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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.

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

Three cysteines in human recombinant folylpoly-gamma-glutamate synthetase (FPGS) that were reactive with iodoacetamide were located in peptides that were highly conserved across species; the functions of two of these peptides, located in the C-terminal domain, were studied by site-directed mutagenesis. When cDNAs containing mutations in each conserved ionic residue on these peptides were transfected into AUXB1 cells, which lack endogenous FPGS activity, one mutant (D335A) did not complement the auxotrophy, and another (R377A) allowed only minimal growth. FPGS activity could not be detected in insect cells expressing abundant levels of these two mutant proteins from recombinant baculoviruses nor from a virus encoding an H338A mutant FPGS. Kinetic analysis of the purified proteins demonstrated that each of these three mutants was quite different from the others. The major kinetic change detected for the H338A mutation was a 600-fold increase in the K(m) for glutamic acid. For the D335A mutation, the binding of all three substrates (aminopterin, ATP, and glutamic acid) was affected. For R377A, the K(m) for glutamic acid was increased by 1500-fold, and there was an approximately 20-fold decrease in the k(cat) of the reaction. The binding of the K(+) ion, a known activator of FPGS, was affected by the D335A and H338A mutations. We conclude that these three amino acids participate in the alignment of glutamic acid in the active site and that Arg-377 is also involved in the mechanism of the reaction.

Previous studies have documented the metabolism of a broad range of folate antimetabolites to polyglutamate derivatives by the enzyme folylpoly-gamma-glutamate synthetase (FPGS). The activity of the more recently developed classes of antifolates directed against thymidylate synthase and de novo purine synthesis is sufficiently dependent on polyglutamation that these compounds should be specifically cytotoxic to any normal or malignant proliferating cell expressing this enzyme. We have studied the patterns of expression of FPGS in mammalian cells and tissues during rapid growth, growth arrest, differentiation, and embryonic development. During embryogenesis in the rat, FPGS levels in liver and brain were higher during the period of proliferative activity and then dropped to a level characteristic of the adult organs. However, the levels in liver were substantially higher than those in brain at any given time. This pattern was mimicked in mouse C3H 10T1/2 embryo fibroblast cells, in which FPGS activity decreased after cessation of growth but then remained at a lower steady state level during an extended period of postconfluent culture. Enzyme activity also dropped after the differentiation of human HL-60 promyelocytic leukemia cells. In a human homolog of these experimental systems, FPGS levels were below the limits of detection in circulating mature human hematopoietic cells of the granulocytic, lymphoblastic, and erythrocytic lineages. In striking contrast, substantial levels of FPGS were found in circulating lymphoblasts from eight patients with acute lymphoblastic leukemia. The levels of FPGS found in these transformed stem cells would help to explain the sensitivity of many acute lymphoblastic leukemias to folate antimetabolites. We concluded that expression of FPGS is regulated by at least two mechanisms, one of which is linked to proliferation and the other of which controls enzyme levels after differentiation and is tissue specific.

We have investigated the mechanism by which reduced folates, such as folinic acid, enhance the cytotoxicity of fluoropyrimidines in L1210 mouse leukemic cells. Exposure of L1210 cells to folinic acid resulted in expansion of intracellular pools of 5,10-CH2-H4PteGlun, delayed the reappearance of catalytically active thymidylate synthase (TS) following 5-fluoro-2′-deoxyuridine exposure, and stabilized inhibited TS complexes over the same concentration range that augmented the cytotoxic effects of fluorodeoxyuridine and 5-fluorouracil. The data showed that, in intact L1210 cells, fluorodeoxyridylate behaves as an inhibitor whose complexes with TS dissociated with a biologically significant rate. However, these complexes become functionally irreversible in cells incubated with high levels of folinic acid. It was also found that bound and total TS levels increased in cells treated with fluorodeoxyuridine to an extent that substantially exceeded the increase in protein content per cell under the same conditions. These results are in accord with the concept that folinic acid augments the effects of the fluoropyrimidines by expansion of cellular 5,10-CH2-H4PteGlun pools with subsequent stabilization of ternary complexes among 5-fluoro-2′-deoxyuridine 5′-monophosphate, TS, and 5,10-CH2-H4PteGlun. In light of the accumulation of TS that occurs following exposure to fluoropyrimidines, this stabilization may be needed for efficient tumor cell killing by these agents.

The diasteromers of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) differing in chirality about carbon 6 were resolved and studied as inhibitors of folate-dependent processes in mouse leukemia cells. Both diastereomers of DDATHF were found to be potent inhibitors of leukemia cell growth due to effects on de novopurine synthesis. Cell growth inhibition by these compounds was prevented by 5-formyltetrahydrofolate in a dose-dependent manner. This indicated that the effects of the DDATHF diastereomers were due to inhibition of folate-dependent processes. Metabolite reversal experiments indicated that 5‵-phosphoribosylglycinamide formyltransferase was the major site of action of these compounds in mouse cells. Another site in de novopurine synthesis was affected at higher concentrations of diastereomer B in L1210 cells. Low concentrations of both diastereomers were found to inhibit pure L1210 5‵-phosphoribosylglycinamide formyltransferase competitively with the folate substrate. The two diastereomers were also efficient substrates for mouse liver folylpolyglutamate synthetase. We conclude that the 6R- and 6S-diastereomers of DDATHF are remarkably similar and equiactive antimetabolites inhibitory to de novopurine synthesis and that the biochemical processes involved in their cytotoxicity display little stereochemical specificity.

The metabolism of 5,10-dideazatetrahydrofolate (DDATHF [lometrexol]) to polyglutamate derivatives by folylpoly-gamma-glutamate synthetase (FPGS) plays a central role in the activity of this compound as an antineoplastic agent. The availability of a series of DDATHF derivatives differing in structure throughout the molecule has allowed a study of the structural requirements for substrate activity with mouse liver and hog liver FPGS. Kinetics of the polyglutamation reaction in vitro have been related to the potency of these compounds as inhibitors of the growth of human CEM leukemic cells. The structure-activity relationships for enzyme from both sources were nearly identical. FPGS from both species showed a broad acceptance for structural changes in the pyridopyrimidine ring, in the phenyl group, and in the intermediate bridge region, with structural changes in these regions being reflected in changes in Km for FPGS but much more modest alterations in Vmax. The data suggested that the phenyl ring was not contributing to any pi-pi hydrophobic interactions. It appeared to function primarily in maintaining a favorable distance between the pyridopyrimidine ring and the glutamate side chain. The lowest Km values were found for DDATHF analogs in which there were small alterations at the 10 position, e.g., 5-deazatetrahydrofolate, 10-methyl-DDATHF, and 10-formyl-5-deazatetrahydrofolate; the first-order rate constants for these substrates were the highest in this series, an indication of the efficiency of polyglutamation at low substrate concentrations. After correction for the intrinsic inhibitory activity of the parent DDATHF analog as an inhibitor of the target enzyme, the first-order rate constants for FPGS were found to be predictive of the potency of tumor cell growth inhibition for most of the compounds in this structural series.

The activity of a series of folic acid analogues as substrates for partially purified mouse liver folylpolyglutamate synthetase was determined and the effects of substituents on the binding to, and catalytic processes of, this enzyme were inferred. A 4-amino group improved substrate activity primarily by decreasing the apparent Km while N10-methyl substitution substantially diminished utilization as a substrate, again, by effects on Km. Isosteric replacement of N-10 altered substrate activity. A free alpha-carboxyl group in the amino acid side chain was required for catalysis as was the presence of the side chain amide carbonyl group. Modification of the amino acid side chain length profoundly affected activity. Several observations were made that may be relevant to chemotherapy with folate antimetabolites: 1) 7-hydroxymethotrexate was a substrate for this enzyme; 2) substrate activity and substrate inhibition were observed with CB 3717, a potent inhibitor of thymidylate synthase; 3) potent classical dihydrofolate reductase inhibitors were identified that were either not substrates for mouse liver folylpolyglutamate synthetase (e.g., 4-amino-4-deoxy-N10-methylpteroyl-L-alpha-aminoadipate) or were much better substrates than methotrexate for this enzyme (e.g., aminopterin); and 4) leucovorin and methotrexate appeared to be substrates for the same synthetase, but leucovorin saturated the reaction at much lower concentrations. These results have implications for the design of folylpolyglutamate synthetase inhibitors and for the selection of dihydrofolate reductase inhibitors that are either not polyglutamated or are efficiently polyglutamated in vivo.

L1210/D3 mouse leukemia cells are resistant to 5, 10-dideazatetrahydrofolate due to expansion of cellular folate pools which block polyglutamation of the drug (Tse, A., and Moran, R. G. (1998) J. Biol. Chem. 273, 25944-25952). These cells were found to have two point mutations in the reduced folate carrier (RFC), resulting in a replacement of isoleucine 48 by phenylalanine and of tryptophan 105 by glycine. Each mutation contributes to the resistance phenotype. Genomic DNA from resistant cells contained both the wild-type and mutant alleles, but wild-type message was not detected. Folic acid was a much better substrate, and 5-formyltetrahydrofolate was a poorer substrate for transport in L1210/D3 cells relative to L1210 cells. Enhanced transport of folic acid was due to a marked, approximately 20-fold, decrease in the influx Km. Influx of methotrexate and 5,10-dideazatetrahydrofolate were minimally altered. Transfection of mutated rfc cDNA into RFC-null L1210/A cells produced the substrate specificity and 5, 10-dideazatetrahydrofolate resistance observed in the L1210/D3 line. Transfection of the mutant cDNA into wild-type cells also conferred resistance to 5,10-dideazatetrahydrofolate. We conclude that the I48F and W105G mutations in RFC caused resistance to 5, 10-dideazatetrahydrofolate, that the region of the RFC protein near these two positions defines the substrate-binding site, that the wild-type allele was silenced during the multistep development of resistance, and that this mutant phenotype represents a genetically dominant trait.

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.

In mammals, folylpoly-gamma-glutamate synthetase (FPGS) activity is found in any cell undergoing sustained proliferative phases, but this enzyme also displays a tissue-specific pattern of expression in differentiated tissues. It is now reported that the steady state levels of FPGS mRNA in normal and neoplastic cells reflect these patterns, supporting the concept that the control mechanisms underlying this distribution are transcriptional. To initiate an understanding of these interacting levels of control, we have determined the position and properties of the minimal FPGS promoter controlling transcription of the FPGS gene in human CEM leukemia cells, a line which expresses high levels of this enzyme and its mRNA. The TATA-less region immediately upstream of the major transcriptional start site previously mapped in human tumor cells, which includes several GC- and Y-boxes, functioned as a remarkably efficient promoter when used to drive expression of a luciferase reporter in transient expression studies in CEM cells. The minimal region of the FPGS promoter required for maximal transcriptional activation in CEM cells included the 80 base pairs over which the multiple transcriptional start sites were located, and the 43 base pairs immediately upstream. DNase I footprint analysis detected the binding of Sp1 at all seven of the consensus sites within the probe used, two of which are contained within the minimal promoter region. The several Sp1 sites immediately upstream of the first major transcriptional start activated transcription in Drosophila cells when cotransfected with an Sp1 construct, including those in the region which functioned as a minimal promoter in CEM cells. An additional region of the minimal promoter, situated between the two translational start codons of the FPGS gene, was bound by protein(s) from HeLa cell nuclear extracts. We conclude that transcription of the FPGS gene in CEM cells involves transactivation events over a limited upstream DNA sequence and that the FPGS promoter used in proliferating human leukemic cells has strong similarity to other TATA-less promoters that utilize tandem, closely spaced Sp1 sites to initiate transcription.

The murine gene encoding dihydrofolate reductase (DHFR) has been localized to a particular mouse chromosome by complementation mapping. Microcells prepared from diploid mouse fibroblasts were fused with mutant hamster cells lacking the dihydrofolate reductase gene (Dhfr),and DHFR+microcell hybrids were selected in medium lacking purines and pyrimidines. The complemented hybrids expressed wild-type levels of DHFR enzyme activity, and selectively retained a single mouse chromosome—chromosome 13. Genomic Southern blots of DNAs prepared from these hybrids and from an independently derived collection of clones isolated without selection on the DHFR phenotype confirmed the assignment of murine Dhfrgene sequences to chromosome 13. This assignment provides further evidence for the existence of genetic homology between regions of mouse chromosome 13 and human chromosome 5. In support of this view, we show that the gene encoding hexosamindase B, a chromosome 5 marker in man, also maps to mouse chromosome 13.

The gene encoding folylpolyglutamyl synthetase (FPGS) was assigned to mouse chromosome 2 by complementation mapping. Chinese hamster ovary cells (AuxBl) deficient in FPGS, and consequently auxotrophic for glycine, adenosine, and thymidine (gat-), were employed as recipients in microcell-mediated chromosome transfer experiments. Mouse chromosomes derived from diploid embryo fibroblasts were introduced into hamster AuxBl cells, and gat+ microcell hybrids were selected in medium lacking adenosine and thymidine. Mouse chromosome 2 was the only donor chromosome whose presence correlated with expression of FPGS activity. Furthermore, every gat+ hybrid clone expressed murine AK-1, a marker previously assigned to chromosome 2. Eight of 20 clones analyzed retained deletion chromosomes derived from mouse chromosome 2. These clones were used to localize murine Fpgs and Ak-1 to a region of this chromosome, namely 2(cen?C1).

Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) from a human leukemic cell line has been purified to homogeneity with one-step affinity column chromatography. The purified enzyme has a specific activity of 3.8 micron/min per mg of protein, which corresponds to a turnover number of 250. These are the highest values reported for a thymidylate synthetase from neoplastic tissue. A ratio of 1.7 mol of 5-fluoro-2'-deoxyuridylate binds per mol of enzyme in the presence of 5,10-methylenetetrahydrofolate. The ternary complex so formed migrates intact on denaturing gels and can be precipitated with trichloroacetic acid; however, urea dissociates the ternary complex. The human thymidylate synthetase is composed of two subunits of 33,000 daltons each. It contains more residues of cysteine, glycine, and arginine and fewer of histidine than the well-studied thymidylate synthetase from Lactobacillus casei.

Several structurally related series of folate analogs were studied as substrates for mouse liver folylpolyglutamate synthetase (FPGS). A comparison of the kinetics of the interaction of this enzyme with folate analogs that contained the quinazoline ring in place of the pteridine ring with those of the analogous pteridines demonstrated that the quinazoline derivatives were more efficient substrates for and tighter binding inhibitors of this enzyme. A series of 2,4-diaminopyrimidine dihydrofolate reductase inhibitors were found to be substrates for FPGS; these are the first known compounds without a fused ring system analogous to the pteridine ring of the folate molecule that are substrates for FPGS. Several 5,8-dideazafolate derivatives that lack the 2-amino group had activity as substrates for FPGS equivalent to that of the corresponding 5,8-dideazafolates. When a homologous series of 5,8-dideazafolic acid analogs with hydrocarbon substituents on the 10-nitrogen were studied, these substituents were found to diminish the efficiency of utilization of these analogs as substrates for FPGS; this effect increased with increasing chain length of the hydrocarbon. It was concluded that neither the 2-amino group nor an intact pyrazine ring of folates and folate analogs are essential for the binding of folates to the active site of mouse liver FPGS but that the pyrazine ring probably serves to position other regions of the folate molecule that interact with amino acid residues in the active site. It was also inferred from these observations that the volume within the active site of FPGS above/below the pyrazine ring or near the 10-position of folate derivatives are regions of limited bulk tolerance; binding of folate analogs with substituents at these positions probably distorts the active site.

Folylpoly-gamma-glutamate synthetase (FPGS) is essential for the survival of proliferating mammalian cells and central to the action of all "classical" folate antimetabolites. We report the isolation of cDNAs corresponding to the 5' ends of FPGS mRNA from both human and hamster cells which include a start codon upstream of and in-frame with the AUG in the previously reported FPGS open reading frame. The predicted hamster and human amino-terminal extension peptides have features consistent with a mitochondrial targeting sequence. Ribonuclease protection and 5'-rapid amplification of cDNA ends assays indicated multiple transcriptional start sites consistent with the sequence of the promoter region of this gene, which was highly GC-rich and did not contain TATA or CCAAT elements. These start sites would generate two classes of transcripts, one including the upstream AUG and one in which only the downstream AUG would be available for translation initiation. Transfection of the full length human cDNA into cells lacking FPGS restored their ability to grow in the absence of glycine, a product of mitochondrial folate metabolism, as well as of thymidine and purines. Therefore, we propose that the mitochondrial and cytosolic forms of FPGS are derived from the same gene, arising from the use of the two different translation initiation codons, and that the translation products differ by the presence of a 42-residue amino-terminal mitochondrial leader peptide.

A single mouse genomic locus encodes proteins catalyzing three steps of purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). This gene has 22 exons and spans 28 kilobases. The existence of a second genetic locus and closely related pseudogenes was ruled out by Southern analysis. Mouse tissues express two related classes of messages encoded by this single locus: a trifunctional GARS-AIRS-GART mRNA and a monofunctional GARS mRNA. These transcripts used the same set of multiple transcriptional start sites, and both used the same first 10 exons. CCAAT and TATA elements were not found for this locus. Exon 11, which represented the last coding sequence of the GARS domain, was differentially utilized for the two messages. The trifunctional mRNA was generated by splicing exon 11 to exon 12, the first coding sequence for the AIRS domain with subsequent use of a polyadenylation signal at the end of exon 22. Genomic sequence corresponding to the 3'-UTR of the monofunctional GARS mRNA was contiguous with exon 11, so that the smaller message arose from the recognition of one of the multiple polyadenylation signals present within the intron between exons 11 and 12. Hence, polyadenylation of the primary transcript at a position corresponding to an intron of the genomic locus was responsible for the generation of the monofunctional GARS class of mRNAs. This utilization of an intronic polyadenylation site without alternative exon usage is comparable to the mechanism whereby both secreted and membrane-bound forms of the immunoglobulin mu heavy chain are made from a single genetic locus.

Previous attempts to design inhibitors of mammalian folylpolyglutamate synthetase (FPGS) have resulted in three classes of active compounds, all of which have charged moieties in the side chain, but structural alteration of the rest of the folate molecule has not seemed to be an avenue for drug discovery. However, groups in the side chain of folate analogs that bear charge distributions different from that of glutamic acid appear to prevent efficient transport into mammalian cells on the reduced folate carrier system. We now report that substituents at the 7-, 2'-, or 3'-position of 2-desamino-2-methyl-4-hydroxyquinazoline antifolates decrease or prevent the catalysis of diglutamate formation by FPGS but are compatible with efficient binding to the reduced folate carrier system. Thus, 5,8-dideazafolates with a 3'-alkyl group had a lower Vmax for FPGS than did the corresponding unsubstituted quinazolines, by a factor of 4-12, but these compounds inhibited the reaction of control FPGS substrates, indicating that the 3'-groups had much larger effects on catalytic activity than on binding to enzyme. A 7-methyl substituent affected the Vmax of a series of 5,8-dideazafolate compounds by a factor of 2-8, but this decrease in the catalytic rate was also accompanied by an increase in the Km of the substituted compounds by a factor of 10-100. The extent of the effect of a 7-methyl substituent on Vmax appeared to be dependent on the size of the substituent at N10. Different substituents at the 2'-position affected the kinetics of the FPGS reaction with one of three patterns, i.e., 1) a 2'-fluoro substituent both increased Vmax and decreased Km slightly, 2) either -OH or -NH2 decreased the Vmax without affecting the Km, and 3) 2'-Cl-, -CH3, -CF3, or -OCH3 substituents were found to both decrease Vmax and increase Km. Substitutions at the 7-, 2'-, or 3'-position had only minor effects on the ability of 2-desamino-2-methyl-4-oxoquinazolines to interfere with the transport of [3H]methotrexate into L1210 cells. Hence, these classes of compounds are likely to be efficiently transported by the reduced folate carrier system. We conclude that the region of the folate molecule bounded by the 7-, 6-, 9-, 10-, 3'-, and 2'-positions, the "bay region," is of major importance both for the binding of folates and folate analogs to FPGS and for the assumption of a conformation of the enzyme-substrate complex compatible with catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Methotrexate (MTX) inhibition of the growth of mouse or human leukemia cells in culture was partially prevented by either thymidine (dThd) or hypoxanthine. 5-Fluoro-2'-deoxyuridine (FdUrd) also decreased the growth-inhibitory potency of MTX in the presence of small concentrations of 5-formyltetrahydrofolate (citrovorum factor) and sufficient exogenous dThd to support the synthesis of thymidylate nucleotides by salvage mechanisms. In addition, citrovorum factor-induced reversal of MTX was several orders of magnitude more efficient in the presence of both FdUrd and dThd than in the presence of dThd alone or in the absence of both nucleosides. Likewise, the presence of FdUrd (3 microM) and dThd (5.6 microM) completely prevented the lethality of 0.3 mM MTX to L1210 cells in culture medium supplemented with micromolar concentrations of citrovorum factor. We propose that this protection against the cytotoxic effects of MTX by dThd, hypoxanthine, and FdUrd have a common biochemical mechanism--namely, inhibition of the de novo synthesis of thymidylate by either a direct [FdUrd; inhibition of thymidylate synthetase (thymidylate synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyl-transferase, EC 2.1.1.45)] or indirect (dThd and hypoxanthine; feedback inhibition by anabolites on ribonucleotide reductase and deoxycytidylate deaminase) effect. The resultant decreased rate of loss of reduced folates due to de novo thymidylate synthesis would allow a higher degree of inhibition of dihydrofolate reductase to be endured without damage to the cell.

5,10-Dideazatetrahydrofolate (DDATHF) is a new antimetabolite designed as an inhibitor of folate metabolism at sites other than dihydrofolate reductase. DDATHF was found to inhibit the growth of L1210 and CCRF-CEM cells in culture at concentrations in the range of 10–30 nM. The inhibitory effect of DDATHF on the growth of L1210 and CCRF-CEM cells was reversed by either hypoxanthine or aminoimidazole carboxamide. Growth inhibition by DDATHF was prevented by addition of both thymidine and hypoxanthine, but not by thymidine alone. 5-Formyltetrahydrofolate reversed the effects of DDATHF in a dose-dependent manner. DDATHF had no appreciable inhibitory activity against either dihydrofolate reductase or thymidylate synthase in vitro, but was found to be an excellent substrate for folylpolyglutamate synthetase. DDATHF had little or no effect on incorporation of either deoxyuridine or thymidine into DNA, in distinct contrast to the effects of the classical dihydrofolate reductase inhibitor, methotrexate. DDATHF was found to deplete cellular ATP and GTP over the same concentrations as those inhibitory to leukemic cell growth, suggesting that the locus of DDATHF action was in the de novopurine biosynthesis pathway. The synthesis of formylglycinamide ribonucleotide in intact L1210 cells was inhibited by DDATHF with the same concentration dependence as inhibition of growth. This suggested that DDATHF inhibited glycinamide ribonucleotide transformylase, the first folate-dependent enzyme of de novopurine synthesis. DDATHF is a potent folate analog which suppresses purine synthesis through direct or indirect inhibition of glycinamide ribonucleotide transformylase.

Starting from the pyrido(4,3‐d)pyrimidinone (I) the derivatives (V) and (VIII) are prepared via their key intermediates (III) and (VII), respectively.

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The pyridopyrimidinecarboxaldehyde (I) is coupled with the aniline (II) and subsequently reduced to give the N‐substituted aniline (III) which is acylated, yielding the N‐acyl derivatives (V).