Folate-requiring Enzymes Research Articles

Abstract 2571: The predictive biomarker for the resistance to antifolate

Abstract Currently, several folate-based antimetabolites are used for cancer treatment. Pemetrexed (MTA) is new generation antifolate which inhibits multiple folate-requiring enzymes, mainly thymidylate synthase (TS). We cultured small lung cell cancer cell line, PC-6 in the presence of MTA and established two MTA resistant cell lines, PC-6/MTA-1.6 and PC-6/MTA-4.0, which grew vigorously in medium containing 1.6uM and 4.0uM, respectively. These cell lines showed cross resistance to methotrexate (MTX), but not to other cytotoxic agents including etoposide, vinorelbine and cisplatin. Among many ATP-binding cassette (ABC) transporters, only ABCC11/MRP8 (ABCC11) gene expression was markedly increased in PC-6/MTA-1.6 cells. In contrast, TS gene expression was markedly increased in PC-6/MTA-4.0 cells. ABCC11 gene expression was not increased in PC-6/MTA-4.0 cells. There were no changes in reduced folate carrier and folylpoly-gamma-glutamate synthetase expression level in MTA resistant cells. To determine if the higher expression of the ABCC11 in the PC-6/MTA-1.6 cells resulted in a greater efflux of drugs, we assayed the intracellular accumulation of MTX, which is known to be a substrate for ABCC11 in the PC-6 and PC-6/MTA-1.6 cells and found that MTX accumulation in PC-6/MTA-1.6 cells was lower than PC-6 cells. We found that decrease of ABCC11 expression by ABCC11 siRNA enhanced MTA and MTX cytotoxicity in PC-6/MTA-1.6 cells. Decreasing the expression of TS by small interfering RNA (siRNA) also enhanced MTA and MTX cytotoxicity in PC-6/MTA-4.0 cells. These results give a concept that resistance for antifolate is advanced as TS gene expression is increased, while ABCC11 may work in early phase of resistance when TS expression is not enough induced. TS and ABCC11 may be useful as predictive biomarkers for the resistance to antifolate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2571.

Dipyridamole potentiates the in vitro activity of MTA (LY231514) by inhibition of thymidine transport.

The novel pyrrolopyrimidine-based antifolate LY231514 (MTA), inhibits multiple folate-requiring enzymes including thymidylate synthase, glycinamide ribonucleotide formyltransferase and dihydrofolate reductase. Both thymidine and hypoxanthine are required to reverse MTA growth inhibition in leukaemia and colon cancer cells. Prevention of MTA growth inhibition by thymidine and/or hypoxanthine was investigated in two human lung (A549, COR L23) and two breast (MCF7, T47D) tumour cell lines, and the effect of the nucleoside/base transport inhibitor dipyridamole (DP) on thymidine and hypoxanthine rescue defined. MTA IC50values (continuous exposure three population doublings) were: A549–640 n M, COR L23–28 n M, MCF7–52 n M and T47D–46 n M. Thymidine (1 μM) completely prevented growth inhibition at the MTA IC50in all cell lines. At 10 × IC50, growth inhibition was only partially reversed by thymidine (≤ 10 μM); both thymidine and hypoxanthine (30 μM) being required for complete reversal, reflecting the multi-targeted nature of MTA. Growth inhibition by MTA was not affected by hypoxanthine alone. A non-toxic concentration (1 μM) of DP prevented thymidine/hypoxanthine rescue of MTA indicating that DP may potentiate MTA activity by preventing nucleoside and/or base salvage. Thymidine transport was inhibited by ≥ 89% by 1 μM DP in all cell lines, whereas hypoxanthine transport was inhibited only in A549 and MCF7 cells. Therefore, prevention of end-product reversal of MTA-induced growth inhibition by DP can be explained by inhibition of thymidine transport alone. © 2000 Cancer Research Campaign

Folic acid-enhanced synergy for the combination of trimetrexate plus the glycinamide ribonucleotide formyltransferase inhibitor 4-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3 H-pyrimidino[5,4,6][1,4]thiazin-6-yl)-( S)-ethyl]-2,5-thienoylamino- l-glutamic acid (AG2034) : Comparison across sensitive and resistant human tumor cell lines

Folic acid (PteGlu)-enhanced intense synergy has been observed between nonpolyglutamylatable dihydrofolate reductase (DHFR) inhibitors and polyglutamylatable inhibitors of other folate-requiring enzymes, such as glycinamide ribonucleotide formyltransferase (GARFT) and thymidylate synthase. Since this phenomenon is potentially therapeutically useful, we explored its universality by examining the combined action of a DHFR inhibitor, trimetrexate (TMQ), with a GARFT inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3 H-pyrimidino[5,4,6][1,4]thiazin-6-yl)-( S)-ethyl]-2,5-thienoylamino- l-glutamic acid (AG2034), in eight human cultured cell lines. Using a 96-well plate cell growth inhibition assay, four ileocecal adenocarcinoma cell lines [HCT-8, HCT-8/DW2 (Tomudex-resistant), HCT-8/DF2 (Tomudex-/FdUrd-resistant), and HCT-8/50 (adapted to 50 nM PteGlu)], three head and neck carcinoma cell lines [A253, FaDu, and Hep-2/500 (FdUrd-resistant)], and a non-small cell lung carcinoma cell line [H460] were treated for 96 hr with TMQ + AG2034 in the presence of 2.3 or 40 μM PteGlu. Cell growth was measured with the sulforhodamine B assay at the end of this period. Drug interactions were assessed by fitting a 7-parameter model including a synergism parameter, α, to data with weighted nonlinear regression. Isobologram analysis was also applied. At 2.3 μM PteGlu, cells exhibited similar intensities of Loewe synergy for the combination of TMQ + AG2034. Loewe synergy was abolished in HCT-8/50 cells cultured and studied in 50 nM PteGlu. At 40 μM PteGlu, the intensity of the combined action in all cell lines was increased. However, the most intense Loewe synergy was seen with HCT-8, HCT-8/DF2, H460, FaDu, A253, and Hep-2/500 cells, whereas the HCT-8/50 subculture showed less of the phenomenon, and PteGlu enhancement was the least with HCT-8/DW2, a subline deficient in folylpolyglutamate synthetase (FPGS). The universality of the PteGlu-enhanced intense synergy phenomenon is suggested. Impaired FPGS activity and low-folate adaptation prior to treatment significantly lessen the degree of PteGlu enhancement.

New generation multi-targeted antifolate active against solid tumours

LY-231514 [Eli Lilly; phase II] is a novel multi-targeted antifolate agent that inhibits a range of folate-requiring enzymes. Its clinical advantages are activity against a variety of solid tumours, a low toxicity profile and non-cross-resistance with platinum drugs. Results of 2 recent clinical trials with LY-231514 were presented at the European Cancer Conference (ECCO) ’97 [ Hamburg, Germany; September 1997 ]. A response rate of 33% was seen in a German trial combining LY-231514 with cisplatin in patients with a variety of solid tumours, and there was a response rate of 23% in a Canadian trial of LY-231514 monotherapy in patients with non-small-cell lung cancer (NSCLC). Myelosuppression was the dose-limiting toxicity in both studies. The German researchers concluded that the maximum tolerated dose of LY-231514 is 600 mg/m 2 when combined with cisplatin 100 mg/m 2.

Methotrexate: An Effective Agent for Treating Cancer and Building Careers. The Polyglutamate Era

This paper chronicles developments in the laboratory of Dr. Bruce Chabner during the period 1978-1981. Initial work demonstrated that methotrexate is taken up by human breast cancer cells by a high-affinity, carrier-mediated, energy-dependent transport system similar to that described in murine leukemia cells. Conversion of methotrexate to a high molecular weight polyglutamate metabolite was also demonstrated to occur in human breast cancer cells. Polyglutamates became the predominant form of intracellular drug, both free in the cytosol and bound to dihydrofolate reductase, during a 24 h exposure to clinically achievable methotrexate concentrations. Intracellular retention of polyglutamates led to prolonged suppression of thymidine synthesis and loss of cell viability after removal of extracellular drug. This work identified methotrexate polyglutamates as biologically active enzyme inhibitors in human tumor cells and launched a series of investigations on the interaction of these derivatives with folate-requiring enzymes.

Methotrexate: an effective agent for treating cancer and building careers. The polyglutamate era.

This paper chronicles developments in the laboratory of Dr. Bruce Chabner during the period 1978-1981. Initial work demonstrated that methotrexate is taken up by human breast cancer cells by a high affinity, carrier-mediated energy-dependent transport system similar to that described in murine leukemia cells. Conversion of methotrexate to a high molecular weight polyglutamate metabolite was also demonstrated to occur in human breast cancer cells. Polyglutamates became the predominant form of intracellular drug, both free in the cytosol and bound to dihydrofolate reductase, during a 24 h exposure to clinically achievable methotrexate concentrations. Intracellular retention of polyglutamates led to prolonged suppression of thymidine synthesis and loss of cell viability after removal of extracellular drug. This work identified methotrexate polyglutamates as biologically active enzyme inhibitors in human tumor cells and launched a series of investigations on the interaction of these derivatives with folate-requiring enzymes.

Newer antifolates in cancer therapy.

Folate vitamins are a class of cofactors that serve as one-carbon donors in biochemical reactions. Folate-requiring enzymes provide excellent targets for cancer chemotherapy due to the close relationship of folic acid metabolism to cell replication. Several of these enzymes are essential for important steps in DNA synthesis, including biosynthesis of purines and conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) (Figure 1). It is interesting to reflect that of the 18 or so known reactions of folate metabolism, exhaustive research of some 30 years has seemingly produced only folate antimetabolites targeted to block dihydrofolate reductase (DHFR). Over recent years, we now see the emergence of new folate antimetabolites with specificities other than DHFR, such as thymidylate synthase (TS) and glycinamide ribonucleotide formyl transferase (GARFT).

Antifolates: the next generation.

A number of promising new antifolates have been entered in clinical trials in recent years. These agents have been rationally designed based on the current understanding of folate transport and metabolism and of the mechanisms by which cells become resistant to methotrexate. Methotrexate-resistant cell lines are generally sensitive to one or more of the newer antifolates, which differ from methotrexate by being either more lipid soluble, more extensively polyglutamated, or by inhibiting folate-requiring enzymes other than dihydrofolate reductase. Five of the agents furthest along in clinical testing, trimetrexate, piritrexim, edatrexate, lometrexol, and D1694, are discussed. These drugs offer exciting opportunities to expand the role of antifolates in cancer chemotherapy, as well as in antimicrobial and antirheumatic therapy.

Synthesis and biological evaluation of 5-deazaisofolic acid, 5-deaza-5,6,7,8-tetrahydroisofolic acid, and their N9-substituted analogues

Prompted by recent disclosures concerning the potent antitumor activities of 5-deaza-5,6,7,8-tetrahydrofolic acid and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), we have prepared 5-deazaisofolic acid (3a) and 5-deaza-5,6,7,8-tetrahydroisofolic acid (4a). Reductive condensation of 2,6-diamino-3,4-dihydro-4- oxopyrido[2,3-d]pyrimidine with di-tert-butyl N-(4-formylbenzoyl)-L-glutamate and subsequent deprotection with trifluoroacetic acid yielded 5-deazaisofolic acid in good yield. Catalytic hydrogenation of this analogue then gave 4a. The 9-CH3 and 9-CHO modifications of 3a and the 9-CH3 derivative of 4a were also synthesized. Each of the new analogues was evaluated with a variety of folate-requiring enzymes as well as MCF-7 cells in culture. Compound 4a had an IC50 of ca. 1 microM against MCF-7 cells and was nearly 100-fold less potent than DDATHF in this regard. The three oxidized isofolate analogues were all poor inhibitors of tumor cell growth.

Inhibition of thymidylate synthase by the diastereoisomers of leucovorin

The clinical formulation of leucovorin (5-CHO-FH4) is a mixture of diastereoisomers with markedly different pharmacologic properties. Comparatively little information is available concerning the cellular pharmacology of reduced folate stereoisomers, due largely to the difficulty in preparing sufficient quantities of these compounds for in vitro use. Recent improvements in HPLC technology have now facilitated this process, enabling studies of folate stereochemistry on a larger scale. Using purified (6R) and (6S) leucovorin, we examined the effects of these compounds on the enzymatic activity of Lactobacillus casei thymidylate synthase (TS) in a cell-free system. The natural (6S), unnatural (6R), and racemic (6R,S) leucovorin preparations inhibited TS activity by 50% at concentrations of 0.11, 2.1, and 0.52 mM, respectively. Dixon plots demonstrated the inhibition to be competitive, with Ki values of 85 microM, 1.59 mM, and 385 microM for (6S), (6R), and (6R,S) leucovorin, respectively. In view of the high doses of leucovorin given clinically and the slow clearance of the unnatural isomer, our observations suggest that leucovorin may have important direct inhibitory effects on folate-requiring enzymes.

Tetrahydrohomofolate polyglutamates as inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase in Lactobacillus casei

In order to determine the mechanism for the effects of homofolates on growth of Lactobacillus casei, polyglutamated derivatives of homofolate (HPteGlu), dihydrohomofolate and tetrahydrohomofolate (H 4HPteGlu) were synthesized and tested as inhibitors of folate-requiring enzymes. The following L. casei enzymes were examined: thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase and dihydrofolate reductase. Polyglutamates of (6 R,S)-H 4HPteGlu are potent inhibitors of TS and GARFT. For example, the IC 50 values of (6 R,S)-H 4HPteGlu 6 are 0.7 μ m for TS and 0.3 μ m for GARFT. By contrast, the value for HPteGlu 6 is > 10 μ m for both TS and GARFT. Inhibition of TS and GARFT by (6 R,S)-H 4HPteGlu derivatives increases with polyglutamate chain length. For TS, the Glu 5 and Glu 6 derivatives of (6 R,S)-H 4HPteGlu are 20 and 30 times more potent than the monoglutamate, respectively. For GARFT, the Glu 2–6 derivatives are 2–3 times more potent than Glu 1. Inhibition of TS and GARFT by (6 R,S)-H 4HPteGlu polyglutamates is almost entirely due to the unnatural (6 R) diastereomer at C-6. Homofolate derivatives are only weak inhibitors of aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase, and dihydrofolate reductase. We conclude that both TS and GARFT are potential targets of (6 R)-H 4HPteGlu polyglutamates.

Further studies on the pharmacologic effects of the 7-hydroxy catabolite of methotrexate in the L1210 murine leukemia cell

This paper describes studies that further explore the pharmacologic activity of the 7-hydroxy catabolite of methotrexate (7-OH-MTX). A 3-hr exposure of L1210 leukemia cells to 100 μM 7-OH-MTX produced negligible suppression of cell growth despite the build-up of intracellular polyglutamyl congeners to levels 2.7 times greater than the dihydrofolate reductase (DHFR) binding capacity. There was no evidence for direct inhibition of DHFR under these conditions based upon measurements of cellular tetrahydrofolate cofactor and dihydrofolate levels, nor was there suppression of [ 3H]deoxyuridine incorporation into DNA or [ 14C]formate incorporation into purines. When the interval of exposure to 100 μM 7-OH-MTX was increased to 6 hr, cell growth was inhibited by 60% and there was mild (~50%) inhibition of purine and thymidylate biosynthesis associated with a small increase in cellular dihydrofolate and a small decline in cellular tetrahydrofolates. Consistent with weak inhibition of DHFR was the absence of significant binding of 7-OH-MTX polyglutamates to DHFR as assessed by gel filtration of cell extracts. Mild direct inhibition of purine biosynthetics by 7-OH-MTX- or MTX-polyglutamyl congeners was demonstrated based upon inhibition of [ 14C]formate incorporation into purines in cells pretreated with fluorodeoxyuridine so as to prevent tetrahydrofolate cofactor depletion or dihydrofolate polyglutamate build-up. Effects of a 6-hr exposure of cells to 100 μM 7-OH MTX on cell growth were reversed completely by 10 μM leucovorin; effects on cells containing comparable levels of MTX polyglutamyl congeners were unaffected by leucovorin. These studies demonstrate very weak inhibition of L1210 leukemia cell growth and purine, pyrimidine and tetrahydrofolate synthesis by the polyglutamyl congeners of 7-OH-MTX. The data suggest that effects of 7-OH-MTX polyglutamates on folate-requiring enzymes are not likely to play an important role in moderate-dose MTX regimens. However, pharmacologic activity may be expressed in high-dose MTX protocols when high blood levels of 7-OH-MTX are sustained over long intervals to the extent to which polyglutamate congeners accumulate in tumor cells and add to the much more potent inhibitory effects of MTX polyglutamates already present. Pharmacologic activity, however, would be diminished, if not completely reversed, by the concurrent administration of leucovorin.

The Influence of Folate Binding Proteins on 1-Carbon Metabolism A Review

The concentration of folylpolyglutamates in liver is 10 - 20 µmol/kg tissue. This concentration is approached by that of the folate-binding sites of several enzymes involved in 1-carbon metabolism, suggesting that a large proportion of cellular folates is protein-bound. Glycine N-methyltransferase and 10-formyl-H4PteGlu dehydrogenase have been identified as "folate binding proteins" because of their strong affinity for folylpolyglutamates during gel chromatography. Serine hydroxymethyltransferase may also prove to be a “folate binding protein.” Although the folate-binding properties of the above proteins in vivo remain virtually untested, it is likely that the proteins have an effect on the activities of other folate-requiring enzymes, such as thymidylate synthase.

ChemInform Abstract: SYNTHESIS AND ANALYSIS OF THE PTEROYLPOLYGLUTAMATES

Publisher Summary This chapter discusses the synthesis and analysis of the Pteroylpolyglutamates. The biological importance of the polyglutamyl chain was recognized on the basis of its universal occurrence and by the fact that, during evolution, the ability to synthesize the peptide chain has been retained, even by organisms that have lost the pathways for folic acid synthesis. The polyglutamyl chain of the folates plays a role in the regulation of one-carbon metabolism. The polyglutamates are the best cosubstrates of the folate-requiring enzymes, and some of these enzymes show a clear preference for certain specific polyglutamyl chain lengths. The part played by the poly-γ-glutamyl chain in affecting the translocation of folates across cellular and subcellular membranes is established. This role acquires special practical significance when considering the pharmacodynamics of polyglutamyl derivatives of folic acid analogs possessing antitumor activity. Changes in the patterns of distribution of polyglutamates of different chain lengths occur under conditions, which alter the steady-state of one-carbon metabolism, a necessary effect when the polyglutamyl chain is involved in regulation. The properties of the enzymes of poly-glutamate biosynthesis and degradation are reviewed in the chapter.

Amplification and organization of dihydrofolate reductase genes in a human leukemic cell line, K-562, resistant to methotrexate.

A subline of human leukemia cells (K-562), highly resistant to methotrexate, was developed by stepwise selection in the presence of increasing concentrations of this drug. The ED50 of these resistant cells was 1 mM compared to 10 nM for the parental line. Comparison of certain folate-requiring enzymes from crude extracts of the parent and resistant cells showed a 240-fold elevation of dihydrofolate reductase activity in the resistant cells with no significant increase in the levels of the other enzymes. Purified dihydrofolate reductase from the resistant cells had the same physical and kinetic properties as the enzyme from the sensitive cells. Southern blot analysis showed a marked increase in the number of dihydrofolate reductase genes in the resistant line. The genomic organization of the human dihydrofolate reductase gene was determined by hybridization with specific cDNA sequences from a human cDNA to DNA fragments from K-562 cells generated by restriction endonucleases. The human dihydrofolate reductase gene contained at least four intervening sequences and was approximately 30 kb in size. Northern blot studies demonstrated an increase of dihydrofolate reductase mRNA species; the predominant message was 3.8 kb. Karyotype analysis revealed three elongated marker chromosomes, derived from chromosomes 5, 6, and 19 which contained homogeneous staining regions, which were not present in the parent cell line.

Synthesis and Analysis of the Pteroylpolyglutamates

Publisher Summary This chapter discusses the synthesis and analysis of the Pteroylpolyglutamates. The biological importance of the polyglutamyl chain was recognized on the basis of its universal occurrence and by the fact that, during evolution, the ability to synthesize the peptide chain has been retained, even by organisms that have lost the pathways for folic acid synthesis. The polyglutamyl chain of the folates plays a role in the regulation of one-carbon metabolism. The polyglutamates are the best cosubstrates of the folate-requiring enzymes, and some of these enzymes show a clear preference for certain specific polyglutamyl chain lengths. The part played by the poly-γ-glutamyl chain in affecting the translocation of folates across cellular and subcellular membranes is established. This role acquires special practical significance when considering the pharmacodynamics of polyglutamyl derivatives of folic acid analogs possessing antitumor activity. Changes in the patterns of distribution of polyglutamates of different chain lengths occur under conditions, which alter the steady-state of one-carbon metabolism, a necessary effect when the polyglutamyl chain is involved in regulation. The properties of the enzymes of poly-glutamate biosynthesis and degradation are reviewed in the chapter.

Characterization of the enzyme complex involving the folate-requiring enzymes of de novo purine biosynthesis.

Evidence is presented for a functional association of GAR TFase and the trifunctional protein within the protein complex consisting of GAR TFase, AICAR TFase, Ser HMase, and trifunctional protein. Resolution of the trifunctional protein from the remaining enzymes in the complex causes a loss of GAR TFase activity which is regained upon recombination. The minimum stoichiometry for GAR TFase reactivation is 3:1 GAR TFase--trifunctional protein. Determination by ultracentrifugation of the sedimentation coefficient as a function of protein concentration disclosed that the complex is in mobile equilibrium with the individual proteins. However, mixed GAR TFase--trifunctional protein species can be detected by trapping with cleavable bifunctional cross-linking reagents. Additional support for their interaction is found in the kinetic coupling of the trifunctional protein and GAR TFase activities that leads to a fourfold more efficient formation of formylglycinamide ribotide commencing with formate rather than with 5,10-methenyl-H4folate triglutamate.

Folyl polyglutamate and folate-requiring enzymes as bacteriophage T4D baseplate structural components

The hexagonal-shaped baseplate on the tail of Escherichia coli bacteriophage T4D has been shown to contain 5–6 molecules of an unusual form of folic acid, dihydropteroyl hexaglutamate. In addition to this viral-induced small molecule, 2 viral-induced folate-requiring enzymes, namely dihydrofolate reductase ( frd) and thymidylate synthetase ( td), have also been found as phage baseplate components. The pteridine portion of the folate has been located near the baseplate frd and td and all 3 components are partially covered by T4D gene product 11 (gp11) which lies largely on the distal surface of the baseplate. On the other hand, the polyglutamate portion of the baseplate folic acid lies under or near the attachment region for the long tail fibers. Recent results have shown that the baseplate frd is a component of the outer wedge-like structures of the baseplate and highly suggestive evidence has now been obtained that the td molecule is a component of the central plug of the baseplate. These findings support the hypothesis that the baseplate folate plays a unique role in linking together the baseplate wedges and central plug by binding simultaneously to the wedge frd and to the plug td. This structural role for the viral folate and folate-requiring enzymes indicates that these baseplate components are not acting primarily in their usual roles as a coenzyme or as enzymes but are participants in a unique biological assembly process. Some molecular properties of dihydropteroyl hexaglutamate are presented which bear on its binding energies as a structural linking element. Finally some speculation is offered on the abiogenic origin of pteridine compounds.

Regulation of dihydrofolate reductase and other folate-requiring enzymes

Regulation of dihydrofolate reductase and other folate-requiring enzymes