Human Folylpolyglutamate Synthetase Research Articles

Maternal genotypes of folate/one-carbon metabolism gene variants and nonsyndromic cleft lip with or without cleft palate risk in Chile.

The aim of this study was to evaluate, in a case-control design, the association between maternal genotypes for variants in 23 genes involved in folate/one-carbon metabolism and nonsyndromic cleft lip with or without cleft palate (NSCL/P) in a Chilean population. After applying several filters to an Illumina array, we extracted 175 single nucleotide polymorphisms (SNPs) from 150 mothers of NSCL/P cases and 150 control women. Association was evaluated using computed odds ratio (OR) with a 95% confidence interval (95% CI) in additive, recessive, and dominant models. After multiple comparison correction, only SNP rs4451422 (A>C), located 237bp downstream of the gene encoding the human folylpolyglutamate synthetase (FPGS), maintained a significant association with NSCL/P in the offspring (OR 3.03; 95% CI 1.69-5.26). The variant rs4451422 is associated with a decrease in FPGS expression according to database annotation. Our results lead to a new hypothesis that a lower activity of FPGS enzyme reduces intracellular folate levels and increases the risk of an offspring having NSCL/P.

Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate

Phosphorus-containing pseudopeptides, racemic at the C-terminal α-carbon, are potent mechanism-based inhibitors of folylpolyglutamate synthetase (FPGS). They are mimics of the tetrahedral intermediate postulated to form during FPGS-catalyzed biosynthesis of poly(γ- l-glutamates). In the present paper, the FPGS inhibitory activity of each diastereomer coupled to three heterocycles is reported. The high R f pseudopeptide containing the 5,10-dideazatetrahydropteroyl (DDAH 4Pte) heterocycle is most potent ( K is = 1.7 nM). While the heterocyclic portion affects absolute FPGS inhibitory potency, the high R f species is more potent in each pair containing the same heterocycle. This species presumably has the same stereochemistry as the natural folate polyglutamate, i.e., ( l-Glu-γ- l-Glu). Unexpectedly, the low R f (presumed l-Glu-γ- d-Glu) species are only slightly less potent (<30-fold) than their diastereomers. Further study of this phenomenon comparing l-Glu-γ- l-Glu and l-Glu-γ- d-Glu dipeptide-containing FPGS substrates shows that <1% contamination of commercial d-Glu precursors by l-Glu may give misleading information if l-Glu-γ- l-Glu substrates have low K m values.

Submitochondrial localization of the mitochondrial isoform of folylpolyglutamate synthetase in CCRF-CEM human T-lymphoblastic leukemia cells

Earlier studies from this laboratory showed that human folylpolyglutamate synthetase (FPGS) exists as cytosolic and mitochondrial (mFPGS) isoforms. Localization of mFPGS within mitochondria may help elucidate how the enzyme functions to maintain the mitochondrial folate pool. A human T-lymphoblastic leukemia CCRF-CEM cell lysate was fractionated by differential centrifugation into cytosolic and mitochondrial fractions. Activity assays for cytosol-and mitochondria-specific enzymes verified the purity and integrity of the fractions. Mitochondria were subfractionated with increasing concentrations of digitonin to successively extract the four submitochondrial compartments. Western analyses of the fractions using protein markers specific for each compartment suggest that mFPGS is distributed in the matrix and/or inner membrane compartments. Further support for an interaction of mFPGS with the inner mitochondrial membrane is provided by localization of about half of the mFPGS in the mitochondrial membrane fraction obtained by freeze–thaw of intact mitochondria; the remaining mFPGS is located in the soluble fraction. Resistance of about half of the mFPGS in whole mitochondria to alkaline carbonate extraction suggests that its interaction with the inner membrane is more similar to an integral, than a peripheral, membrane protein. The data suggest that human mFPGS is at least in part strongly associated with the inner mitochondrial membrane.

Escherichia coli FolC Structure Reveals an Unexpected Dihydrofolate Binding Site Providing an Attractive Target for Anti-microbial Therapy

In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.

Potent inhibition of human folylpolyglutamate synthetase by a phosphinic acid mimic of the tetrahedral reaction intermediate

A phosphorous-containing pseudopeptide folate analog (Valiaeva et al., J Org Chem 2001;66:5146–54) was designed to mimic the tetrahedral intermediate formed in the ATP-dependent reaction catalyzed by folylpolyglutamate synthetase (FPGS). This analog, methotrexate-phosphinate (MTX-phosphinate; 4-amino-4-deoxy-10-methylpteroyl- l-Glu-γ-[Ψ{P(O)(OH)CH 2}]glutarate), is a highly potent ( K is , 3.1±0.5 nM), competitive inhibitor of recombinant human cytosolic FPGS. Within experimental limits, FPGS inhibition was not time-dependent, and preincubation of FPGS, inhibitor, and ATP did not potentiate the inhibition. These results suggest that slow phosphorylation to produce a more potent inhibitor form is not involved. MTX-phosphinate was not growth inhibitory to human CCRF-CEM leukemia cells at 1 μM (70-fold above the concentration of MTX giving 50% growth inhibition), probably because of poor transport. Because of its exceedingly high potency as an FPGS inhibitor, MTX-phosphinate represents a lead structure from which cell-permeable analogs may be developed to test the hypothesis that FPGS inhibition is therapeutically efficacious.

Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania.

Folates are polyglutamylated in most organisms by the enzyme folylpolyglutamate synthetase (FPGS). The Leishmania tarentolae FPGS gene was isolated. Its predicted product contains 538 amino acids and shows 33 and 30% identity with the human and yeast FPGS proteins, respectively. The level of folate polygtutamylation was studied in L. tarentolae promastigotes and in Leishmania infantum promastigotes and axenic amastigotes. In all species examined, folates were found predominantly as pentaglutamates, although monoglutamates were found in higher proportion in L. infantum axenic amastigote cells. Leishmania cells transfected with a FPGS containing plasmid (FPGS transfectant) exhibited a 6-fold increase in FPGS activity (32.7 pmol mg(-1) h(-1)) compared with wild-type cells (4.7 pmol mg(-1) h(-1)). HPLC analysis of the polyglutamylated forms of folates indicated a 2-fold increase of hexaglutamates in the FPGS transfectant compared with wild-type cells, while cells with one FPGS allele interrupted showed a higher proportion of short chain glutamates. The long-term accumulation of folates was greatly increased in the FPGS transfectant. Overall, this work indicates that FPGS activity is expressed in all forms of the parasite, and modulates the retention of folate, thereby possibly playing an important role in physiology.

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.

Effect of C9-methyl substitution and C8-C9 conformational restriction on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines.

N-[4-[1-methyl-2-(2,4-diaminofuro[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (5) and its C8-C9 conformationally restricted E- and Z-isomers (6 and 7) were designed and synthesized in order to investigate the effect of incorporating a methyl group at the C9 position and of conformational restriction at the C8-C9 bridge of N-[4-[2-(2,4-diaminofuro[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (1) with respect to dihydrofolate reductase (DHFR) inhibitory activity as well as antitumor activity. The compounds were synthesized by a Wittig reaction of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with ethyl 4-acetylbenzoate followed by catalytic reduction, hydrolysis, and standard peptide coupling with diethyl L-glutamate. The biological results indicated that the addition of a 9-methyl group to the C8-C9 bridge, as in 5, increased recombinant human (rh) DHFR inhibitory potency (IC(50) = 0.42 microM) as well as the potency against the growth inhibition of tumor cells in culture (CCRF-CEM EC(50) = 29 nM, A253 EC(50) = 28.5 nM, and FaDu EC(50) = 17.5 nM) compared with the 9-desmethyl analogue 1. However, the conformationally restricted 4:1 Z/E mixture of 7 and 6 was less potent than 5 in both assays, and the pure E-isomer 6 was essentially inactive. These three classical analogues were also evaluated as inhibitors of Lactobacillus casei, Escherichia coli, and rat and rh thymidylate synthase (TS) and were found to be weak inhibitors. All three analogues 5-7 were good substrates for human folylpolyglutamate synthetase (FPGS). These data suggested that FPGS is relatively tolerant to different conformations in the bridge region. Further evaluation of the cytotoxicity of 5 and 7 in methotrexate (MTX)-resistant CCRF-CEM cell sublines suggested that polyglutamylation was crucial for their mechanism of action. Metabolite protection studies of 5 implicated DHFR as the primary intracellular target. Compound 5 showed GI(50) values in 10(-9)-10(-7) M range against more than 30 tumor cell lines in culture.

Human Cytosolic and Mitochondrial Folylpolyglutamate Synthetase Are Electrophoretically Distinct: EXPRESSION IN ANTIFOLATE-SENSITIVE AND -RESISTANT HUMAN CELL LINES

Folylpolyglutamate synthetase (FPGS) activity in CCRF-CEM human leukemia cells was found in the cytosolic ( approximately 67% of total) and mitochondrial ( approximately 22%) fractions. A polyclonal antipeptide antibody (430Ab) to human FPGS specifically recognized distinct immunoreactive bands ( approximately 60 kDa) present in each subcellular fraction. Human cytosolic FPGS (hcFPGS) migrated more rapidly than mitochondrial FPGS (hmFPGS); their estimated difference in molecular mass was 1 kDa. The human K562 acute nonlymphocytic leukemia and the A253 and FaDu head and neck cancer cell lines also expressed the two FPGS isoforms, and the ratio of hcFPGS to hmFPGS protein in each cell line was similar. Since K562 and A253 cells are intrinsically resistant to pulse methotrexate (MTX) exposure relative to CCRF-CEM and FaDu cells, respectively, because of decreased MTX polyglutamate synthesis (despite having similar levels of total FPGS activity expression), these data suggest that the natural difference in drug sensitivity cannot be explained by compartmentalization of FPGS activity. Higher expression of hmFPGS relative to hcFPGS was observed in some sublines of CCRF-CEM with acquired MTX resistance suggesting that differential expression of the hmFPGS isoform may contribute to MTX resistance caused by decreased FPGS activity.

Mechanism-Based Inhibition of Human Folylpolyglutamate Synthetase: Design, Synthesis, and Biochemical Characterization of a Phosphapeptide Mimic of the Tetrahedral Intermediate

Folylpolyglutamate synthetase (FPGS) catalyzes anATP-dependent ligation reaction that results in the synthesis of poly(γ-glutamate) metabolites of folates and some antifolates. We have synthesized and characterized the prototype of a new class of mechanism-based FPGS inhibitor in which a phosphonate moiety mimics the tetrahedral intermediate formed during the ligation reaction.This phosphonate, 4-amino-4-deoxy-10-methyl-pteroyl-l-glutamyl-γ-[Ψ{P(O)(OH)-O}]glutarate (4-NH2-10-CH3-Pte-l-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate), is not a substrate for human FPGS, but is a linear, competitive inhibitor (Kis= 46 nM) with respect to methotrexate as the variable substrate. Inhibition is not time-dependent and preincubation of FPGS with this phosphonate does not increase the degree of inhibition, suggesting that it is not a slow, tight-binding inhibitor involving a time-dependent isomerization, EI → EI*. Substructures containing the phosphonate moiety but lacking the pterin are much less inhibitory to FPGS, indicating that a significant portion of the inhibitor binding energy is derived from the pterin moiety, a feature also observed in substrate binding. 4-NH2-10-CH3-Pte-l-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate is also an analog of a proposed tetrahedral intermediate in the reaction catalyzed by γ-glutamyl hydrolase (γ-GH), another enzyme of importance in controlling folate homeostasis in cells. This intermediate would arise from direct attack of H2O on the dipeptide, 4-NH2-10-CH3-Pte-l-Glu-γ-l-Glu. The fact that 4-NH2-10-CH3-Pte-l-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate is not an inhibitor of γ-GH strongly suggests that hydrolysis of poly-γ-glutamates catalyzed by γ-GH does not involve the direct attack of water at the scissile amide bond. Methotrexate, its γ-glutamyl dipeptide metabolite, and 4-NH2-10-CH3-Pte-l-Glu-γ-[Ψ{P(O)(OH)-O}]glutarate are equipotent as inhibitors of human dihydrofolate reductase (the primary target of methotrexate), but the phosphonate does not significantly inhibit another important folate-dependent enzyme, thymidylate synthase. Thus, the phosphonate moiety in this analog represents an important new lead in the development of FPGS inhibitors.

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.

Purification and Properties of Human Cytosolic Folylpoly-γ-glutamate Synthetase and Organization, Localization, and Differential Splicing of Its Gene

Human cytosolic folylpolyglutamate synthetase (FPGS) was expressed in Escherichia coli and purified to homogeneity. Tetrahydrofolate and dihydrofolate were the most effective substrates, while 5-substituted folates were poor substrates. Most pteroyldiglutamates were better substrates than monoglutamates. The human FPGS gene spans 12 kilobases and contains 15 exons and 14 introns. A single FPGS gene was located to chromosome region 9q34.1. Four exon 1 variants were identified, each of which was spliced to exon 2. The exon 1 variant corresponding to the isolated cDNA contains two ATG codons and multiple transcription start sites in this region generates mitochondrial and cytosolic FPGS (Freemantle, S. J., Taylor, S. M., Krystal, G., and Moran, R. G. (1995) J. Biol. Chem. 270, 9579-9584). Exons 1B and 1C, generated by alternate splicing in intron 1, and exon 1A, which is 5' to exon 1 and may encode an additional mitochondrial isoform, are preceded by a number of potential promoter sites. Chinese hamster ovary cell transfectants expressing FPGS activity in the mitochondria contained normal mitochondrial and low cytosolic folylpolyglutamate pools. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, while cytosolic FPGS activity is needed for establishment of normal cytosolic folate pools. The reconstructed FPGS gene restored normal cytosolic and mitochondrial folate metabolism in hamster cells.

Chapter 5 Molecular Biology In Nutrition Research: Modeling Of Folate Metabolism

Model CHO cells obtained by transfecting CHO mutants with the E. coli and human folylpolyglutamate synthetase genes have proven useful for assessing the role of folylpolyglutamates in one carbon metabolism and for delineating how folate intracellular stores are regulated. Cells expressing enzymes in specific subcellular compartments, expressing enzymes with different substrate specificity's, and expressing enzyme activity at different levels, all in a common background, in this case the CHO cell, has allowed the development of kinetic models for assessing the role of folypolyglutamate synthetase in folate retention and in the cytotoxicity of antifolates.

Effect of bridge region variation on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines.

Variation of the bridge linking the heterocyclic ring and p-aminobenzoyl-L-glutamate portions of our previously described classical 2,4-diaminofuro[2,3-d]pyrimidines 1 and 2 are reported as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and as antitumor agents. Specifically -CH2CH2- and -CH2NHCH2- bridged analogues, N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) ethyl]benzoyl]-L-glutamic acid (3) and N-[4-[[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) methyl]amino]methyl]benzoyl]-L-glutamic acid (4), respectively, were synthesized. Compound 3 was obtained via a Wittig reaction of the tributylphosphonium salt of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine (5) and methyl 4-formylbenzoate (6) followed by reduction and coupling with the diethyl ester of L-glutamic acid. Compound 4 was synthesized by the nucleophilic displacement of 5 with diethyl N-[4-(aminomethyl)benzoyl]-L-glutamate (15) and saponification. Both analogues were evaluated in vitro as inhibitors of DHFRs from (recombinant) human, human CCRF-CEM cells, and Lactobacillus casei. Compound 3 showed moderate activity (IC50 10(-6)-10(-7) M). Compound 4 was essentially inactive (IC50 10(-5) M, CCRF-CEM). The compounds were also evaluated against TS from (recombinant) human and L. casei and were of low activity (IC50 10(-5) M). The three-atom-bridged analogue 4 was somewhat more inhibitory to human TS than methotrexate (MTX). Compound 3 inhibited the growth of tumor cells in culture (IC50 10(-7) M) while 4 showed a low level of growth inhibitory activity. The inhibition of the growth of leukemia CCRF-CEM cells by both compounds parallels their inhibition of CCRF-CEM DHFR. Analogue 3 was a good substrate for human folylpolyglutamate synthetase (FPGS) derived from CCRF-CEM cells (Km 8.5 microM). Further evaluation of the growth inhibitory activity of 3 against the MTX-resistant subline of CCRF-CEM cells (R30dm) with decreased FPGS indicated that poly-gamma-glutamylation was important for its action. Protection studies with 3 in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin [(6R,S-5-formyltetrahydrofolate] or by a combination of thymidine and hypoxanthine, suggesting an antifolate effect directed at DHFR.

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 2,4-diamino-5-substituted-pyrrolo[2,3-d]pyrimidines as classical and nonclassical antifolate inhibitors of dihydrofolate reductases.

Eight novel, nonclassical, antifolate 2,4-diamino-5-(anilinomethyl)pyrrolo[2,3-d]pyrimidines, 1-8, with 3',4',5'-trimethoxyphenyl, 3',4'-dimethoxyphenyl, 2',5'-dimethoxyphenyl, 4'-methoxyphenyl, 2',5'-diethoxyphenyl, 3',4'-dichlorophenyl, 1'naphthyl, and phenyl substituents were synthesized as potential inhibitors of dihydrofolate reductases (DHFRs). The classical analogue N-[4-[N-[(2,4-diaminopyrrolo[2,3-d]pyrimidin- 5-yl)methyl]amino]benzoyl]-L-glutamic acid (9) was also synthesized as an inhibitor of DHFR and an antitumor agent. The classical and nonclassical analogues were obtained via reductive condensations of the key intermediate 2,4-diamino-5-cyanopyrrolo[2,3-d]pyrimidine (12) with the appropriate substituted aniline or (p-aminobenzoyl)-L-glutamate followed by reduction of the intermediate Schiff bases with NaCNBH3. Compounds 1-9 were evaluated in vitro as inhibitors of rat liver (rl), Pneumocystis carinii (pc), and Toxoplasma gondii (tg) DHFRs. The nonclassical analogues were significantly selective against tgDHFR (vs rat liver DHFR), ranging from 7- to 92-fold. The inhibitory activity was lower in pcDHFR and rlDHFR (IC50s > 10(-5) M) than in tgDHFR (IC50s = 10(-6) M). The classical analogue had inhibitory activity similar to that of methotrexate (MTX) against the growth of human leukemia CCRF-CEM, A253, and FaDu squamous cell carcinoma (SCC) of the head and neck cell lines. Further evaluation of 9 against CCRF-CEM and its sublines having defined mechanisms of MTX resistance demonstrated that the analogue utilizes the reduced folate/MTX-transport system and primarily inhibits DHFR and poly-gamma-glutamylation plays a role in its mechanism of action. Compound 9 was found to be 3-fold more efficient than aminopterin as a substrate for human folylpolyglutamate synthetase.

Role of folylpolyglutamate synthetase in the metabolism and cytotoxicity of 5-deazaacyclotetrahydrofolate, an anti-purine drug

Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpolyglutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of 5-deazaacyclotetrahydrofolate (DATHF), an anti-purine agent that requires conversion to polyglutamate forms to be a potent inhibitor of its target enzymes. The sensitivity of cells continuously exposed to DATHF was not influenced by FPGS activity. After short term exposure to DATHF, cells expressing higher levels of human FPGS were more sensitive to the drug while cells expressing low levels were resistant. Cells expressing E. coli FPGS solely in the cytosol were resistant to DATHF and accumulated only low levels of the drug. Expression of E. coli FPGS in the mitochondria of these cells restored drug sensitivity and cytosolic drug accumulation. DATHF is extensively metabolized in the mitochondria of mammalian cells and, despite an inability to enter the mitochondria, folylpolyglutamates and DATHF polyglutamates formed in the mitochondria are released into the cytosol without prior hydrolysis. DATHF was not solely an inhibitor of de novo purine biosynthesis but also inhibited glycine synthesis in the cell.

Purification and general properties of human folylpolyglutamate synthetase.

We recently cloned a human cDNA encoding folylpolyglutamate synthetase (FPGS) by complementation of an E. coli strain (SF4) deficient in FPGS activity1. The open reading frame of the cDNA predicted a protein having 545 amino acids, a Mr of 60 kDa, and an estimated pI of 6.95. In this report we describe the plasmid construct used for the expression of unfused human FPGS in E. coli. In addition, general characteristics of the purified enzyme are discussed as well as its specificity for selected folyl and analog substrates.KeywordsDEAE CelluloseMonovalent CationAnalog SubstrateCrude Enzyme PreparationDEAE Cellulose ChromatographyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Activation of mammalian folylpolyglutamate synthetase by sodium bicarbonate

NaHCO 3 activated the folylpolyglutamate synthetase (FPGS) from rat liver and the human leukemia cell lines K562 and CCRF-CEM by 1.7- to 2.0-fold. Optimal activation was achieved by 10 m m NaHCO 3 in all cases; NaCl, sodium formate, sodium acetate, NaN 3, and Na 2SO 3 at 10 m m did not cause activation. Activation could be masked if assay solutions which had extensively absorbed atmospheric CO 2 were used. Activation of the human CCRF-CEM FPGS was examined in detail. K m and V max values for pteroyl substrates (aminopterin or methotrexate) and l-glutamate increased proportionally in the presence of NaHCO 3; there was thus no apparent change in the catalytic efficiency ( V max K m ) of the FPGS reaction with these substrates. However, NaHCO 3 increased the efficiency of the reaction with respect to ATP by decreasing its apparent K m while increasing the V max of the reaction. NaHCO 3 also activated FPGS activity when folic acid, dihydrofolic acid and tetrahydrofolic acid were substrates. The relative distribution of products synthesized from methotrexate or tetrahydrofolate by FPGS was not altered by addition of NaHCO 3. The potency of 5,8-dideazapteroylornithine, an FPGS-specific inhibitor, was not changed by the presence of NaHCO 3 (IC 50 = 0.4 μ m). These results suggest that FPGS activity with folates and classical antifolates may be activated at physiological concentrations of NaHCO 3. In addition, inadvertent contamination of assay solutions with bicarbonate from atmospheric CO 2 may cause artifacts in the determination of activity levels and kinetic constants of FPGS.

Structural specificity of inhibition of human folylpolyglutamate synthetase by ornithinecontaining folate analogs

A series of folate analogs containing ornithine instead of glutamate was synthesized and tested for inhibition of folylpolyglutamate synthetase (FPGS) and other folate-dependent enzymes of human leukemia cell lines. Reduced derivatives of 2-amino-4-oxo-10-methyl-pteroyl-ornithine had dramatically increased inhibitory potency against FPGS compared to the oxidized parent. The aminopterin analog (2,4-diamino-pteroylornithine) was a potent inhibitor of both dihydrofolate reductase and FPGS. It was a much more potent linear competitive inhibitor of human FPGS than the corresponding methotrexate derivative previously described ( K i = 0.15–0.26 and 3 μM respectively). A quinazoline folate analog, 2-amino-4-oxo-5,8-dideazapteroyl-ornithine, was a relatively poor inhibitor of isolated dihydrofolate reductase and thymidylate synthase; however, it is the most potent human FPGS inhibitor identified to date ( K i = 100–150 nM). Because of the lack of appreciable interaction with other folatedependent enzymes, structures incorporating the 2-amino-4-oxo-5,8-dideazapteroate nucleus may thus lead to selective inhibition of FPGS. Substitution of ornithine for glutamate caused a profound decrease in cytotoxic potency for these analogs; this was apparently the result of poor transport. Together with earlier studies, these data indicate that the potency of FPGS inhibition by an analog containing ornithine closely parallels the relative substrate activity of its glutamate-containing counterpart. The substitution of ornithine apparently does not perturb the pterin specificity of FPGS. The close parallel between substrate and inhibitor specificity may thus allow the use of currently available structure-activity studies on FPGS to design more potent and more selective inhibitors of FPGS.