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PubMed1991

Synthesis and biological activity of the 2-desamino and 2-desamino-2-methyl analogues of aminopterin and methotrexate.

The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.

Aminopterin↗
PubMed1991

Thymidylate synthase inhibitors: the in vitro activity of a series of heterocyclic benzoyl ring modified 2-desamino-2-methyl-N10-substituted-5,8-dideazafolates.

Heterocyclic para-aminobenzoate modifications of 2-desamino-2-methyl-5,8-dideazafolic acid and a series of its N10-substituted analogs have produced a number of interesting compounds that have enabled a deeper understanding of the biochemical events required for activity in this class of antimetabolite. There is a relationship that has become apparent between compound potency and both uptake via the reduced-folate carrier and FPGS substrate activity. Rapid cellular uptake and metabolism of polyglutamate forms that are approximately 100-fold more potent as inhibitors of TS can translate a modest TS inhibitor such as ICI D1694 into a very potent inhibitor of cell growth (approximately 500- and approximately 10-fold more potent than CB3717 or ICI 198583, respectively). Polyglutamation may therefore act as an almost essential activation step and ICI D1694 may be highly specific for tumors expressing both the reduced-folate carrier and FPGS. Polyglutamation of folate analogs also leads to drug retention which may play a major role in the pharmacodynamics of TS inhibition by ICI D1694 in vivo. Current studies with 3H-ICI D1694 are aimed at demonstrating metabolism to polyglutamates in tumor cells. The serious toxic limitations of CB3717, i.e., liver and kidney toxicities, are not seen with ICI D1694 reflecting the good water solubility of the drug compared with CB3717. The toxicities observed in mice are however to hematological tissues and are due to its TS inhibitory effects. Thus ICI D1694 may elicit toxicities in man more typical of an antimetabolite than of CB3717. The clinical evaluation of ICI D1694 may further our understanding of the role that metabolism to polyglutamates may have in therapeutic activity.

Animals↗
PubMed1993

Gamma-linked dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8- dideazafolate as antitumour agents.

Our search for water-soluble quinazoline TS inhibitors that are transported into cells via the RFC, but are not substrates for FPGS, led us to the synthesis of dipeptide analogues of ICI 198583 diglutamate. Although a number of dipeptide analogues were active against isolated TS and L1210 cells in vitro, lack of in vivo stability was a problem. This was circumvented by the synthesis of modified dipeptides where either the alpha-carboxyl of the second amino acid was removed (alpha'-COOH) e.g. -L-glu-GABA or where the second amino acid was the unnatural D-enantiomer e.g.-L-glu-D-glu. Further studies were performed with the -L-glu-D-glu and its 7-CH3, 2'F modified analogue, demonstrating that they use the RFC for cell entry but are not active through polyglutamate formation. The latter compound was tested against experimental tumour models and found to have good activity.

Animals↗
PubMed1984

Studies on the polyglutamate specificity of methylenetetrahydrofolate dehydrogenase from pig liver.

Methylenetetrahydrofolate dehydrogenase, which is one of the activities of a trifunctional folate-dependent enzyme isolated from pig liver, displays an ordered bi-bi kinetic mechanism when methylenetetrahydropteroylmonoglutamate is used as the folate substrate [Cohen, L., & MacKenzie, R. E. (1978) Biochim. Biophys. Acta 522, 311-317]. We have studied the inhibition of this activity by a series of pteroylglutamates containing one to seven glutamyl residues. Inhibitors with one to four glutamyl residues exhibit a kinetically determined KD of about 56 microM for binding at the folate site of the enzyme, while inhibitors with five to seven glutamyl residues exhibit a KD of about 16 microM. These results suggest that folylpolyglutamates are bound to the trifunctional enzyme relatively weakly, with the major interaction involving the fifth glutamyl residue of the polyglutamate "tail". A free energy decrease of about 0.74 kcal (3.1 kJ) is associated with this interaction. The possibility of a swinging arm mechanism for the trifunctional enzyme is discussed. We have also measured the kinetic parameters Vmax and the Km values for NADP+ and the folate substrate associated with catalysis using a series of methylenetetrahydropteroylpolyglutamate substrates. The variation in these parameters with the length of the polyglutamate tail is small.

Animals↗
PubMed1990

Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells.

We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 micron MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 micron MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.

Breast Neoplasms↗
PubMed2000

Molecular analysis of murine leukemia cell lines resistant to 5, 10-dideazatetrahydrofolate identifies several amino acids critical to the function of folylpolyglutamate synthetase.

Four L1210 murine leukemia cell lines resistant to 5, 10-dideazatetrahydrofolate (DDATHF) and other folate analogs, but sensitive to continuous exposure to methotrexate, were developed by chemical mutagenesis followed by DDATHF selective pressure. Endogenous folate pools were modestly reduced but polyglutamate derivatives of DDATHF and ALIMTA (LY231514, MTA) were markedly decreased in these mutant cell lines. Membrane transport was not a factor in drug resistance; rather, folypolyglutamate synthetase (FPGS) activity was decreased by >98%. In each cell line, FPGS mRNA expression was unchanged but both alleles of the FPGS gene bore a point mutation in highly conserved domains of the coding region. Four mutations were in the predicted ATP-, folate-, and/or glutamate-binding sites of FPGS, and two others were clustered in a peptide predicted to be beta sheet 5, based on the crystal structure of the Lactobacillus casei enzyme. Transfection of cDNAs for three mutant enzymes into FPGS-null Chinese hamster ovary cells restored a reduced level of clonal growth, whereas a T339I mutant supported growth at a level comparable to that of the wild-type enzyme. The two mutations predicted to be in beta sheet 5, and one in the loop between NH(2)- and COOH-terminal domains did not support cell growth. When sets of mutated cDNAs were co-transfected into FPGS-null cells to mimic the genotype of drug-selected resistant cells, clonal growth was restored. These results demonstrate for the first time that single amino acid substitutions in several critical regions of FPGS can cause marked resistance to tetrahydrofolate antimetabolites, while still allowing cell survival.

Amino Acid Sequence↗
PubMed1994

Folate Metabolism in Datura innoxia (In Vivo and in Vitro Folylpolyglutamate Synthesis in Wild-Type and Methotrexate-Resistant Cells).

In vivo folylpolyglutamate pools of the wild-type (Px4) and methotrexate-resistant (MTX161) Datura innoxia cell lines were detected by incorporation of [14C]p-aminobenzoate into folates. The folylpolyglutamate derivatives were cleaved to p-aminobenzoylpolyglutamates and separated according to glutamyl chain length by high-performance liquid chromatography. Hexaglutamates were the predominant form in both Datura cell lines. The proportions of individual folylpolyglutamates were unaffected by culturing the cells in medium containing products of one-carbon metabolism such as glycine, adenine, thymidine, or methionine. Radiolabeling of the hexaglutamates was greatly reduced in the presence of 10-8 M methotrexate (MTX) in the Px4 cells but not in the MTX161 cells. Tetrahydrofolate, 5, 10-methylenetetrahydrofolate, and folinic acid were effective substrates for the folylpolyglutamate synthetase from Datura cells in vitro, whereas MTX and folate were poor substrates. In vivo, MTX can be slowly converted into its polyglutamate derivatives up to MTXGlu4 or MTXGlu5 in Datura cells in the longer term. Significantly lower levels of MTX polyglutamates in MTX161 cells were found compared with those of Px4 cells during prolonged (10 d) exposure to MTX. Although in vivo and in vitro folylpolyglutamate synthesis was found to be similar in both cell lines, about a 4-fold increase in specific activity of [gamma]-glutamyl hydrolase (GGH) was detected in the MTX161 cell line. The increase in GGH in the resistant cells suggested that breakdown of polyglutamylated forms of MTX may play a role in acquired MTX resistance.

Journal Article↗
PubMed1987

Mechanism of action of 5,8-dideazaisofolic acid and other quinazoline antifols in human colon carcinoma cells.

The clonal cytotoxic effects and mechanism of action of a new series of 2-amino-4-hydroxyquinazoline folate analogues (5,8-dideazafolates) have been assessed using the human colon tumor cell line HCT-8. Of these compounds only 5-methyl-5,8-dideazafolate was potentially more effective than a compound previously identified, 5,8-dideazaisofolate (H-338, NSC 289517). HCT-8 sublines resistant to methotrexate, 5-fluorodeoxyuridine, and H-338 were either minimally or not cross-resistant to the other agents. The cytotoxicity of H-338 was strongly dependent on the time of exposure; at exposure times shorter than 8 h it was essentially nontoxic. Thymidine alone, as well as leucovorin or folic acid, protected against the cytotoxic effects of H-338. This is consistent with thymidylate synthase (TS) as its only locus of action. Studies with dihydrofolate reductase and TS isolated from HCT-8 cells indicated that these quinazolines were weaker inhibitors of dihydrofolate reductase than was methotrexate, but they were not particularly potent TS inhibitors. However, synthetic poly-gamma-glutamate derivatives of quinazolines showed dramatically increased TS, but not dihydrofolate reductase, inhibition. TS inhibition increased as the polyglutamate chain length increased. Using isolated HCT-8 folylpolyglutamate synthetase, all the parent quinazolines containing L-glutamate were found to be substrates. With H-338, the results indicated that tetraglutamate or longer derivatives could be synthesized intracellularly. These results are consistent with our hypothesis that cytotoxicity by such quinazolines necessarily involves "lethal synthesis" from a prodrug; i.e., the nontoxic parent drug must be converted to polyglutamates before TS inhibition and subsequent cytotoxicity can occur.

Cell Survival↗
PubMed1985

Protein binding as a component of drug interaction in cellular pharmacokinetic studies. Effects of probenecid on transport and accumulation of methotrexate in Ehrlich ascites tumor cells in vitro.

The organic acid probenecid has been shown to interfere with the active extrusion of methotrexate (MTX) from L1210 tumor cells in vitro leading to enhanced MTX accumulation and increased formation of MTX polyglutamate derivatives. In the presence of serum albumin (4 g/100 ml), to which probenecid is bound, the inhibition by probenecid of [3H]MTX efflux from the Ehrlich ascites tumor cell was reduced markedly. While half-maximal inhibition of MTX efflux occurred with 0.12 mM probenecid in the absence of albumin, 1.45 mM probenecid was required in its presence. The presence of albumin also modified the probenecid-induced elevation of steady-state MTX levels in the tumor cell. Maximal elevation of cellular MTX levels occurred with 0.5 mM probenecid in the absence of albumin, and 3 mM probenecid in its presence. Serum albumin further reversed the effects of probenecid on MTX influx. While probenecid inhibited influx of 1 microM [3H]MTX in the absence of albumin (half-maximal inhibition at approximately 1 mM probenecid), probenecid stimulated MTX influx in its presence (half-maximal effect at 0.5 to 1 mM). Equilibrium dialysis studies demonstrated that probenecid displaced MTX from albumin, increasing the effective free concentration of MTX in the incubation medium, and hence the rate of MTX influx. Therefore, probenecid may enhance the accumulation of MTX in the tumor cells by increasing the level of free (as opposed to albumin bound) MTX in the extracellular medium as well as by direct inhibition of MTX efflux. These observations may provide an additional explanation for probenecid enhancement of the therapeutic efficacy of MTX in tumor bearing mice and highlight the importance of assessing drug-protein interactions in an in vitro experimental model.

Animals↗
PubMed2003

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line.

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.

Animals↗
PubMed1994

Folylpolyglutamate synthetase in Lactobacillus leichmannii: in vitro synthesis and characterization of polyglutamylfolates.

In vitro synthesis of folylpolyglutamates by folylpolyglutamate synthetase from Lactobacillus leichmannii has been studied and optimal conditions for enzyme activity determined. It is found that while ATP (5 mM) is essential for the synthesis of folylpolyglutamates homocysteine augments the same. Replacement of vitamin B12 (2 ng/ml) with deoxyuridine (20 micrograms/ml) in growth medium does not alter the enzymatic parameters studied. DEAE-cellulose column chromatography of in vitro synthesised folylpolyglutamates indicates that folylpolyglutamate synthetase of L. leichmannii can synthesize polyglutamates up to a chain length of four glutamate residues.

Lactobacillus↗
PubMed2002

Intestinal absorption of different types of folate in healthy subjects with an ileostomy.

Our knowledge on the absorption of folate is incomplete. The deconjugation process as a possible limiting factor in the absorption of folates was investigated. The study also attempted to validate the use of the area under the serum response curve (AUC) from food compared with folic acid as a proxy variable for food folate bioavailability. Folate absorption was determined in healthy ileostomy volunteers (n 11) using a single-dose short-term protocol. In a randomised crossover design, volunteers received spinach meals and a supplement. Based on analysis of test meals and ileostomy effluents, there was no difference in folate absorption between spinach with a mono-:polyglutamate ratio 40:60 and the same spinach with a 100:0 ratio. The absolute absorption of spinach folate (79 %) calculated from the difference between folate intake and folate content of ileostomy effluents was approximately equal to the relative absorption (81 %) calculated from the AUC after consumption of spinach meals in relation to the AUC after consumption of the folic acid supplement. We conclude that the deconjugation process is not a limiting factor in the absorption of spinach folates. Comparison of AUC of food folate v. folic acid in a short-term protocol may be suitable for assessing food folate bioavailability.

Adult↗
PubMed1984

The metabolism of [3H] pteroylglutamic acid in rat liver during regeneration after partial hepatectomy.

The rate of [3H] PteGlu uptake and of its incorporation into pteroylpolyglutamate derivatives at different times after label administration, was studied in the regenerating liver of rats 36 hr after partial hepatectomy. While 1 hr after administration of labelled PteGlu the radioactivity levels in regenerating livers are quite similar to controls, they are markedly higher 6, 12, 24 hr after label injection, which is mostly due to the high polyglutamate content. If the regenerating process, on the one hand does not modify the folate uptake rate, it does, on the other, increase the capacity for retaining folates, thanks to a more powerful ability to convert them into pteroylpolyglutamates.

Animals↗
PubMed1994

Crystal structure of thymidylate synthase from T4 phage: component of a deoxynucleoside triphosphate-synthesizing complex.

Thymidylate synthase from phage T4 (T4TS) is part of a complex of several enzymes required for coordinate DNA synthesis in infected Escherichia coli cells. It has been proposed that similar complexes of enzymes related to DNA synthesis are also functional in eukaryotes [Pardee, A. B. (1989) Science 246, 603-608]. To delineate the role of structure in the function of this complex, we have solved the structure of T4TS as a basis for mapping the complex by mutagenesis. The 3.1 A structure of the unliganded enzyme was determined by molecular replacement and refined to 19.9% for all data. Three inserts and one deletion in the coding region are unique to T4TS, and all sites lie on one side of the enzyme surface, possibly encoding unique T4 specific intermolecular interactions during the infective cycle. The crystal structure is generally in the open, unliganded conformation seen in unliganded E. coli TS, as opposed to the closed, ternary complex conformation, except that the critically important C-terminus is inserted into the active site hydrogen bonded to residue Asn85, as seen in functional ternary complex structures. Other differences between E. coli TS and T4TS appear to explain the enhanced binding of folyl polyglutamate to the latter.

Amino Acid Sequence↗
PubMed1996

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.

Animals↗
PubMed1990

Multiple membrane transport systems for the uptake of folate-based thymidylate synthase inhibitors.

N10-Propargyl-5,8-dideazafolic acid (CB3717) and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI-198,583) are potent folate-based inhibitors of thymidylate synthase. We studied the membrane transport and the growth-inhibitory effects of the two thymidylate synthase inhibitors on human CCRF-CEM leukemia cells with different transport properties for folic acid, reduced folates, and methotrexate (MTX). Membrane transport of [3H]ICI-198,583 can proceed via the high affinity/low capacity reduced folate carrier as supported by findings that (a) uptake of [3H]ICI-198,583 was significantly impaired in CEM cells which have a transport defect for MTX, (b) variants of CEM cells which overproduce the reduced folate carrier system showed a concomitant increase in the uptake of [3H]ICI-198,583 as for [3H]MTX, (c) MTX inhibited transport of [3H]ICI-198,583, and (d) uptake of [3H]ICI-198,583 was inhibited after treatment of CEM cells with an N-hydroxysuccinimide ester of MTX, which is a potent inhibitor of MTX transport. However, a membrane-associated folate-binding protein (FBP) offers another route for entry of CB3717 and ICI-198,583. CEM-FBP cells that have an elevated amount of FBP and do not have a functional reduced folate carrier were 640- and 61-fold more sensitive to CB3717 and ICI-198,583, respectively, compared to control CEM cells expressing the reduced folate/MTX carrier. This high sensitivity was related to a high affinity of the FBP for CB3717 and ICI-198,583 (Kd 2-3 nM), which is only 3-fold lower than for folic acid (Kd 1 nM) but significantly higher than for MTX (Kd 100 nM). Furthermore, after incubation of CEM-FBP cells for 24 h at 10 nM [3H]ICI-198,583, the high affinity binding of the FBP for ICI-198,583 allowed a 600-fold concentrative uptake of [3H]ICI-198,583 and its conversion to polyglutamate forms. These results indicate that multiple folate transport systems may be involved in the uptake of folate-based thymidylate synthase inhibitors.

Antineoplastic Agents↗