Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trimetrexate is a 2,4-diaminoquinazoline inhibitor of dihydrofolate reductase (DHFR) which is cytotoxic in vitro and in vivo to several tumors resistant to methotrexate. It is more lipophilic than the parent antifolate, and is not transported by the reduced folate carrier. These features promise activity greater than that of methotrexate in the clinic; its inability to undergo polyglutamylation may also enhance the therapeutic index. In preclinical models, the activity of trimetrexate was highly schedule dependent, being superior on repeated dose schedules. Phase I studies have demonstrated that myelosuppression is the major toxic effect of trimetrexate on all schedules tested in man. Phase II studies will evaluate a 5-day schedule initially; trials in multiple tumor types and examination of the role of schedule are already under way.
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PMID:Trimetrexate: clinical development of a nonclassical antifolate. 296 28

Cl-920 is a structurally novel antitumor antibiotic which has activity against a wide spectrum of tumor cells in vitro and is curative in L1210 leukemia in vivo. Several lines of evidence indicate that this drug penetrates L1210 cells via the reduced folate carrier system. Reduced folates (100 microM) including leucovorin and 5-methyltetrahydrofolate completely protected L1210 cells from growth inhibition by Cl-920. Protective effects were not observed, however, with folic acid, a compound which is transported by a process distinct from that for reduced folates. Cl-920 was a potent inhibitor of methotrexate influx exhibiting a mixture of competitive and noncompetitive inhibition and having a Ki (slope) of 30.0 microM and a Ki (intercept) of 58.8 microM. The inhibition appeared to be irreversible since, after cells were preincubated with drug, the inhibitory effects persisted after cells were washed in drug-free media. The irreversibility could be eliminated, however, by dithiothreitol, suggesting that Cl-920 may interact with a thiol which is essential to this transport system. Cells made 71-fold resistant to Cl-920 by continuous exposure to increasing concentrations of this drug were 245-fold cross-resistant to methotrexate but were collaterally sensitive to the lipophilic antifolate trimetrexate and contained normal levels of dihydrofolate reductase. This mutant cell line had a severely impaired reduced folate carrier system exhibiting methotrexate influx rates of less than 1% of control cells. Finally, inhibition of methotrexate influx by a number of Cl-920 analogues showed that the intact lactone ring and the presence of the phosphate ester were required for maximum interaction with the carrier system and that the degree of inhibition correlated with relative antitumor potency. These observations are compatible with the concept that Cl-920 utilizes the folate carrier system and could be of fundamental importance for understanding the cytotoxicity and selectivity of Cl-920.
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PMID:Transport of the antitumor antibiotic Cl-920 into L1210 leukemia cells by the reduced folate carrier system. 654 36

Studies on the mode of action of PT523 [N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine], a potent nonpolyglutamatable antifolate, were carried out in sensitive and resistant H35 rat hepatoma cell lines in culture, to compare it with other antifolates, including three dihydrofolate reductase (DHFR) inhibitors, i.e., methotrexate (MTX), gamma-fluoro-MTX, and trimetrexate (TMQ), two thymidylate synthase inhibitors, i.e., N10-propargyl-5,8- dideazafolate (PDDF) and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (dmPDDF), and the glycinamide ribonucleotide formyltransferase inhibitor 5,10-dideaza-5,6,7,8-tetrahydrofolate. PT523 was the most active compound in this group against the parental H35 cells, with an IC50 ranging from 2.5 nM for 72 hr of treatment to 0.21 microM for 2 hr of treatment. Sublines resistant to MTX by virtue of a transport defect or a combination of defective transport and increased DHFR activity were resistant to PT523 and MTX but not to PDDF, whereas sublines resistant to fluoropyrimidines by virtue of increased thymidylate synthase activity were resistant to PDDF but not to PT523, TMQ, or MTX. Inhibition of H35 cell growth by PT523 was associated with a concentration- and time-related decrease in de novo dTMP and purine biosynthesis. Growth inhibition by PT523, MTX, and TMQ was prevented by leucovorin or a combination of thymidine (dThd) and hypoxanthine but not by dThd or hypoxanthine alone; in contrast, growth inhibition by dmPDDF was prevented by dThd alone. Intracellular reduced folate polyglutamate pools were markedly altered by PT523 treatment, with the most pronounced effect being an increase in 7,8-dihydrofolate mono- and polyglutamates and a decrease in 5,10-methylene-5,6,7,8-tetrahydrofolate mono- and polyglutamates, 5,6,7,8-tetrahydrofolate mono- and polyglutamates, and 10-formyl-5,6,7,8-tetrahydrofolate mono- and polyglutamates. This pattern was qualitatively similar to that observed with MTX and TMQ but different from that observed with dmPDDF or 5,10-dideaza-5,6,7,8-tetrahydrofolate, which resulted in little or no change in the folate species. Uptake of [3H]MTX and [3H]folinic acid, but not [3H]folic acid, by H35 cells was inhibited in a dose-related manner by PT523, suggesting that penetration of the cell probably involves, at least in part, active transport by the MTX/reduced folate carrier. To determine whether the potent cellular effects of PT523 might be due to chemical or enzymic clevage to N'-(4-amino-4-deoxypteroyl)-L-ornithine, a potent inhibitor of folylpolyglutamate synthetase, the formation of [3H]MTX polyglutamates in CCRF-CEM lymphoblasts pulsed with [3H]MTX after preincubation with PT523 was examined.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biochemical studies on PT523, a potent nonpolyglutamatable antifolate, in cultured cells. 751 64

The transport properties and growth-inhibitory potential of 37 classic and novel antifolate compounds have been tested in vitro against human and murine cell lines expressing different levels of the reduced folate carrier (RFC), the membrane-associated folate binding protein (mFBP), or both. The intracellular targets of these drugs were dihydrofolate reductase (DHFR), glycinamide ribonucleotide transformylase (GARTF), folylpolyglutamate synthetase (FPGS), and thymidylate synthase (TS). Parameters that were investigated included the affinity of both folate-transport systems for the antifolate drugs, their growth-inhibitory potential as a function of cellular RFC/mFBP expression, and the protective effect of either FA or leucovorin against growth inhibition. Methotrexate, aminopterin, N10-propargyl-5,8-dideazafolic acid (CB3717), ZD1694, 5,8-dideazaisofolic acid (IAHQ), 5,10-dideazatetrahydrofolic acid (DDATHF), and 5-deazafolic acid (efficient substrate for FPGS) were used as the basic structures in the present study, from which modifications were introduced in the pteridine/quinazoline ring, the C9-N10 bridge, the benzoyl ring, and the glutamate side chain. It was observed that RFC exhibited an efficient substrate affinity for all analogues except CB3717, 2-NH2-ZD1694, and glutamate side-chain-modified FPGS inhibitors. Substitutions at the 2-position (e.g., 2-CH3) improved the RFC substrate affinity for methotrexate and aminopterin. Other good substrates included PT523 (N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine), 10-ethyl-10-deazaaminopterin, and DDATHF. With respect to mFBP, modifications at the N-3 and 4-oxo positions resulted in a substantial loss of binding affinity. Modifications at other sites of the molecule were well tolerated. Growth-inhibition studies identified a series of drugs that were preferentially transported via RFC (2,4-diamino structures) or mFBP (CB3717, 2-NH-ZD1694, or 5,8-dideazaisofolic acid), whereas other drugs were efficiently transported via both transport pathways (e.g., DDATHF, ZD1694, BW1843U89, or LY231514). Given the fact that for an increasing number of normal and neoplastic cells and tissue, different expression levels of RFC and mFBP are being recognized, this folate antagonist structure-activity relationship can be of value for predicting drug sensitivity and resistance of tumor cells or drug-related toxicity to normal cells and for the rational design and development of novel antifolates.
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PMID:Carrier- and receptor-mediated transport of folate antagonists targeting folate-dependent enzymes: correlates of molecular-structure and biological activity. 756 26

Either the alpha- or gamma-carboxyl group of the glutamic acid moiety of N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (1b, TNP-351) and its related compound (1a) was replaced with a 1H-tetrazole ring, and the inhibitory effects of the resulting compounds on dihydrofolate reductase (DHFR) and the growth of murine fibrosarcoma Meth A cells were examined. The gamma-tetrazole analogs (2) were found to be much more potent DHFR inhibitors than TNP-351, and strongly inhibited the growth of Meth A cells. On the other hand, the alpha-tetrazole analogs (3) were much less active against Meth A cells, even though their DHFR-inhibitory activity was comparable to that of TNP-351. These findings suggest that the alpha-carboxyl group plays an important role in effective uptake via the reduced folate carrier, and a novel DHFR inhibitor could be obtained by chemically modifying the gamma-carboxyl moiety while leaving the alpha-carboxyl group intact.
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PMID:Non-glutamate type pyrrolo[2,3-d]pyrimidine antifolates. I: Synthesis and biological properties of pyrrolo[2,3-d]pyrimidine antifolates containing tetrazole congener of glutamic acid. 772 29

The biological activity and cellular metabolism of ZD1694, a novel folate-based thymidylate synthase (TS) inhibitor, were analyzed in a human leukemia cell line, MOLT-3, and its antifolate-resistant sublines with different mechanisms of resistance to methotrexate (MTX), trimetrexate (TMQ) and N10-propargyl-5,8-dideazafolic acid (CB3717). MOLT-3/CB3717(40), which was selected for CB3717 resistance, demonstrated impaired membrane drug transport via reduced folate carrier (RFC) and lower accumulation of [3H]ZD1694-polyglutamates in the cells with a shift in the polyglutamate distribution profile to shorter chain length polyglutamates, indicating an alteration in polyglutamation capacity in this subline. Impaired RFC and reduced rate of polyglutamation could explain the cross-resistance (12-fold) of this subline to ZD1694. On the other hand, there was little or no cross-resistance to this drug in a subline (MOLT-3/TMQ800) reportedly resistant to TMQ through impaired membrane transport for TMQ and an increase in dihydrofolate reductase (DHFR) activity. Total amount of ZD1694 polyglutamated to a level higher than diglutamate was approximately 1.7-fold higher in the TMQ-resistant cells than that in the parent cells, but a low degree of increase in TS activity in the cells counteracted the supposed increase in sensitivity to ZD1694. MOLT-3/TMQ800-MTX10000 cells, which were established by sequential exposure of the TMQ-resistant cells to MTX and were previously shown to amplify mutated DHFR with low affinity for MTX, showed a decreased accumulation of polyglutamated ZD1694 as compared with the parent line and this was consistent with cross-resistance to ZD1694 in this subline. Overproduction of variant DHFR scarcely influenced the sensitivity to this drug. These results indicate that ZD1694 could overcome antifolate resistance through a mechanism such as amplified DHFR activity, and the biological activity of this drug against the cells paralleled the amount of polyglutamated drug inside the cells. Determination of polyglutamation capacity in tumor cells may allow prediction of sensitivity to this drug.
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PMID:Biological activity and intracellular metabolism of ZD1694 in human leukemia cell lines with different resistance mechanisms to antifolate drugs. 869 29

We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.
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PMID:Comparison of methotrexate polyglutamylation in L1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor. Lack of evidence for influx route-specific effects. 876 68

The glutamic acid moiety of N-[4-[3-(2,4-diamino-7H-pyrrolo[2, 3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid (1b, TNP-351) and related compounds was replaced with some N5-substituted glutamines. Antifolates (4A-S) were effectively prepared by coupling pyrrolo[2,3-d]pyrimidine carboxylic acids (11a, b) with some properly protected N5-substituted glutamine derivatives (10A-S), which were prepared by coupling Boc-Glu-OMe (7) with various amines (8A-S) using a suitable condensing reagent, followed by hydrolysis. The inhibitory effects of the resulting products on dihydrofolate reductase (DHFR), thymidylate synthetase (TS) and the growth of murine fibrosarcoma Meth A cells in culture were examined. All N5-substituted glutamine analogs (4A-S) inhibited DHFR much more strongly than TNP-351 and some analogs exhibited the same potent growth inhibition of Meth A cells as TNP-351. Some typical analogs (4Bb, 4Db, 4F, 4Oa) were also examined for inhibitory effects on the growth of methotrexate (MTX)-resistant human CCRF-CEM cells in culture and for in vivo antitumor activities against murine leukemia and solid tumors. MTX-resistant cells, with a defect in transport and decreased polyglutamylation activity, showed little cross resistance to the analog (4Oa) having a tetrazole moiety as a substituent of glutamine, which exhibited potent antitumor activities. These results demonstrate that the antifolate analogs (4) with N5-substituted glutamine in place of glutamic acid are novel potent DHFR inhibitors with activity against MTX-resistant tumors. The potent antitumor activity of these analogs (4) may result from their effective uptake via reduced folate carrier in combination with their potent inhibition of DHFR.
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PMID:Non-glutamate type pyrrolo[2,3-d]pyrimidine antifolates. II. Synthesis and antitumor activity of N5-substituted glutamine analogs. 879 69

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 microM) inhibitor of FPGS partially purified from CCRF-CEM human leukemia cells than is bromosulfophthalein (IC50, 17 microM), the first reported nonsubstrate-analog inhibitor of FPGS (J. J. McGuire et 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 (K(ii) = 0.9 microM; K(is) = 1.1 microM) and glutamic acid (K(ii) = 1.0 microM; K(is) = 5.2 microM) as the variable substrates; suramin inhibition tends toward being competitive with respect to the third FPGS substrate, ATP (K(ii) = 3.4 microM; K(is) = 0.35 microM), since the major effect is on its K(m). Suramin is a much less potent inhibitor of two other folate-dependent enzymes, dihydrofolate reductase (IC50, 38 microM; methotrexate (MTX), 0.6 nM) and thymidylate synthase (IC50, 87 microM; MTX, 48 microM). 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 Rideout et al. (Int. J. Cancer 61, 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.
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PMID:Potent inhibition of human folylpolyglutamate synthetase by suramin. 891 44

N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.
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PMID:LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. 906 81


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