Substrate For Folylpolyglutamate Synthetase Research Articles

Synthesis of classical, four-carbon bridged 5-substituted furo[2,3-d]pyrimidine and 6-substituted pyrrolo[2,3-d]pyrimidine analogues as antifolates.

We report, for the first time, the biological activities of four-carbon-atom bridged classical antifolates on dihydrofolate reductase (DHFR), thymidylate synthase (TS), and folylpolyglutamate synthetase (FPGS) as well as antitumor activity. Extension of the bridge homologation studies of classical two-carbon bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine (2), afforded two four-carbon bridged antifolates, analogues 5 and 6, with enhanced FPGS substrate activity and inhibitory activity against tumor cells in culture (EC(50) < or = 10(-7) M) compared with the two-carbon bridged analogues. These results support our original hypothesis that the distance and orientation of the side chain p-aminobenzoyl-L-glutamate moiety with respect to the pyrimidine ring are a crucial determinant of biological activity. In addition, this study demonstrates that, for classical antifolates that are substrates for FPGS, poor inhibitory activity against isolated target enzymes is not necessarily a predictor of a lack of antitumor activity.

Synthesis of N-[4-[1-ethyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid as an antifolate.

N-[4-[1-Ethyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid 3 was designed and synthesized to investigate the effect of homologation of a C9-methyl to an ethyl on dihydrofolate reductase (DHFR) inhibition and on antitumor activity. Compound 3 was obtained via a concise seven step synthesis starting from palladium-catalyzed carbonylation of 4-propionylphenol, followed by a Wittig reaction with 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine (6), catalytic hydrogenation, hydrolysis, and standard peptide coupling with diethyl L-glutamate. The biological results indicated that extending the C9-methyl group to an ethyl on the C8-C9 bridge region (analogue 3) doubled the inhibitory potency against recombinant human (rh) DHFR (IC(50) = 0.21 microM) as compared to the C9-methyl analogue 1 and was 4-fold more potent than the C9-H analogue 2. As compared to 1, compound 3 demonstrated increased growth inhibitory potency against several human tumor cell lines in culture with GI(50) values < 1.0 x 10(-8) M. Compound 3 was also a weak inhibitor of rh thymidylate synthase. Compounds 1 and 3 were efficient substrates of human folylpolyglutamate synthetase (FPGS). Further evaluation of the cytotoxicity of 3 in methotrexate-resistant CCRF-CEM cell sublines and metabolite protection studies implicated DHFR as the primary intracelluar target. Thus, alkylation of the C9 position in the C8-C9 bridge of the classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine is highly conducive to DHFR and tumor inhibitory activity as well as FPGS substrate efficiency.

5-deazafolate analogues with a rotationally restricted glutamate or ornithine side chain: synthesis and binding interaction with folylpolyglutamate synthetase.

Rotationally restricted analogues of 5-deazapteroyl-L-glutamate and (6R,6S)-5-deaza-5,6,7,8-tetrahydropteroyl-L-glutamate with a one-carbon bridge between the amide nitrogen and the 6'-position of the p-aminobenzoyl moiety were synthesized and tested as substrates for folylpolyglutamate synthetase (FPGS), a key enzyme in folate metabolism and an important determinant of the therapeutic potency and selectivity of classical antifolates. The corresponding bridged analogues of 5-deazapteroyl-L-ornithine and (6R,6S)-5-deaza-5,6,7, 8-tetrahydropteroyl-L-ornithine were also synthesized as potential inhibitors. Condensation of diethyl L-glutamate with methyl 2-bromomethyl-4-nitrobenzoate followed by catalytic reduction of the nitro group, reductive coupling with 2-acetamido-6-formylpyrido[2, 3-d]pyrimidin-4(3H)-one in the presence of dimethylaminoborane, and acidolysis with HBr/AcOH yielded 2-L-[5-[N-(2-acetamido-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]glutaric acid (1). When acidolysis was preceded by catalytic hydrogenation, the final product was the corresponding (6R,6S)-tetrahydro derivative 2. A similar sequence starting from methyl N(delta)-benzyloxycarbonyl-L-ornithine led to 2-L-[5-[N-(2-amino-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]-5-aminopentanoic acid (3) and the (6R,6S)-tetrahydro derivative 4. Compounds 3 and 4 were powerful inhibitors of recombinant human FPGS, whereas 1 and 2 were exceptionally efficient FPGS substrates, with the reduced compound 2 giving a K(m) (0.018 microM) lower than that of any other substrate identified to date. (6R,6S)-5-Deazatetrahydrofolate, in which the side chain is free to rotate, was rapidly converted to long-chain polyglutamates. In contrast, the reaction of 1 and 2 was limited to the addition of a single molecule of glutamic acid. Hence rotational restriction of the side chain did not interfere with the initial FPGS-catalyzed reaction and indeed seemed to facilitate it, but the ensuing gamma-glutamyl adduct was no longer an efficient substrate for the enzyme.

A new analogue of 10-deazaaminopterin with markedly enhanced curative effects against human tumor xenografts in mice.

These studies sought to evaluate the biochemical and cellular pharmacokinetic properties, cytotoxicity and antitumor efficacy of a new analogue of 10-deaza-aminopterin (PDX) against human tumors. Studies were conducted with a group of human tumor cell lines in culture examining PDX and other folate analogues as permeants for mediated membrane transport, as inhibitors of dihdrofolate reductase and as substrates for folylpolyglutamate synthetase. These same analogues were examined for their cytotoxicity following a 3-h pulse exposure, in experiments providing a value for IC50. Other studies with these analogues were conducted in nude mice bearing subcutaneously implanted human tumors. Treatment of the mice was initiated 4 days after implantation of the tumor using a schedule of administration of one dose per day for 5 days. The tumors were measured 6 days after cessation of therapy and compared to controls for assessment of response. In the CCRF-CEM cell system, PDX was 2- to 3-fold less effective as an inhibitor of dihydrofolate reductase than aminopterin (AMT), methotrexate (MTX) or edatrexate (EDX) but much more effective as a permeant for one-carbon, reduced folate transport inward (PDX > AMT approximately equal to EDX > MTX) and substrate for folylpolyglutamate synthetase (PDX > AMT > EDX > MTX). As predicted by these results, PDX was 15- to 40-fold more cytotoxic than MTX and 3- to 4-fold more cytotoxic than the highly potent EDX following a 3-h pulse exposure in culture of CCRF-CEM cells and cells from a panel of three human breast and two human nonsmall-cell (NSC) lung cancers. The same relative differences were shown for the therapeutic efficacy of these three analogues at equitoxic doses in studies with the human MX-1 and LX-1 tumors and the human A549 NSC lung tumor xenografted in nude mice. On a schedule of qd x 5 given 3-4 days posttransplant, MTX was minimally active (modest tumor growth delay) against all three tumors. EDX was highly active (25-35% complete regressions and 5-10% cures) against the MX-1 and LX-1 tumors but very modestly active (no regressions) against the A549 tumor. In contrast, PDX was even more active (75-85% complete regressions and 25-30% cures) than EDX against the MX-1 and LX-1 tumors and highly active (30% complete regressions and 20% cures) against the A549 tumor. These studies showed significantly enhanced antitumor properties of PDX compared with MTX and EDX. Based upon these results, clinical trials of PDX in patients with metastatic breast and NSC lung cancer appear to be warranted.

Dietary folate and folylpolyglutamate synthetase activity in normal and neoplastic murine tissues and human tumor xenografts

The importance of polyglutamation for the activation of natural folates and classical antifolates and recent evidence for the role of dietary folate as a biochemical modulator of antifolate efficacy led us to investigate the influence of changes in dietary folate on folylpolyglutamate synthetase (FPGS) activity. Activities were measured using lometrexol (6R-5,10-dideazatetrahydrofolic acid) as a substrate for FPGS with extracts of murine tissues, murine tumors, and human tumor xenografts from mice on standard diet or low folate diet. Tissues and tumors from mice on standard diet exhibited a 6-fold range of FPGS activity. Kidney had the lowest activity (36 pmol/hr · mg protein), followed by the human xenograft PANC-1 pancreatic carcinoma (46 pmol/ hr · mg protein), liver (109 pmol/hr · mg protein), murine C3H mammary tumor (112 pmol/hr · mg protein), and the human xenograft MX-1 mammary carcinoma (224 pmol/hr · mg protein). In response to restricted dietary folate, four out of five tissues had significantly increased (25–50%) FPGS activity. Only the tumor with highest FPGS activity under standard diet conditions (MX-1 mammary) did not respond to low folate diet. The results indicate that changes in dietary folate intake can modulate FPGS activity significantly in vivo and suggest that the tissue distribution and toxicities of classical antifolates requiring polyglutamation for activation and cellular retention will be influenced significantly by folate status of the host.

Potent Inhibition of Human Folylpolyglutamate Synthetase by Suramin

Suramin, a bis-hexasulfonated napthylurea, was studied as an inhibitor of human folylpolyglutamate synthetase (FPGS), a crucial enzyme in folate metabolism. Suramin is a more potent (IC50, 0.9 μM) inhibitor of FPGS partially purified from CCRF-CEM human leukemia cells than is bromosulfophthalein (IC50, 17 μM), the first reported nonsubstrate-analog inhibitor of FPGS (J. J. McGuireet al., Adv. Exptl. Med. Biol.163, 199, 1983). FPGS inhibition by suramin is reversed by bovine serum albumin (which binds suramin). Suramin is a noncompetitive inhibitor with aminopterin (Kii= 0.9 μM;Kis= 1.1 μM) and glutamic acid (Kii= 1.0 μM;Kis= 5.2 μM) as the variable substrates; suramin inhibition tends toward being competitive with respect to the third FPGS substrate, ATP (Kii= 3.4 μM;Kis= 0.35 μM), since the major effect is on itsKm. Suramin is a much less potent inhibitor of two other folate-dependent enzymes, dihydrofolate reductase (IC50, 38 μM; methotrexate (MTX), 0.6 nM) and thymidylate synthase (IC50, 87 μM; MTX, 48 μM). The effects of suramin on growth of CCRF-CEM cells and a MTX-resistant subline (R30dm) expressing low levels of FPGS activity were determined. R30dm is slightly collaterally sensitive to suramin consistent with FPGS inhibition contributing to the cytotoxic mechanism. These data, and those of Rideoutet al.(Int. J. Cancer61, 840, 1995), demonstrating that the reduced folate carrier system of CCRF-CEM is inhibited, suggest that inhibition of folate metabolism could be involved in the mechanism of action of suramin.

DL-β,β-Difluoroglutamic Acid Mediates Position-Dependent Enhancement or Termination of Pteroylpoly(γ-Glutamate) Synthesis Catalyzed by Folylpolyglutamate Synthetase

Poly(γ-glutamylation) of glutamate (L-Glu)-containing antifolates and natural folates is important in pharmacological mechanisms and in physiological processes. Based on previous work from our laboratories, we hypothesized that replacement of the L-Glu moiety in parent molecules with DL-β,β-difluoroglutamic acid (DL-β,β-F 2Glu) might be a generic means of increasing polyglutamylation by both increasing the synthesis rate and decreasing the degradation rate (J. J. McGuire et al., J. Biol. Chem. 265, 14073-14079 (1990)); thus biological potency might be increased without other biochemical properties being altered. DL-β,β-F 2Glu, synthesized by an improved route (B. P. Hart and J. K. Coward, Tetrahedron Lett. 34, 4917-4920 (1993)), has been incorporated into a methotrexate (MTX) homolog, β,β-difluoromethotrexate (β,β-F 2MTX), and a folic acid (PteGlu) homolog, β,β-difluorofolic acid (β,β-PteF 2Glu). Biochemical properties of β,β F 2MTX (e.g., inhibition of isolated dihydrofolate reductase, transport in whole cells) are similar to those of MTX except that, in accord with our hypothesis, apparent substrate efficiency for rat and human folylpolyglutamate synthetase (FPGS) is 4- to 7.5-fold higher, respectively, for β,β-F 2MTX than for MTX. Analysis of the products synthesized by purified FPGS, however, suggests that while addition of the first γ-Glu to β,β-F 2MTX is highly efficient, subsequent additions occur at a negligible rate; this premise was confirmed by directly comparing the in vitro FPGS substrate activity of MTX-γ-Glu and β,β-F 2MTX-γ-Glu. Furthermore, the dramatically diminished in vitro growth inhibitory potency of β,β-F 2MTX as compared to MTX when exposure time to drug is decreased (despite otherwise similar biochemical properties) suggests that polyglutamylation is also impaired in intact cells. Similar results with FPGS have been obtained with oxidized and reduced forms of β,β-PteF 2Glu. These data suggest that the effect of β,β-F 2Glu on polyglutamylation by FPGS is dependent on its position relative to the point of L-Glu ligation. When β,β-F 2Glu is the acceptor amino acid (i.e., point of attachment), ligation of Glu is enhanced; however, if β,β-F 2Glu is one residue distal to the acceptor amino acid, further elongation is blocked.

Novel pyrrolo[2,3-d]pyrimidine antifolate TNP-351: cytotoxic effect on methotrexate-resistant CCRF-CEM cells and inhibition of transformylases of de novo purine biosynthesis

N-[4-[3-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (TNP-351), characterized by a pyrrolo[2,3-d]pyrimidine ring, is a novel antifolate that exhibits potent antitumor activities against mammalian solid tumors. The mechanism of action of TNP-351 was evaluated using some methotrexate-resistant CCRF-CEM human lymphoblastic leukemia cell lines as well as partially purified enzymes folylpolyglutamate synthetase (FPGS), aminoimidazolecarboxamide ribonucleotide transformylase (AICARTFase), and glycinamide ribonucleotide transformylase (GARTFase) from parent CCRF-CEM cells. TNP-351 was found to inhibit the growth of L1210 and CCRF-CEM cells in culture, with the doses effective against 50% of the cells (ED50 values) being 0.79 and 2.7 nM, respectively. The growth inhibition caused by TNF-351 was reversed by leucovorin or a combination of hypoxanthine and thymidine. The methotrexate-resistant CCRF-CEM cell line, which has an impaired methotrexate transport, showed less resistance to TNP-351 than to methotrexate. TNP-351 was also found to be an excellent substrate for FPGS with a Michaelis constant (Km) of 1.45 microM and a maximum of velocity (Vmax) of 1,925 pmol h-1 mg-1. Inhibitory activities of TNF-351-Gn (n = 1-6) for AICARTFase were found to be significantly enhanced with increasing glutamyl chain length [inhibition constants (Ki): G1, 52 microM; G6, 0.07 microM]. Neither TNP-351 nor its polyglutamates were very strong inhibitors of GARTFase. These findings have significant implications regarding the mechanism of action of TNP-351.

The role of the reduced-folate carrier and metabolism to intracellular polyglutamates for the activity of ICI D1694.

The uptake of ICI D1694 into L1210 cells is very rapid and evidence strongly suggests that transport is via the reduced-folate/MTX cell membrane carrier (RFC); for example a cell line with a greatly impaired RFC is highly resistant to ICI D1694. Polyglutamates can be found intracellularly within a few minutes, so that experiments initially designed to measure transport were actually measuring transport and polyglutamation. After 30mins, in normal serum-containing tissue culture medium, the concentration of polyglutamates (di, tri and tetra) exceeded that of the parent drug 6-fold. Studies where cells were resuspended in drug-free medium demonstrated that the parent drug and its diglutamate could readily leave the cell. Folinic acid could markedly decrease the polyglutamation of ICI D1694, but had to be given simultaneously with the drug as a 4hr delayed rescue was less effective because substantial polyglutamation had already occurred. This effect was translated into considerable antagonism for cell growth inhibition by simultaneous folinic acid. The importance of the metabolism of ICI D1694 to polyglutamates to its potent cytotoxic activity is demonstrated by compounds related in structure to ICI D1694 but with different properties for the RFC and FPGS. For example, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (ICI 198583) owes its less potent cytotoxic activity to its poorer FPGS substrate activity (Km 40 microM compared with 1.3 microM for ICI D1694). Replacing the 2-methyl of either compound with amino, which appears to prevent use of the RFC, has a deleterious effect on growth inhibitory activity presumably by limiting the transport of the parent compounds into the cells, thereby slowing the rate of polyglutamate formation. Again a single change to another part of the molecule, that is methylation of the 7-position can have serious consequences on cytotoxic potency, particularly for the ICI D1694 molecule. The 7-methylated compounds are apparently poor or non-substrates for FPGS and therefore retain activity against a cell line unable to polyglutamate antifolates. These same compounds are only slightly affected by coincubation with folinic acid in L1210 tissue culture, consistent with the failure of these compounds to form intracellular polyglutamates. The results of short-exposure assays and in situ TS assays confirms that 7-methylation largely prevents the formation of a retained drug-form (polyglutamates), continuous exposure being necessary to maintain TS inhibition and cause a cytotoxic effect after removal of extracellular drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and biological activity of open-chain analogues of 5,6,7,8-tetrahydrofolic acid--potential antitumor agents.

This study describes the synthesis and in vitro antitumor activity of inhibitors of purine de novo biosynthesis that are analogues of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl) propyl]amino]benzoyl-L-glutamic acid (5-DACTHF). Benzene ring substituted analogues were synthesized from a protected pyrimidinyl propionaldehyde and a substituted benzoyl glutamate moiety by a key reductive amination step. Pyrimidine and linking chain substituted analogues were built up stepwise from p-aminobenzoic acid or analogues. The compounds were tested as inhibitors of methotrexate uptake as a measure of binding to the reduced folate transport system, as inhibitors of glycinamide ribonucleotide transformylase, as substrates for folylpolyglutamate synthetase, and as inhibitors of tumor cell growth in cell culture. With the exception of 2'-F substituent, the ring-substituted analogues are less active than the parent compound. Replacement of the 10-nitrogen by carbon, sulfur, or oxygen produced less than 2-fold changes to biological activity in vitro. A four-atom linking chain and an amino group at the 2-position on the pyrimidine ring are important for good activity.

Folic acid analogs lacking the 2-carbon are substrates for folylpolyglutamate synthetase and inhibit cell growth

Folic acid analogs lacking the 2-carbon are substrates for folylpolyglutamate synthetase and inhibit cell growth

6‐aza‐5,8,10‐trideaza analogues of tetrahydrofolic acid and tetrahydroaminopterin: Synthesis and biological studies

Abstract6‐Aza‐5,8,10‐trideaza‐5,6,7,8‐tetrahydrofolic acid (3) and 6‐aza‐5,8,10‐trideaza‐5,6,7,8‐tetrahydroamino‐pterin (4) were synthesized from 6‐aza‐5,8,10‐trideaza‐5,6,7,8‐tetrahydropteroic acid (5) and 4‐amino‐6‐aza‐5,8,10‐trideaza‐4‐deoxy‐5,6,7,8‐tetrahydropteroic acid (6), respectively, by mixed carboxylic‐carbonic anhydride condensation with dimethyl L‐glutamate followed by ester hydrolysis. The pteroic acid analogues 5 and 6 were prepared in several steps from 1‐benzyl‐3‐carbethoxypiperidin‐4‐one via 2‐amino‐6‐benzyl‐5,6,7,8‐te‐trahydropyrido[4,3‐d]pyrimidin‐4(3H)‐one (7). Compound 3 did not inhibit the growth of L1210 mouse leukemia cells in culture, and was not an inhibitor of dihydrofolate reductase (DHFR) or thymidylate synthase (TS). It was a very poor substrate for mouse liver folylpolyglutamate synthetase (FPGS). The 2,4‐diamino analogue 4 was only a marginal substrate for FPGS, yet showed activity comparable to methotrexate as a DHFR inhibitor and as an inhibitor of tumor cell growth. The cytotoxicity of 4 is noteworthy because this compound is, to our knowledge, the first example of a classical antifolate which forms polyglutamates poorly even though it contains an intact p‐aminobenzoyl‐L‐glutamic acid side‐chain. The inability of 3 and 4 to form polyglutamates indicates that a basic nitrogen at position 6 is highly unfavorable for binding to FPGS.

Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates.

The transfer of 17O and/or 18O from (COOH-17O or -18O) enriched substrates to inorganic phosphate (Pi) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-18O-labeled folate, methotrexate, and dihydropteroate, in addition to [17O]-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. Pi was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for 17O and 18O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate 18O-enriched carboxyl group to Pi occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of Pi obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that 18O transfer had occurred.

ChemInform Abstract: A MECHANISM FOR THE ADDITION OF MULTIPLE MOLES OF GLUTAMATE BY FOLYLPOLYGLUTAMATE SYNTHETASE

The role of the alpha-carboxyl group in methotrexate (MeAPA-Glu) and the gamma-glutamate derivative of methotrexate (MeAPA-Glu-Glu) in the reaction catalyzed by folylpolyglutamate synthetase (FPGS) has been investigated. MeAPA-Glu and MeAPA-Glu-Glu were accepted as substrates by the same FPGS species contained in an (NH4)2SO4 precipitate of mouse liver protein, as judged by a lack of additivity of product formation at saturating concentrations of both substrates. MeAPA-Gaba, the MeAPA-Glu analogue lacking an alpha-carboxyl, was inactive as a substrate for this enzyme as was MeAPA-Glu-Gaba, the analogue of MeAPA-Glu-Glu that lacked the alpha-carboxyl of the terminal glutamic acid. However, MeAPA-Gaba-Glu, the analogue of MeAPA-Glu-Glu without an alpha-carboxyl on the first glutamic acid, had activity as a substrate for FPGS that approached that of MeAPA-Glu-Glu. These results suggest that the alpha-carboxyl is essential for the binding of folyl monoglutamates to FPGS in the correct orientation to allow catalysis. Moreover, the binding of the terminal alpha-carboxyl of folyl oligoglutamates to the same residue(s) responsible for the binding of the alpha-carboxyl of folyl monoglutamates would allow correct positioning of the terminal gamma-carboxyl of the chain for reaction. This binding mechanism would be compatible with the utilization of a single enzyme species for the addition of glutamate to the monoglutamate or oligoglutamate forms of folates and folate analogues.

A mechanism for the addition of multiple moles of glutamate by folylpolyglutamate synthetase.

The role of the alpha-carboxyl group in methotrexate (MeAPA-Glu) and the gamma-glutamate derivative of methotrexate (MeAPA-Glu-Glu) in the reaction catalyzed by folylpolyglutamate synthetase (FPGS) has been investigated. MeAPA-Glu and MeAPA-Glu-Glu were accepted as substrates by the same FPGS species contained in an (NH4)2SO4 precipitate of mouse liver protein, as judged by a lack of additivity of product formation at saturating concentrations of both substrates. MeAPA-Gaba, the MeAPA-Glu analogue lacking an alpha-carboxyl, was inactive as a substrate for this enzyme as was MeAPA-Glu-Gaba, the analogue of MeAPA-Glu-Glu that lacked the alpha-carboxyl of the terminal glutamic acid. However, MeAPA-Gaba-Glu, the analogue of MeAPA-Glu-Glu without an alpha-carboxyl on the first glutamic acid, had activity as a substrate for FPGS that approached that of MeAPA-Glu-Glu. These results suggest that the alpha-carboxyl is essential for the binding of folyl monoglutamates to FPGS in the correct orientation to allow catalysis. Moreover, the binding of the terminal alpha-carboxyl of folyl oligoglutamates to the same residue(s) responsible for the binding of the alpha-carboxyl of folyl monoglutamates would allow correct positioning of the terminal gamma-carboxyl of the chain for reaction. This binding mechanism would be compatible with the utilization of a single enzyme species for the addition of glutamate to the monoglutamate or oligoglutamate forms of folates and folate analogues.

Enzymatic synthesis of polyglutamate derivatives of 7-hydroxymethotrexate

7-Hydroxymethotrexate, an important metabolite of methotrexate, is a substrate for folylpolyglutamate synthetase (FPGS) isolated from rat liver and several human leukemia cell lines. The substrate activity it displays over a wide range of concentrations (0–200 μM) is nearly equivalent to that of methotrexate. The 7-hydroxy derivative of dichloromethotrexate is also a substrate for FPGS. The pattern of polyglutamate products synthesized by rat liver FPGS was nearly identical with both 7-hydroxymethotrexate and methotrexate. In addition, conversion of MTX polyglutamates to the corresponding 7-hydroxy compounds was demonstrated using partially purified rabbit liver aldehyde oxidase. The rate of conversion was concentration dependent, and the relative rate decreased as the MTX polyglutamate chain length increased. We propose that 7-hydroxymethotrexate polyglutamates may be formed by initial hydroxylation of methotrexate and subsequent polyglutamate formation or by direct hydroxylation of methotrexate polyglutamates. It was further shown that the relative substrate activity of folate analogs for folylpolyglutamate synthetase is dependent on the source of the enzyme.

Effects of 5,8-dideazaisopteroylglutamate and its possible tri-gamma-glutamyl metabolite (5,8-dideazaisoPteGlu3) on colon adenocarcinoma, and the folate dependent enzymes thymidylate synthase and dihydrofolate reductase.

A series of 2-amino-4-hydroxy-quinazolines was synthesized and evaluated as inhibitors of colon adenocarcinoma and the folate-dependent enzymes, thymidylate synthase and dihydrofolate reductase. Of the quinazolines tested, 5,8-dideazaisopteroylglutamate, (IAHQ), when administered at 85 mg/kg on days 2 and 10 after tumor implantation delayed the growth of colon tumor No. 38, and resulted in 6 of 20 tumor-free animals at 90 days. In contrast, methotrexate had no effect on the growth of colon tumor No. 38 at maximally tolerated doses. IAHQ was also active against human colon adenocarcinoma cells (HCT-8) in tissue culture, requiring a concentration of 5 X 10(-7) M to inhibit cell growth 50% after 72 hours continuous exposure. Since IAHQ was an effective substrate for folylpolyglutamate synthetase, we examined the effects of IAHQ and its possible tri-gamma-glutamyl metabolite, 5,8-dideazaisoPteGLu3, on thymidylate synthase and dihydrofolate reductase. Neither IAHQ nor 5,8-dideazaisoPteGlu3 stimulated significant binding of 5-fluorodeoxyuridylate to thymidylate synthase. This was consistent with the observation that IAHQ antagonized the killing of HCT-8 cells by 5-fluorouracil. 5,8 DideazaisoPteGlu3 bound more tightly to thymidylate synthase than dihydrofolate reductase as indicated by Kis of 0.09 and 0.7 microM when deoxyuridylate and dihydropteroylglutamate, respectively, were the variable substrates. Inhibition studies also revealed that binding of IAHQ and 5,8-dideazaisoPteGlu3 to thymidylate synthase is promoted and not antagonized by deoxyuridylate. The data suggests that the biochemical basis for the antitumor effects of IAHQ is the intracellular conversion of IAHQ to poly-gamma-glutamyl metabolites, which inhibit thymidylate synthase via formation of an inhibitor-deoxyuridylate-enzyme complex.