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Enhancement of biological activity by conjugation reactions.

Conjugation reactions, such as the formation of sulfates, glucuronides and amino acid derivatives of biologically active compounds, are classically considered as detoxication processes. In essence, common nonpolar compounds are rendered less active by the formation of water-soluble metabolites, which then can readily be excreted in the urine. In the past decade, however, it has become clear that some natural metabolites become more active rather than less so by the formation of such conjugates. Several compounds in this special group will be considered in this article; namely, glucuronide and hydroxyphenylamide conjugates of retinoic acid, glutathione and cysteine conjugates of halogenated hydrocarbons, glucuronide conjugates of various xenobiotics and polyglutamates of folic acid and its inhibitors. The metabolism, biological activity and toxicity of the parent molecule is profoundly affected in complex ways by such reactions. Thus, the historical concept of conjugation reactions as general detoxication processes is no longer tenable.

Animals↗

The poly-alpha- and -beta-1,4-glucuronic acid moiety of teichuronopeptide from the cell wall of the alkalophilic Bacillus strain C-125.

Teichuronopeptide is a structural component of the cell wall of alkalophilic Bacillus strain C-125 and is a complex composed of polyglutamate and polyglucuronate. A structural analysis of the polyglucuronic acid moiety was carried out. Periodate oxidation and Smith degradation of the moiety, and enzymic analysis after reduction of glucuronic acid to glucose, revealed that glucuronic acid bound together with alternately alpha- and beta-1,4-linkages.

Bacillus↗

Regulation of drosophila folylpolyglutamates during development.

The polyglutamate status of reduced folates during the larval, pupal and adult stages of Drosophila melanogaster development was investigated. The chain length distribution is very similar and is predominantly pentaglutamate. Half-life estimates of the hydrolytic degradation to the monoglutamate showed larva less than pupa less than adult. This raises the possibility that polyglutamate hydrolase may have a role in regulating the total intracellular reduced folate content of the different developmental stages.

Animals↗

Cellular folates prevent polyglutamation of 5, 10-dideazatetrahydrofolate. A novel mechanism of resistance to folate antimetabolites.

Mouse L1210 cell variants were selected for resistance to 5, 10-dideazatetrahydrofolate, a potent inhibitor of the first folate-dependent enzyme in de novo purine synthesis, glycinamide ribonucleotide formyltransferase. The drug-resistant phenotype selected was conditional to the folate compound used to support growth: grown on folic acid cells were 400-fold resistant, whereas they were 2.5-fold more sensitive to 5,10-dideazatetrahydrofolate than wild-type L1210 cells when grown on folinic acid. In folic acid-containing media, polyglutamation of 5, 10-dideazatetrahydrofolate was markedly reduced, yet folylpolyglutamate synthetase activity was not different from that in parental L1210 cells. Resistance was due to two changes in membrane transport: a minor increase in the Km for 5, 10-dideazatetrahydrofolate influx, and a major increase in folic acid transport. Enhanced folic acid transport resulted in an expanded cellular content of folates which blocked polyglutamation of 5,10-dideazatetrahydrofolate. We propose that polyglutamation of 5,10-dideazatetrahydrofolate is limited by feedback inhibition by cellular folates on folylpolyglutamate synthetase, an effect which reflects a mechanism in place to control the level of cellular folates. Although the primary alteration causative of resistance is different from those reported previously, all 5, 10-dideazatetrahydrofolate resistance phenotypes result in decreased drug polyglutamation, reflecting the centrality of this reaction to the action of 5,10-dideazatetrahydrofolate.

Animals↗

Autoantibodies-like antigen binding characteristics of induced antibodies against polylysine-polyglutamate complex.

Rabbits immunized with polylysine-polyglutamate complex induced high titer antibodies. The immune IgG exhibited high degree of specificity towards the immunogen. In a competition assay, nDNA, heat denatured DNA and total buffalo thymus RNA were inhibitory. An appreciable binding with polyglutamate, poly(rG).poly(dC) poly(dG).poly(dC), cardiolipin, poly(dA-dU). Poly(dA-dU) and Z- or Z-like conformations demonstrates the polyspecificity of induced antibodies. The immunological specificity of induced antibodies was comparable to autoantibodies derived from SLE. The results might be of some help in understanding the etiology of autoimmune process.

Animals↗

Characterization of human folylpolyglutamate synthetase expressed in Chinese hamster ovary cells.

Chinese hamster AUX B1 cells lack the enzyme folylpolyglutamate synthetase (FPGS) responsible for adding polyglutamates to folic acid. The human gene for FPGS was introduced into AUX B1 cells by cotransfection with human genomic DNA and either pSV2neo or pSV2gpt plasmid DNA. Cells were coselected for FPGS expression by growth in the absence of glycine, adenosine, and thymidine, and for drug resistance by growth in geneticin or mycophenolic acid. The presence of human enzyme in transfected cells was identified by relative enzyme activity determinations with ATP or dATP as substrates. The human HeLa cell enzyme displayed a 50% higher activity with dATP, and the hamster enzyme showed a 50% higher activity with ATP. Hamster and human enzymes also differed in the chain length of polyglutamates on cellular folate. Only monoglutamates were detected in AUX B1 cells, while wild-type hamster cells had predominantly hexa- and heptaglutamates, and human HeLa cells had predominantly hepta- and octaglutamates. Transformants with FPGS activity that showed a human enzyme preference for dATP also had folate polyglutamate chain lengths characteristic of the human enzyme.

Animals↗

Zinc deficiency and dietary folate metabolism in pregnant rats.

Five groups of pregnant Wistar rats (zinc-deficient diet without folate supplementation; folinic acid, folate monoglutamate, folate polyglutamate-supplemented groups receiving zinc-deficient diet; pair-fed groups as controls) were fed from day one of fertilization with a semisynthetic zinc-deficient diet containing 0.2 mg/kg of Zn in the diet for the 4 deficient groups and 100 mg/kg for the pair-fed group. After 20 days, the zinc status (plasma, liver, femoral bone) was significantly decreased in the zinc-deficient groups. The liver and plasma folate levels were lower in the zinc-deficient groups compared to the pair-fed group. Moreover, the folinic acid and the polyglutamate folate supplementations (100 mg/kg diet) did not normalize the folate status of the animals. Only the supplementation with folate monoglutamate led to correct folate levels in the pregnant rats. Nevertheless, no form of folate supplementation prevented fetal growth retardation in any of the zinc-deficient groups. These results indicate that zinc deficiency in pregnant rats decreases folate bioavailability of folinic acid, folate polyglutamates and, to a lesser extent, that of folate monoglutamate. However, no form of folate supplementation (i.e., folate monoglutamate) prevents fetal growth defect and the incidence of malformation in zinc-deficient rats.

Animals↗

Folate analogues as substrates of mammalian folylpolyglutamate synthetase.

The antifolate drugs methotrexate (MTX) and aminopterin (AM) have been tested as substrates for folylpolyglutamate synthetase (FPGS) partially purified from beef liver. The Km for MTX is 100 microM, and that for AM is 25 microM. These values are considerably higher than those for either tetrahydrofolate or folinic acid. Based on their ratios of Vmax to Km, AM is a better substrate than is MTX for the beef liver FPGS. Both are poorer substrates than tetrahydrofolate. The 7-hydroxy metabolites of MTX and AM also are substrates for FPGS. The reactivity of 7-hydroxymethotrexate is similar to that of MTX, but 7-hydroxyaminopterin is a poorer substrate than AM. Folinic acid, often used as the rescue agent in high-dose MTX therapy, has a low Km with mammalian FPGS (7 microM). Its activity is comparable to that of the best substrate, tetrahydrofolate. Low concentrations of folinic acid prevent the formation of polyglutamates of MTX. This inhibition is competitive, presumably because folinic acid and MTX are competing substrates for FPGS. The activities of folate and antifolate substrates also have been determined with rat liver FPGS. With near-saturating concentrations of AM, MTX, or 7-hydroxymethotrexate, the reaction velocity exceeds that with an optimal concentration of tetrahydrofolate. However, the Km values of the folate analogues all are greater than those of the tetrahydrofolate coenzymes. In contrast to the formation of long-chain polyglutamates observed when tetrahydrofolate or folinic acid was the substrate, beef liver FPGS, under our reaction conditions, cannot catalyze the formation from MTX monoglutamate of polyglutamates longer than the triglutamate. MTX di- and triglutamates are poorer substrates than is MTX itself. Longer polyglutamates of MTX, while having no activity as substrates, must bind to the enzyme, because they are inhibitors. Our observations using MTX and AM with the enzymatic FPGS system help to rationalize the therapeutic use of antifolates.

Aminopterin↗

The effect of methotrexate on intracellular folate pools in human MCF-7 breast cancer cells. Evidence for direct inhibition of purine synthesis.

This report details the effects of methotrexate on the intracellular folate pools of the MCF-7 human breast cancer cell line. To achieve this goal, we designed a high-pressure liquid chromatography system capable of separating the physiologic folates. The folate pools were quantitated following growth and equilibration in 2.25 microM radiolabeled folic acid. Each of the intracellular folates was identified by coelution with standard folates and by chemical/biochemical tests unique to each of the various folates. The 10-formyl-H4PteGlu (where H4PteGlu represents dl-tetrahydrofolic acid) pool accounted for 20.5% of the total intracellular folate pool in untreated cells, whereas 5-formyl-H4PteGlu and H4PteGlu accounted for 6.5 and 10.6%, respectively. The levels of these three folates remained stable throughout cell growth. The 5-methyl-H4PteGlu pool accounted for less than 10% in early growth phase cells but assumed greater than 60% of the total pool by the mid- and late-log phases of cell growth. When the MCF-7 cells were exposed to 1 microM methotrexate, de novo purine synthesis and de novo thymidylate synthesis were rapidly inhibited to less than 20% of control within 3 h. During this time period, rapid alterations in the folate pools also occurred such that dihydrofolic acid levels rose from less than 1% in untreated cells to greater than 30% of the total pool. This rise was accompanied by a parallel fall in 5-methyl-H4PteGlu. H4PteGlu and 5-formyl-H4PteGlu were undetectable following 2 h of methotrexate exposure, but 10-formyl-H4PteGlu, the required cosubstrate for de novo purine synthesis, was preserved at greater than 80% of pretreatment values following a 1 microM methotrexate exposure of up to 21 h. The rapid inhibition of de novo purine synthesis in these cells following methotrexate exposure coupled with a relatively preserved 10-formyl-H4PteGlu pool suggests direct inhibition of this synthetic pathway by the temporally coincident accumulation of dihydrofolic acid and/or methotrexate polyglutamates. This inhibition cannot be ascribed to depletion of the folate cofactor 10-formyl-H4PteGlu.

Breast Neoplasms↗

Folylpolyglutamates as substrates and inhibitors of folate-dependent enzymes.

The true intracellular substrates for folate-dependent enzymes are folylpolyglutamates. We have used measurements of the Ki values of folylpolyglutamate dead end inhibitors to assess the relative affinities of folate-dependent enzymes for folate derivatives of different polyglutamate chain lengths. Studies of four enzymes from pig liver, methylenetetrahydrofolate reductase, serine hydroxymethyltransferase, methylenetetrahydrofolate dehydrogenase and thymidylate synthase, have indicated that folylpolyglutamate inhibitors are bound 3-500 fold more tightly than the corresponding monoglutamates. The individual enzymes differ in their selectivity for polyglutamate vs. monoglutamate inhibitors, and in the chain length associated with the greatest affinity of enzyme for inhibitor. We have also examined the effect of polyglutamate chain length on the catalytic parameters associated with folate substrates. Two enzymes, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase, show decreases in Km values for folylpolyglutamate substrates. Methylenetetrahydrofolate dehydrogenase shows no detectable differences in the catalytic parameters of polyglutamate vs. monoglutamate substrates and no change in the order of substrate addition or product release. Thymidylate synthase shows small effects of Km and Vmax values, but the order of addition of substrates and of release of products is reversed with polyglutamate as compared with monoglutamate substrates. Our studies with thymidylate synthase from L. casei have shown that the bacterial enzyme also exhibits a greatly increased affinity for polyglutamate vs. monoglutamate derivatives of folic acid, and that reversal in the order of substrate addition and product release also occurs with polyglutamate as compared with monoglutamate substrates. We have also studied the polyglutamate specificity of methionine synthase, which is responsible for the conversion of CH3-H4PteGlu1 into H4PteGlu1. This reaction is required for the incorporation of plasma folate into the cellular folate pool, because methyltetrahydrofolate is a poor substrate for folylpolyglutamate synthetase. Our studies demonstrate that CH3-H4PteGlu6, and suggest that incorporation of plasma CH3-H4PteGlu1 will only occur when methylenetetrahydrofolate reductase is inhibited by adenosylmethionine and cellular pools of CH3-H4PteGlu6 are at very low levels.

Animals↗

Synthesis and biological evaluation of N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine.

A novel folic acid analogue, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine, 3, was prepared via a multistep synthetic sequence. The key steps involved the conversion of 5-deazapteroic acid to its N10-formyl derivative followed by catalytic hydrogenation of the pyridine ring and subsequent heating in dilute sodium hydroxide to afford the new 5-deaza-5,6,7,8-tetrahydropteroic acid. After trifluoroacetylation, this compound was coupled to N delta-(tert-butyl-oxycarbonyl)-L-ornithine using conventional peptide bond forming conditions. Deprotection first in base and then in acid gave the title compound. Compound 3 was an effective inhibitor of hog liver folylpolyglutamate synthetase (Kis, estimated = 64 nM), and was shown to retard the formation of polyglutamates of a structurally related folic acid analogue in HCT-8 cells in vitro.

Adenocarcinoma↗

Methotrexate analogues--XVII. Antitumor activity of 4-amino-4-deoxy-N10-methylpteroyl-D,L-homocysteic acid and its dual inhibition of dihydrofolate reductase and folyl polyglutamate synthetase.

A new analogue of methotrexate was synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and D,L-homocysteic acid. The product (mAPA-HCysA) was bound tightly to L1210 mouse leukemia dihydrofolate reductase (IC50 = 1 nM), inhibited L1210 cell proliferation in culture (IC50 = 0.3 microM), and prolonged the survival of L1210 leukemic mice (98% increase in lifespan at 120 mg/kg, qdx9). Studies on the interaction of mAPA-HCysA with partially purified mouse liver folyl polyglutamate synthetase revealed that mAPA-HCysA was not a substrate. Hence, the increased dose of mAPA-HCysA required to inhibit tumor growth in vitro and in vivo relative to methotrexate may reflect, in part, the inability of this compound to form non-effluxing polyglutamates. Folyl polyglutamate synthetase was competitively inhibited by mAPA-HCysA (K1 = 190 +/- 70 microM) when folate was the variable substrate. Thus, mAPA-HCysA is the first known compound to inhibit both mammalian dihydrofolate reductase and mammalian folyl polyglutamate synthetase.

Animals↗

Simultaneous determination of methotrexate and its eight metabolites in human whole blood by capillary zone electrophoresis.

A simple and selective capillary electrophoretic method was established for the simultaneous determination of methotrexate (MTX), 7-hydroxymethotrexate (7-OHMTX), 2,4-diamino-N10-methylpteroic acid (DAMPA), and polyglutamate derivatives [MTX-(Glu)n, n=2-7] in whole blood. After extraction, those analytes were separated by fused-silica capillary and a running buffer containing glycine (1.2 M, pH 9.3). The quantitative ranges were 10-50 microM for each analyte. The intra- and inter-day RSD and RE values were all less than 6 and 11%, respectively. The limits of detection (S/N= 3, injection 5 s) were found to be 1 microM for MTX, 7-OHMTX, MTX-(Glu)2, MTX-(Glu)3, and MTX-(Glu)4; 3 microM for MTX-(Glu)5 and MTX-(Glu)6; 5 microM for MTX-(Glu)7, and 8 microM for DAMPA. All recoveries were greater than 94%. This method was applied to blood MTX monitoring in a patient with acute lymphoblastic leukemia.

Antimetabolites, Antineoplastic↗

Dose dependent retention and polyglutamation of N10-propargyl-5,8-dideazafolic acid (CB 3717) in the tumour-bearing mouse.

The antifolate drug, CB 3717, when given to the tumour-bearing mouse, was retained in its liver and kidneys and to a smaller extent (50-200 fold) in EAC-cells. The amount of the drug retained in the tumour cells, but not in the host organs, depended on the route of its administration, being distinctly higher after i.p. than after s.c. injection. The extensive polyglutamation of CB 3717 occurred in the liver only after administration of the drug at 20 but not at 100 mg per kg. CB 3717 polyglutamation in the kidneys was low and in EAC-cells negligible.

Animals↗

Biological properties of fluoroglutamate-containing analogs of folates and methotrexate with altered capacities to form poly (gamma-glutamate) metabolites.

Fluoroglutamate-containing analogs of folates and methotrexate (MTX) with altered capacities for poly (gamma-glutamate) metabolism were synthesized to probe the biological roles of polyglutamates. Compared to folic acid, DL-e,t-gamma-fluorofolic acid, a compound that is a poor substrate for polyglutamylation, was approximately 25-fold less potent in promoting growth of folate-depleted H35 rat hepatoma cells. DL-beta,beta-Difluorofolic acid, a compound that forms diglutamates more readily than does folic acid, was at least equivalent to folic acid in potency. Leucovorin (LV), a reduced folate, was 30-fold more potent than folic acid in promoting growth, whereas the analogous form of DL-e,t-gamma-fluorofolate, DL-e,t-gamma-fluoroleucovorin (DL-e,t-gamma-FLV) was only 4-fold more potent than folic acid. Both LV and DL-e,t-gamma-FLV protected or "rescued" cells from the growth inhibitory effects of MTX; however a 37- to 46-fold higher concentration of the fluoro analog was required. Folic acid, DL-e,t-gamma-fluorofolic acid, LV, and DL-e,t-gamma-FLV each potentiated the growth inhibitory effect of 5-fluoro-2'-deoxyuridine on CCRF-CEM human leukemia cells; higher concentrations of fluorinated analogs again were required. Stereochemically pure L-t-gamma-fluoromethotrexate (L-t-gamma-FMTX), a poor substrate for polyglutamylation, was evaluated as a cell growth inhibitor. In continuous exposure, L-t-gamma-FMTX), was 7-fold less potent than MTX as an inhibitor of CCRF-CEM growth. Results with these fluorinated folate and MTX analogs offer insight into the importance of polyglutamate metabolism to these biological and pharmacological effects.

Animals↗

A study of folate absorption and metabolism in man utilizing carbon-14--labeled polyglutamates synthesized by the solid phase method.

The absorption and metabolism of synthetic polyglutamates of folic acid have been compared with free pteroylglutamic acid in four subjects having chronic lymphatic leukemia and one with Hodgkin's granuloma. Pteroylpolyglutamates containing either three or seven glutamate residues were prepared by the solid-phase method permitting placement of carbon-14 labels in either the pteridine ring or in a selected glutamate unit of the gamma peptide chain. Complete dissociation was observed between biological folate activity and radioactivity of plasma after ingestion of pteroyltriglutamate labeled in the middle glutamate. This indicates cleavage to the monoglutamate form at the time of absorption from the intestine or very soon thereafter. A large portion of radioactivity liberated from the middle glutamate is recoverable as carbon dioxide in the exhaled air. Fecal losses of folate tended to be greater with increasing length of the poly-gamma-glutamyl chain. Higher blood levels and greater urinary losses of folate tended to occur after ingestion of mono- and triglutamates than with the heptaglutamate. Calculations based on radioactivity determinations in feces plus urinary folate losses, judged by either radioactivity or microbiological assays, indicated net retention of 37-67% of the dose irrespective of chain length ingested and major avenue of loss. During the peak of absorption the folate circulating in plasma was active for both Streptococcus fecalis and Lactobacillus casei and carried specific radioactivity which was virtually identical with that of the administered dose. This suggests that neither methylation, conjugation, nor displacement of nonradioactive folate occurred to any significant extent during the 1st 2 hr. The specific radioactivity of 24-hr urine specimens as measured with L. casei corresponded closely with that of the administered dose. Evidence exists that methylation of the radioactive folate may occur, but significant displacement of nonradioactive methylfolate was not observed under the conditions of this study. Since 50-75% of administered heptaglutamate appears to be absorbable in man, estimates of dietary intake should include this fraction as well as the "free" folate.

Adult↗

Methenyltetrahydrofolate synthetase regulates folate turnover and accumulation.

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation.

Biological Transport↗