracil). These studies provide clear evidence for a potent inhibition of thymidylate synthase catalytic activity andcomplex formation by the MTXPGs. MATERIALS ANDMETHODS
Chemicals-Dextran (clinical grade), dUMP, bovine albumin fraction V, and acid-washed activated charcoal were purchased from Sigma. [5-3H]dUMP (14.8 Ci/mmol) was obtained from Amersham Corp., and [6-3H]5-FdUMP (19 Ci/mmol) from Moravek Biochemicals Co. (Brea, CA). Methotrexate was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD) and purified by DEAE-cellulose chromatography with elution along a linear gradient of0.1-0.4 NH4HC03 (24). Purified synthetic MTX-GIU~.~ and purified PteGlu, were provided by Dr. John Montgomery (Southern Research Institute, Birmingham, AL) and Dr. C. M. Baugh (Department of Biochemistry, University of South Alabama, Mobile, AL), and gave single peaks on high-pressure liquid chromatography (25). dl-L-Tetrahydrofolate-Glul (H4PteGlul) was purchased from Sigma, and HzPteGlu, was reduced fromPteGlu, as previously described (26, 27). dl-~-5,10-Methylenetetrahydrofolate-Glul (CHz-H4PteGlul)was prepared prior to each experiment by adding 50 p1 of 1M ascorbate, pH 6.5,5pl of 37% (v/v) formaldehyde, and 10 ml of a buffer solution (0.5 ml of 1 M KHzP04,pH 7.2, 70 pl of 2-mercaptoethanol type I, 10 mg of bovine albumin, and 9.5 ml of water) to 1.8 mg of H4PteGlul. Experimentalcalculations were based on the assumption that the biologically active d-L-methylene tetrahydrofolate constituted 50% of the racemic mixture. d-I,-Methylene tetrahydrofolate-Glu, (CHZ-H4PteGlu5)was prepared by chemical reduction of PteGluo to HzPteGlu6 as previously described (26, 27) followedby enzymatic reduction to H4PteGlu5. For the enzymatic reduction, a mixture of HZPteGlu5(50 mg) and NADPH (125 mg) in 20 ml of Tris/HCl buffer, 0.05 M, pH 7.4, wasincubated at 37 "C with lhctobacillw casei dihydrofolate reductase (New England Enzyme Center, Boston, MA). The reaction was followed spectrophotometrically at 340 nm until no additional NADPH was metabolized. The H4PteGlusthus formed was purified by elution from a DEAE-cellulose column using a linear gradient of ammonium acetate, pH 6.0, from 0.01 to 1.5 M, in the presence of 1%2-mercaptoethanol, and was converted to CHz-H4PteGlu6prior to each experiment as described above for the synthesis of the monoglutamate. The time required for the formation of CH2-H4PteGlul or -Glu5 from its corresponding H4PteGlul or -Gh5 was determined by measuring the ability of the product to form a stable ternary complex with FdUMP and thymidylate synthase (see below). The rateof formation of CH*-H,PteGlu, was found to be time- and temperature-dependent, reaching maximum product formation in 4 h at 4 "C and 1.5 h at 21 "C. All other chemicals were of reagent grade and were purchased from Sigma. Enzyme Source-A human breast cancer cell line, MCF-7, was used as the source of thymidylate synthase. The characteristics of this cell line have been previously described (28). The cells, grown in continuous monolayer culture by HEM Laboratories (Rockville,MD) and supplied in aliquots containing 5-7 g of packed cells, were stored at -40 "C. Thymidylate synthase was purified 2000-fold to a specific activity of 1.5 units/mg of protein (a unitof activity being defined as the amount of enzyme required to form 1 nM TMP/min at 37 "C) according to themethod of Dolnick and Cheng (29). Thymidylate Synthase Assay-Thymidylate synthase was assayed by a modification of the tritium release procedure of Roberts (30). The assay was performed in a total volume of200 pl containing varying concentrations of CHz-H4PteGlulor -Glu5, 1 x M [5-3H] dUMP (specific activity, 14.8 Ci/mmol), 100 mM 2-mercaptoethanol, and 50 mM KHzP04,pH 7.2; the reaction was initiated by adding 20 p1 (0.03 unit) of purified enzyme. The sample was then incubated for up to 30 min at 37 "C, and the reaction was stopped by adding 100 pl of ice-cold 20% trichloroacetic acid. Residual [5-3H]dUMP was removed by adding 200 pl of an albumin-coated charcoal slurry, pH 7.2 (prepared by mixing 10 g of acid-washed activated charcoal with 2.5 g of bovine albumin, 0.25 g of dextran, and 100 ml of ice-cold water). The suspension was allowedto stand at room temperature for 10 min, and the charcoal was sedimented by centrifugation at 10,000 X g for 30 min. A 250-pl sample of the supernatantwas then assayed for [3H] Hz0 radioactivity. All assays were performed in duplicate. Reaction velocities were found to be linear for 10 rnin with CHZ-HdPteGIu5and
9721
for 30 min with CHZ-H4PteGlulas substrate. Thymidylate Synthase-FdUMP-Folate Complex Formation-The formation of a ternary complex consisting of thymidylate synthasefolate-5-FdUMP andthe effects of MTX-Glul and MTX-Glu6 on this process were studied using a binding assay described by Moran et al. (31). The assay was performed in a total volume of 200 p1 containing 1 p~ CHz-H4PteGlueor 75 p~ CH2-H4PteG1uIor 10 p~ HzPteGlu6, 3.0 pmol of [6-3H]5-FdUMP (specific activity, 19 Ci/mmol), and 50 mM KHzP04,pH 7.2. The binding was initiated by adding 20 p1 (0.01 unit) of the purified MCF-7 enzyme. Samples were incubated at 37 "C for various time intervals, following which 1 ml of a 4:l dilution of water to albumin-coated charcoal slurry (prepared as above) was added; the mixture was allowed to stand atroom temperature for 10 min and centrifuged at 10,000 X g for 30 min. An 800-pl sample of the supernatant containing the enzyme-bound [6-3H]5-FdUMPwas then assayed for radioactivity. All assays were performed in duplicate. Dissociation of the ternary complex was measured by allowing the complex to form over a 30-min incubation period using the same conditions listed above for the association experiments; 10" M unlabeled FdUMP was then added to the solution which contained 0.2 pmol of complex in 50 mM K H z P O buffer, ~ pH 7.2, in a total volume of200 pl, and dissociation of the preformed complexwas then monitored at intervals over a period of 90 min at 37 "C. At designated times, 1 mlof ice-cold charcoal slurry was added and, after centrifugation, 800 pl of the supernatant was assayed for radioactivity as above. Association and dissociation experiments were performed in the absence or presence of MTX or MTXPGs. DATA ANALYSIS
Enzyme Kinetics-Data were first analyzed by conventional double reciprocal and Dixon plots. The graphical estimates of parameters were then used as initial estimatesfor refinement by curve fitting, using an integrated nonlinear least-squares method. We have developed a new program, designated"ENZYME," which employs the BASIC language on the DEC-10 computer to perform curve fitting by a Marquart-Levenberg algorithm (32). The program permits testing and selection of any of nine kinetic models (331, including Michaelian (for estimates of K,,, and V,, in the absence of inhibitor) kinetics and inhibition by substrate or by a non-substrate inhibitor displaying competitive, uncompetitive, or noncompetitive kinetics. Preliminaryanalysis of theerrorstructure of the experimental data indicated a constant percentage error (less than 10%) superimposed on a small constant error, and this relationship was used as a basis for weighting (34). Selection among models involvingdifferent numbersof parameters was made by an F-testbased on the extra-sum-of-squares principle (35). Selectionamong models withthe same number of parameters was based on the root mean square error, on tests for randomness of residuals and on demonstration of reproducibilitybetween independentexperiments.Plots of velocity versus log inhibitor concentrations were sigmoidal, and Is, values were calculated by the program ALLFIT (36), using weighted nonlinearleast-squares curve fitting.Multiple curves were fittedsimultaneously with appropriateconstraints. Kinetics of Ternary Compkx Formation-Dissociation curves were analyzed by the program EXPFIT (method A), allowing useof weighted nonlinear least-squarescurve fitting were also similar to thatof ALLFIT.' Dissociation rates (kff) calculated according to the following equation (method B):
where [X] = ternary complex concentration at time t (min) and [X,] = concentration of preformed complex at time 0 (38). Results from both methods were essentially identical.
v. Guardabaso, P. J. Munson, and D. Rodbard, personal communication.
Enhanced Inhibition of Thymidylate Synthase
9722
Association curves were also analyzed by two methods, both yielding compatible results. Results were first analyzed (method C)assuming irreversible second-order kinetics using the linear relationship:
where [Eo] = initial concentration of enzyme binding sites, t = time (min), [FdUMPo] = initial concentration of tritiated FdUMP, and [X]= concentration of ternary complex at time t (38).[Eo]was calculated on thebasis of each mole ofenzyme binding 1.7 mol of[3H]FdUMPat equilibrium in thepresence of saturating folate and [3H]FdUMP concentrations(38, 39). Results were also analyzed (method D) using pseudo-firstorder kinetics, which are appropriate in view of the ratio of [FdUMPo]/[Eo] > 10. This provided an improved estimate of the initial velocity ( Vo),an estimate of [FdUMP] bound a t equilibrium ([FdUMP,]), andanapparentrateconstant (P P P ) according to thefunction: [FdUMP] = [FdUMP,] (1 - e-wq)
(3)
where [FdUMP] = concentration of FdUMP at time t. When [FdUMPoI >> [Eo], Pm = & [FdUMPo] - k,n. (4) The latterrelationship permitscalculating the relative change in Vo and k,, (provided [E0] is constant) in the presence and absence of inhibitor. This estimate of the relative change in k,, makes the fewest assumptions since it does not require an estimate of [Eo] when k.,~is directly available. Dissociation constants ( K D )were calculated as thequotient of kff/kOn. RESULTS
Characteristics of MCF-7 Thymidylate Synthase-We first determined the kinetic properties of thymidylate synthase purified from MCF-7 human breast cancer cell extracts. The Michaelis constant (K,) was 22.6 & 4.5 pM with CH2H,PteGlul as substrate, with a Vmx of 1.68 -+ 0.18 nmol of TMP formed per mg/min; the K , decreased to 0.63 k 0.15 FM for CHp-H4PteGlu5with a V,, of2.40 f 0.16 nmol of TMP formed per mg/min. The uninhibited reaction followed Michaelian kinetics and was convertible to linear plots over thesubstrate concentration range used inthis study. At higher concentrations of CH2-H4PteGlulor (>3 x M), a marked decrease in reaction velocity occurred, and this was likely the result of inhibition by the reaction product dihydrofolate, a known potent inhibitor of the reaction (40). MTXPGs as Inhibitors of MCF-7 Thymidylate SynthuseWe studied the effects of MTX-Glul and MTXPGs having two to five glutamyl groups as direct inhibitorsof thymidylate synthase. Inhibition constantsof each MTX metabolite were determined at a constant dUMP concentrationof 1 X lo-' M and at variable concentrations of the mono- or pentaglutamated folate (Table I).While MTX-Glul was a weak inhibitor of thymidylate synthase (Ki= 13 p ~ ) the , addition of 1 glutamyl residue led to a 76-fold increase in the inhibitory capacity of the drug(MTX-Glu2 Ki = 0.17 p M ) . Further additions of glutamyl residues ( M T X - G ~ U ~led - ~to ) further decreases in the observed Ki values. MTX-Glu5 exhibited the most potent inhibition (Ki = 0.047 p ~ ) Inhibition . constants for the MTXPGs in the presence of the CH2-H4PteGlulwere identical to the constant in the presence of CH2-H4PteGlu6. Also illustrated in Table I are the IWvalues for each of the inhibitors. These values parallel and substantiate the Ki determinations. Representative experiments that examine inhibition of thymidylate synthase by MTX-Glul and MTX-
Glus in competition with the monoglutamated folate are illustrated in Fig. 1. MTX-Glu, was found to be an uncompetitive inhibitor of thymidylate synthase with either CH2-H4PteGlu, or CH2-H4PteGlusas co-substrate. The marked increase in inhibitory potency of MTXPGs was accompanied by a change in the inhibition pattern, which became noncompetitive for MTX-Glu2through -Glus. This change in patternof inhibition from uncompetitive to noncompetitive occurred with either the mono- or pentaglutamated folate as co-substrate. The change in pattern of inhibition from uncompetitive to noncompetitive is further verified by an analysis of the relationship of Ki to Iw. Accordingto Cheng and Prusoff (41), the Ki should equal the 1, if the inhibitor follows noncompetitive kinetics, and this relationship is apparent from Table I. In contrast, the 160 for MTX-Glu1 is approximately 2-fold higher than its Ki. This difference is consistent with uncompetitive kinetics wherein the IWshould equal Ki (1 K,/[S]). In these experiments, the substrate concentration ([SI)was approximately equal to the K,. These consistencies further support the choice of kinetic models for each of the inhibitors. Influence of MTXPGs on FdUMP Binding to Thymidylate Synthase-Methotrexate and 5-fluorouracil (5-FU) are frequently used together in combination chemotherapy and have complex interactions. The 5-FU metabolite FdUMP forms a covalent complex with thymidylate synthase in the presence of CH2-H4PteGlu,, butthis cofactor maybe depleted by MTX-Glul inhibition of dihydrofolate reductase. We examined the direct effect of MTX-Glul and its polyglutamate derivatives on the ternary complex formed by thymidylate synthase, folate, and FdUMP. To determine the concentrations of CH2-H4PteGlulor -Glus required to achieve maximal formation of the complex of enzyme-folate-FdUMP, we defined dose-response curves for complex formation as a function of folate concentration. One-half maximal complex formation occurred at a concentrationof 2.8 PM CH2-H4PteGlul or 6.5 X lo-' M CH2-H4PteGlu5 (Fig.2). In view of the potent direct inhibition of thymidylate synthase catalytic activity by MTXPGs, we examined the effect of MTX-Glul and MTX-Glu5 on the binding of 5-FdUMP to thymidylate synthase.NeitherMTX-Glul nor MTX-GIu5 could form a charcoal-stable complex with the enzyme and 5FdUMP when drug concentrations up to M were incubated with 1.2 X lo-' M FdUMP and 0.23 pmol of purified MCF-7 thymidylate synthase, as determined by the absence of protein-bound radioactivity in the supernatant after charcoal treatment. MTX-Glul did not inhibit the formation of complex by thymidylate synthase, CH2-H4PteGlul, and FdUMP, even a t high ( M) drug concentrations. However, MTX-Glu5 retarded complex formation (Fig. 3). The k,for FdUMP binding to thymidylate synthase in the presence of CH2-H,PteGlul and in the absence of MTXPGs was 16.7 X lo6 M-' min" as compared to a k,, of 10.0 X lo6 M-' min" in the presence of both folate and 10 PM MTX-Gl%. As shown in Fig.3,MTX-Glu6 inhibited the initial rate of complex formation (first 5 min) in a noncompetitive fashion with 160 of20 p ~ Despite . the reduction in association rate induced by MTX-Glu5, the total amount of complex formed beyond 30 min of incubation with MTX-Glu5 was identical to control incubations performed in the absence of MTX-Glu5 (Fig. 4). To further define the effect of MTX-Glul and MTXPGs on the ternary complex, we examined the kinetics of 5-FdUMPthymidylate synthase-CH2-H4PteG1u5dissociation in the presence and absence of MTX-Glu, and MTX-Glu5 (Fig. 5). The dissociation of complex in the absence of MTX-Glu5 was found to have a t%of 161 min and a k,n of 0.43 X lo-' rnin". As shown in Table 11, the dissociation rate of preformed
+
9723
Enhanced Inhibitionof Thymidylate Synthase
. . ~ ~
using computerized nonlinear least-squares curve fitting as-described under ‘‘Data Analysis”. Folate cofactor
CHn-H4PteClul CH2-H,PteG1u6
K,
KP
KbL
V,.
@M
nnol
IrM
22.6 f 4.5‘ 0.63 2.40? 0.15
1.68 2 0.18 14.3 2 0.16
13.0 f 1.4
K; MTX-Glul ~
MTX-Glul
Glu,
Gllh
”“
Glu.
Glun
K, MTX-Glu,
0.13 f 0.006
0.047 i 0.002 255 f 0.004
277
ZM
0.17 f 0.01 0.14
_t
0.007
0.056f 3.6
Iso MTX-Glu, Im M T X - G I s
L d
PM
27.7
CHt-H,PteGlul 22.2 f 4.8 0.15 f 0.07 0.15 k 0.02 CH2-H.PteGlua 0.059 f 3.7 Based on uncompetitive model. Based on noncompetitive model. e S.E. Determined by computer-assisted analysis (see “Data Analysis”).
I
0
0.005
l
l
0.015
1
0.13 f 0.02
470
0.054 f 0.006 f 0.005
411
1
0.025
1/s O !o-4 MTXGLU.Zx10”M
MTX GLU, 1.5x10”M
mo
MTX GLU. 1 x 10-7 M
10-6
10-1 FOLATE /M)
-
3
10-7
10-8
FIG. 2. Formation of ternary complex with L3H]FdUMP, thymidylate synthase, and either CHz-H4PteGlul(0) or CHzH4PteGlu6(0). For each data point, 3 pmol of [6-3H]5-FdUMP,0.01 unit of purified MCF-7 thymidylate synthase (specific activity, 1.5 units/mg of protein), 50 mM KHzP04,pH 7.2, and the indicated concentrations of either CH2-H4PteGlulor -Glu5 were incubated for 1 h at 37 “C. The amount of pentaglutamated folate (6.5 X lo-’ M) required for one-half enzyme saturation was40-foldless than for monoglutamate (2.8 X M).
glutamated folate (I50= 4 pM at 5 min). Dissociation rates of the ternary complex incorporating the monoglutamated folate also were not affected by the presence of MTX-Glu5 (Fig. 5 ) . The KD values for the binding of FdUMP to thymidylate synthase in the presence of either the mono- or pentaglutamated folate were calculated from the ratio of the on and off 1IS 11. This binding affinity was FIG. 1. Representative double reciprocal plotsof thymidyl- rates and are presented in Table ate synthase inhibition by MTX-Glul (panel A ) a n d MTX-Glu5 10-fold tighter in the presence of the pentaglutamate folate (panel B ) . Thymidylate synthase activity was assayed in the presas compared to monoglutamate folate and was markedlydeence of variable CHz-H4PteGlul concentrationswith no inhibitor and creased by the inhibitor. wit,h four concentrations each of MTX-Glul @anel A ) and MTXWe next studied the effect of MTX-Glu5 on 5-FdUMPG l u ~(panel B). Similar plots forMTX-Glu,.,were qualitatively the presence of identical to that for MTX-GluS, illustrating noncompetitive inhibi- thymidylate synthase complex formation in tion. V , moles of TMP formed per minute X lo6;S , concentration of HpPteGlu5as the folate cofactor. This compound, which acCH2-H4PteGlul X lo6. cumulates in the presenceof dihydrofolate reductase inhibition (42), has previously been shown to inhibit thymidylate complexes was not affected by the presence of MTX-Glu, synthase catalytic activity directly (K, = 6 p ~ ( 2) 2 ) a n d t o (tl,z= 151 min)inconcentrationsupto 1X M. Identical participate in ternary complex formation with thymidylate as the synthaseandFdUMP (43). MTX-Glu5 ( 2 X experimentswereperformedusingCH2-H4PteGlu, M ) noncomfolate cofactor and qualitatively similar results were obtained, petitively slowed the initial rate of FdUMP-H,PteGlu,-thyalbeit the complex association rate was slower (ken = 6.9 X midylate synthase complex formation by 50% when incubated IO6M” rnin”) and the abilityof MTX-Glu, to interfere with simultaneously with 10 p~ H2PteGlu5 for 5 min. However, complex formation was more marked than with the pentaultimately the amount of complex formed in the presence of NO INHIBITOR
Enhanced Inhibition of Thymidylate Synthase
20
40
60
MINUTES
FIG. 3. Inhibition of FdUMP-CHz-H4PteGlua-thymidylate I I I I 1 synthase complex formation by 10 WM MTX-Glu,. Purified 125 75 loo 25 50 MCF-7 thymidylate synthase was incubated at 37 “C with 1.2 X lo-’ INCUBATION TIME (MINUTES) M [6-3H]5-FdUMP,1 FM CHn-H4PteGluS with10 PM (0)or without (0)MTX-Glu5 in 50 mM KHzP04, pH 7.2. At the end of various FIG. 5. Influence of MTX-Glu, on the dissociation of periods of incubation, the amount of complex formed wasdetermined FdUMP-CHz-H4PteGlu,-thymidylatesynthase or FdUMPas indicated under “Materials and Methods.” Complex formation was CH~-H4PteGlul-thymidylate synthase complex. FdUMP-CH,initiatedwiththeaddition of enzyme. Bothsets of points were H4PteGlue or -Gh-thymidylate synthase complex was preformed by determined simultaneously. a 30-min incubation at 37 “C at pH 7.2 with 3.0 pmol of [6-3H]5FdUMP, 38 FM CHz-H4PteGlulor 1 p~ CHZ-H4PteGlu5, and0.01 unit of purified thymidylate synthase in 50 mM KHzP04. Thedissociation of the labeledcomplex was thenmonitored by adding M unlabeled FdUMP at time 0 and measuring residualradiolabeled complex at theindicated time points in the presence or absence of 60 FM MTX-Glu5. The method for measuring residual complex is described under “Materials and Methods.” Illustrated are dissociation and CHz-H4PteGlu5 curves for complex including CH,-H,PteGlu, (0) (O), eitherinthe absence of inhibitor (open symbols) orinthe presence of inhihitor (closed symbols). MTX G U S 1x10-5
DISCUSSION
3~10-~
6~10-~
I 1 CH, -H,
2
R e GLU, (pM)
FIG. 4. Kinetics of the inhibition of FdUMP-CHzH4PteGlua-thymidylatesynthase complex formation by MTXGlua. Thymidylate synthase-FdUMP-CHz-H4PteGlu5complexformation was measured after 5-min incubations a t 37 “C at pH 7.2 in the absence ( 0 )and presence of MTX-Glus at concentrations of 10 p~ (A), 30 PM (O), and 60 PM (0). Each assay included MTX-Glus, the indicated concentrationsof CH,-H,PteGlu,, 0.01 unit of purified thymidylate synthase, and 50 mM KHzP04.
MTX-Glus was identicaltocontrolscontainingnoMTXGlu6, aresult similar to that obtained with the reduced folate co-substrates.
These studies have shown that polyglutamate metabolites of methotrexate have greatly enhanced inhibitory effects on the catalytic activityof thymidylate synthase,as compared to the parent compound (MTX-Glul), and provide further evidence that these metabolites alter thepharmacologic properties of this antineoplastic agent. Previous work has demonstrated that the polyglutamates are preferentially retained in human breast cancercells, particularly thosederivatives with three and four additional glutamates, and thereby extend the duration of inhibition of dihydrofolate reductaseandthe cytotoxic action of thedrug (17). Thepresent work has provided strong evidence foran additional site of direct action of the polyglutamates, specifically thymidylate synthase, and has shown a nearly 300-fold enhancement of inhibitory activity for the MTX-Glu5derivative, as compared to MTX-Glul. While this finding confirms theincreased potency of thymidylatesynthaseinhibition by MTX-Glu2noted in earlier reports (23, 44),the current studies reveal a complex kinetic pattern of thymidylate synthase inhibition by these metabolites. Theuncompetitiveinhibitionpattern observed for MTX-Glu, is closely akintothe noncompetitive patterns previously described (22, 45) using highly purified enzyme, but differentfrom the competitive inhibition initially reported by BorsaandWhitmore (46), who used partially purified
Inhibition Enhanced
of Thymidylate Synthase
TABLE I1 Influence of MTX-Glu5 on apparentrate of association and dissociation of FdUMP The rates of ternary complex formationand dissociation were measured in the presence and absence of 10 p~ MTX-Glu5using three different folate cofactors. Experiments for each folate, with and withoutinhibitor, were performedsimultaneously,and all experiments were performed using identical experimental conditions, allowingdirectcomparisons of the effect of inhibitorontheternary complex. No charcoal-stable ternary complexes were isolated when using MTX-Glul or MTX-Glub in place of the folate cofactors. All parameters were calculated according to "Materials and Methods" as indicated, and the equilibrium constant (KO) was derived from the ratio of k,ff/k,,. Kinetic constants
MTX-
Folate cofactor
Glu6
min"
M"
/.lM
MTX-Glul (1 X MTX-Glu5 ( 5 X CHZ-H4PteGlul(38 p
~
)
0 10 0 10 0 10
x
10-6
0 0 6.90 0.86 16.7 10.0 2.70 0.22
&fib
tm
KO
min" x
min
RM
m m
1.31 k 1.26 & 0.43 & 0.46 ?
0.10" 53 .10 55 0.06 161 0.07 151
1.89 14.65 0.26 0.46
9725
Since thymidylate synthase has been considered to have two nonequivalent binding sites for folate (21), it is possible that binding of the folate substrate to the first site is required in order toallow MTX-Glul bindingto the second catalytic site. An alternative explanationis that the bindingof the inhibitor requires the prior binding of theproduct,dTMP,tothe enzyme. In contrast, the polyglutamatederivatives of methotrexate display a noncompetitive inhibition of thymidylate synthase, the specific form of which is represented by K, = K,' and, in this case, the inhibitor binding does not. require the prior binding of either substrate or product. +S E-ES+E
k2
+P
EI -ESl
This ability to bind to thymidylate synthase in the absence of other ligands may be theresult of the presence of a polyglutamatetailthat markedlyincreases the affinity of H2PteG1u5(10 pM) binding to theunoccupied enzyme. In addition to their enhancedpotency for inhibition of the ' Calculated according to methodC (Equation 2). thymidylate synthase catalyticreaction, the MTXPGsreduce Calculated according to method A. the rate of ternary complex formation by FdUMP and thyS.E. midylate synthase in the presence of a folate, CHz-H4PteGlul, enzymederived fromEhrlichascitescarcinoma cells and CH,-H,PteGlu,, or H2PteGluB.Although the amount of terEscherichia coli. These differences may be partly explained nary complex formed in the presence of MTX-Glu, was ultiby temperature-dependent conformational changes thathave mately equal to that formed in the absence of inhibitor (Fig. been described with the humanenzyme (as),as well as differ- 31, the initial rate of complex formation was depressed in a ent sources anddegrees of purity of the enzyme. The present noncompetitive manner. These results can be explained by studies were performed at 37 "C, and conformational changes in that occur below 35 "C may alter the inhibition kinetics(29). the noncompetitive pattern of inhibition(Equation7), The noncompetitive pattern of inhibition observed with the which a given concentration of inhibitor will reversibly bind polyglutamated metabolites of MTX is consistent with that a constant fractionof the available free enzyme, independent reported for MTX-Glu2 by Szeto et al. (44),whose experi- of substrate concentration.However, as covalent ternary complex is formed by interaction offolate and FdUMP with ments were also performed at 37 "C. Through the use of a general kinetic model for enzyme uninhibited enzyme, thymidylatesynthase is progressively inhibition,itis possible to explainthechangeinkinetic removed from the unbound pool and enters the irreversible pattern from uncompetitive to noncompetitive with the ad- complex. dition of glutamyl residues to MTX-Glul. A general analysis The foregoing findings have important implications for the of the possible interactions of enzyme, substrate, and inhibitor mechanism of action of methotrexate and its interactions may be expressed by the following model: with 5-fluorouracil, a second antineoplastic drug that is freis kz quently used in combination with the antifolate. This work E-ES-E i P suggests that, in addition to its inhibitory effect on dihydrofolate reductase, methotrexate may inhibit the synthesis of +I K, +I K! (5) thymidylate by the direct inhibitory effect of its polyglutamate derivatives on thymidylate synthase. The concentrations of +s k; MTXPG formed in tumor cells in uitro exceed the K, values E I -ESI +EI +P determined with purified enzymes. For example, exposure of where E = enzyme, S = CH2-H,PteGlulor -Glu6, P = MCF-7 cells to 2 p~ MTX results in the formationof 1.6 ~ L M H,PteGlu,, and I = MTX-Glu,. (Since the second substrate intracellular concentrationsof "I'XPGs a t 6 h and 4 @M a t dUMP is always used in vast excess in these studies,we may 12 h.Theconcentration of MTX-Glusat 12 h, 2.5 X M, consider the kinetic reaction as a single substrate catalysis.) is 5-fold higher than its K , for thymidylate synthase. InhibiThe specific uncompetitive inhibition pattern observedfor tion of thymidylate synthase by MTXPGs may have particMTX-Glul is representedby a model in which the binding of ular importance in understanding the process of Leucovorin inhibitor requires the prior binding of either a substrate or (5-CHO-H,PteGlul) rescue. Leucovorinwhen administered product, such thatK,>> KI (KJK: = m) and k; = 0. This case following methotrexate aborts thecytotoxic action of methosimplifies to the following. trexate in a competitive manner (47). Since Leucovorin by+S kz passes the block in dihydrofolate reductase activity, thecomE+ES+E+P petitive character of rescue cannot be explained simply by repletion of reduced folate pools. However, it could be ex+I K: (6) plained by the competition of Leucovorin and methotrexate for transmembrane transport or by the competitionof reduced ESI folate and MTXPGs for inhibition of thymidylate synthase CHp-H4PteGlu6(1 pM)
1 I I
9726
Inhibition Enhanced
of Thymidylate Synthase
or other enzymatic sites. Finally, these results suggest that the MTXPGs may have potent inhibitory effects on other folate-dependent enzymes, such as those involved in purine biosynthesis or folate interconversions; effects would be particularly likely on enzymes that show enhanced affinity for polyglutamated folates, such as AICAR transformylase and methylenetetrahydrofolate reductase (20, 21). With respect to methotrexate interaction with 5-FU, our results suggest that pretreatment of cells with the antifolate would tendtonegatetheimportance of 5-FU effects on thymidylate synthase, particularly in cells that readily form MTXPGs, since the polyglutamates themselves inhibit thymidylate synthase catalytic activity and retard ternary complex formation among thymidylate synthase, FdUMP, and the folate cofactors. In experimental chemotherapy, pretreatment of cells or tumor-bearing animals with MTX appears enhance 5-FU activity. This enhancement likely results from increased activation of 5-FU to 5-fluorouridine 5”triphosphate and incorporation into RNA (37, 48, 49), rather than enhancement of effects on thymidylate synthase. The inhibition of thymidylate synthase catalytic activity and FdUMP-thymidylate synthase-folatecomplex formation by MTXPGs is highly influenced by the stateof glutamation of thefolate cofactor andissubstantially reduced inthe presence of CH2-H4PteGlu5. Thus,in order to better understand the importance of MTXPGs and their effects on thymidylate synthase, itwill be important to determine the effect of MTX on the composition and state of glutamation of intracellular folatepools. REFERENCES 1. Goldman, I. D., Chabner, B. A,, and Bertino, J. R. (eds) (1983) Folyl and Antifolyl Polyglutamates, Plenum Press, New York 2. Jolivet, J., Cowan, K. H., Curt, G. A., Clendeninn, N. J., and Chabner, B. A. (1983) N . E&. J. Med. 3 0 9 , 1094-1104 3. Baugh, C. M., Drumdieck, C. L., and Nair, M. G. (1973) Biochem. Biophys. Res. Commun. 5 2 , 27-34 4. Whitehead, V. M., Perrault, M. M., and Stelcner,S. (1975) Cancer Res. 35,2985-2990 5. Whitehead, V. M. (1977) Cancer Res. 37, 408-412 6. Rosenblatt, D. S., Whitehead, V. M., Dupont, M. M., Vuchich, M. J., and Vera, J. (1978) Mol. Pharmacol. 14,210-214 7. Rosenblatt, D. S., Whitehead, V. M., Vera, N., Pottier, A., Dupont, M., and Vuchich, M. J. (1978) Mol. Pharmacol. 14,11431147 G. (1977) Biochem. 8. Jacobs, S. A,, Derr, C. J., and Johns, D. PhQrmQCOl.2 6 , 2310-2313 9. Poser, R. G., Sirotnak, F. M., and Chello, P. L. (1980) Biochem. Pharmacol. 2 9 , 2701-2704 10. Poser, R. G., Sirotnak, F. M., and Chello, P. L. (1981) Cancer Res. 41,4441-4446 11. Witte, A., Whitehead, V. M., Rosenblatt, D. S., and Vuchich, M. J. (1980) Deu. Pharmacal. Ther. 1,40-46 12. Gewirtz, D. A,, White, J. C., Randolph, J. K., and Goldman, I. D. (1980) Cancer Res. 40,573-578 13. Galivan, J. (1980) Mol. Pharmacol. 1 7 , 105-110 14. Schilsky, R. L., Bailey, B. D., and Chabner, B. A. (1980) Proc. Natl. Acad. Sei. U. S. A. 77,2919-2922 15. Fry, D. W., Yalowich, J. C., and Goldman, I. D. (1982) J. Bid. Chem. 2 5 7 , 1890-1896 16. Krakower, G. R., Nylen, P. A., and Kamen, E. A. (1982) Anal. Biochem. 1 2 2 , 412-416
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