Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

5,10-Dideazatetrahydrofolic acid (DDATHF) is a new potent antitumor agent that specifically inhibits purine biosynthesis, primarily through inhibition of glycinamide ribonucleotide transformylase, the first of the tetrahydrofolate-requiring enzymes in the de novo synthesis pathway. DDATHF has been shown to be an excellent substrate for mouse liver folylpolyglutamate synthetase in vitro, suggesting that intracellular conversion to polyglutamates could play an important role in the action of this antifolate. In this report, metabolic studies of the 6R-diastereomer of DDATHF in the cultured human leukemia cell lines CCRF-CEM and HL-60 are presented. At both 1 and 10 microM (6R)-DDATHF was rapidly converted to polyglutamates in both cell lines. DDATHF(Glu)5 and DDATHF(Glu)6 were the main intracellular metabolites. After incubation in drug-free medium, (6R)-DDATHF polyglutamates were better retained intracellularly with increasing glutamate chain length. (6R)-DDATHF showed reduced cytotoxicity toward a folylpolyglutamate synthetase-deficient cell line, CCRF-CEM30/6 related to a dramatically diminished accumulation of polyglutamates. The activity of (6R)-DDATHF in CCRF-CEM30/6 cells was decreased after both short and prolonged exposures. These results suggest that polyglutamylation of (6R)-DDATHF not only represents a mechanism for trapping the drug inside the cells but also produces a more potent inhibitor of the target enzyme.
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PMID:Intracellular metabolism of 5,10-dideazatetrahydrofolic acid in human leukemia cell lines. 170 76

Five analogues of methotrextate (MTX), 10-deazaaminopterin (10-DAM), and 10-ethyl-10-deazaaminopterin (10-EDAM) in which the glutamate moiety was replaced by either a gamma-methyleneglutamate or beta-hydroxyglutamate were synthesized and evaluated for their antifolate activity. These analogous are 4-amino-4-deoxy-N10-methylpteroyl-beta-hydroxyglutamic acid (1), 4-amino-4-deoxy-10-deazapteroyl-beta-hydroxyglutamic acid (2), 4-amino-4-deoxy-N10-methylpteroyl-gamma-methyleneglutamic acid (3, MMTX), 4-amino-4-deoxy-10-deazapteroyl-gamma-methyleneglutamic acid (4, MDAM), and 4-amino-4-deoxy-10-ethyl-10-deazapteroyl-gamma-methyleneglutamic acid (5, MEDAM). None of these compounds were metabolized to the respective polyglutamate derivative as judged by their inability to serve as substrates for CCRF-CEM human leukemia cell folylpolyglutamate synthetase (FPGS) in vitro. All compounds inhibited recombinant human-dihydrofolate reductase (DHFR) at nearly equivalent magnitude as MTX. Growth-inhibition studies with H35 hepatoma, Manca human lymphoma, and CCRF-CEM human leukemia cells established greater cytotoxic effects with compounds 3-5 than with compounds 1 and 2. gamma-Methyleneglutamate derivatives 3-5 were transported to H35 hepatoma cells better than MTX or beta-hydroxyglutamate derivatives 1 and 2. Compound 3 was 2.5 times better than MTX in competing with folinic acid transport in H35 hepatoma cells. Compound 1 did not have a significant inhibitory effect on folinic acid transport even at 50 microM under identical conditions. The IC50 for compound 1 against H35-hepatoma cell growth was 8.5-fold higher than MTX. Compounds with the gamma-methyleneglutamate moiety (3-5) exhibited almost equal or lower IC50 values than MTX against the growth of CCRF-CEM human leukemia cells. These studies show that on continuous exposure, the non-polyglutamylatable inhibitors DHFR (3-5) can exhibit superior antifolate activity compared to the polyglutamylatable methotrexate, presumably due to their enhanced transport to these cell lines. Compounds 3-5 appear to be excellent models to study the role of polyglutamylation of antifolates in antitumor activity and host toxicity.
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PMID:Folate analogues. 34. Synthesis and antitumor activity of non-polyglutamylatable inhibitors of dihydrofolate reductase. 199 21

Determinants of methotrexate (MTX) resistance in cell lines resistant to short, but not continuous, MTX exposure were investigated since such lines may have relevance to clinical resistance. CCRF-CEM R30dm (R30dm), cloned from CCRF-CEM R30/6 (a MTX-resistant subline of the CCRF-CEM human leukemia cell line), had growth characteristics similar to CCRF-CEM. R30dm was resistant to a 24-h exposure to levels as high as 300 microM MTX but was as sensitive as CCRF-CEM to continuous MTX exposure. MTX resistance of R30dm was stable for greater than 68 weeks in the absence of selective pressure. Initial velocities of MTX transport were comparable for R30dm and CCRF-CEM, as were dihydrofolate reductase specific activity and MTX binding. A 2-fold thymidylate synthase activity decrease for R30dm from that of CCRF-CEM was not a significant factor in R30dm MTX resistance. Decreased MTX poly(gamma-glutamate) synthesis resulted in lower levels of drug accumulation by R30dm. Decreased polyglutamylation was attributable to folylpolyglutamate synthetase (FPGS) activity in R30dm extracts which was 1, 2, and less than or equal to 10% of CCRF-CEM extracts with the substrates MTX, aminopterin, and naturally occurring folates, respectively. Comparison of cell lines with varying levels of resistance to short term MTX exposure indicated that the extent of MTX resistance was proportional to the reduction of FPGS activity. The evidence supported decreased FPGS activity as the mechanism of resistance to short MTX exposure in the cell lines investigated.
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PMID:Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines. 200 75

NaHCO3 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 mM NaHCO3 in all cases; NaCl, sodium formate, sodium acetate, NaN3, and Na2SO3 at 10 mM did not cause activation. Activation could be masked if assay solutions which had extensively absorbed atmospheric CO2 were used. Activation of the human CCRF-CEM FPGS was examined in detail. Km and Vmax values for pteroyl substrates (aminopterin or methotrexate) and L-glutamate increased proportionally in the presence of NaHCO3; there was thus no apparent change in the catalytic efficiency (Vmax/Km) of the FPGS reaction with these substrates. However, NaHCO3 increased the efficiency of the reaction with respect to ATP by decreasing its apparent Km while increasing the Vmax of the reaction. NaHCO3 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 NaHCO3. The potency of 5,8-dideazapteroylornithine, an FPGS-specific inhibitor, was not changed by the presence of NaHCO3 (IC50 = 0.4 microM). These results suggest that FPGS activity with folates and classical antifolates may be activated at physiological concentrations of NaHCO3. In addition, inadvertent contamination of assay solutions with bicarbonate from atmospheric CO2 may cause artifacts in the determination of activity levels and kinetic constants of FPGS.
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PMID:Activation of mammalian folylpolyglutamate synthetase by sodium bicarbonate. 216 55

Acquired resistance of the L1210 leukemia in mice developed with less rapidity during therapy with edatrexate (10-ethyl-10-deazaaminopterin, EDX) than with MTX. Since this was explained only partially by the somewhat greater antitumor activity of EDX, this result may also reflect a difference in biochemical phenotypes selected in each case. Among 20 sublines selected for resistance to MTX, a reduction in influx, an elevation of DHFR, and a reduction of DHFR inhibition by MTX were all delineated. Among 14 sublines selected for resistance to EDX, both a reduction in influx and an elevation in level of DHFR were also commonly found. In addition, however, 7 of 14 EDX-resistant sublines exhibited a reduction in the level of folylpolyglutamate synthetase (FPGS) activity. Clonal derivatives of these 7 EDX-resistant cell lines exhibited 2- to 28-fold reductions in FPGS activity and a commensurate reduction in [3H]-MTX polyglutamate formation in situ following exposure to [3H]-MTX during growth in mice. An analysis of the kinetics and relative substrate preferences for FPGS from variant and parental L1210 cells revealed that the various changes in FPGS activity were at the level of the Vmax rather than Km. These results derived from an in vivo tumor model provide further evidence for a role of FPGS as a determinant of cytotoxicity and acquired resistance to classical folate analogs. They also provide evidence in the same pharmacologic model for a manifestation of resistance to 4-aminofolates in vivo that involves all of the alterations of its primary target, transport, and metabolism that have ever been associated with acquired resistance in cell culture systems.
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PMID:Preferential selection during therapy in vivo by edatrexate compared to methotrexate of resistant L1210 cell variants with decreased folylpolyglutamate synthetase activity. 220 78

The biological activities of novel analogues of methotrexate (MTX) and aminopterin (AMT) in which the gamma-carboxyl was replaced by a 1H-tetrazol-5-yl ring, an isosteric group with acidic properties similar to a carboxyl group, were investigated. The tetrazolyl analogues of MTX and AMT were more potent inhibitors of the growth of CCRF-CEM and K562 human leukemia cell lines during continuous (120 h) and 24-h pulse exposure than were the respective parent drugs; only when the exposure time was reduced to 6 h were the parent drugs more potent. These inhibitory effects on growth correlated with the onset of and recovery from inhibition of de novo thymidylate biosynthesis. Growth inhibition by the analogues was protectable by leucovorin. MTX-resistant CCRF-CEM sublines with decreased transport or increased dihydrofolate reductase (DHFR) levels were cross-resistant to the analogues. The analogues were as potent as their parent drugs in inhibiting DHFR activity in vitro and at displacing [3H]MTX from intracellular DHFR. Each analogue was more effective than its parent drug at inhibiting uptake of [3H]MTX into CCRF-CEM cells. The tetrazole analogue of AMT was a linear competitive inhibitor (Kis = 50 microM) of CCRF-CEM folylpolyglutamate synthetase, while the tetrazole analogue of MTX, unlike all other inhibitors, was linear noncompetitive (Kis = 51 microM, Kii = 321 microM). The data suggest that, compared with MTX or AMT, the tetrazole substituent, in place of the gamma-carboxyl group, allows more efficient transport into cells via the reduced folate/MTX carrier and the resulting greater uptake of the analogues leads to inhibition of DNA synthesis and cell death at lower extracellular concentrations during long exposures. The mechanism of cell death could involve inhibition at folypolyglutamate synthetase, but DHFR is the primary target. The low potency of the analogues during short exposure is presumably related to the inability to form the poly-gamma-glutamyl metabolites required for intracellular retention.
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PMID:Biochemical and growth inhibition studies of methotrexate and aminopterin analogues containing a tetrazole ring in place of the gamma-carboxyl group. 230 27

The properties of a series of methotrexate analogs containing 2,omega-diaminoalkanoic acids have been investigated. The compounds were potent inhibitors of dihydrofolate reductase but, unlike methotrexate, they were also inhibitors of mammalian folylpolyglutamate synthetases. The potency of synthetase and reductase inhibition increased with increasing length of the 2,omega-diaminoalkanoate moiety. The most cytotoxic compound and the most potent inhibitor of both dihydrofolate reductase (I50 = 2.5 to 4 nM) and folylpolyglutamate synthetase (Ki ca. 4 microM) contained 2,5-diaminopentanoic acid (ornithine). These compounds were 70- to 100-fold less cytotoxic than methotrexate to human leukemia cell lines; however, they retained their potency against sublines resistant to methotrexate via defective transport. Their dual loci of enzyme inhibition and their efficacy against methotrexate transport-defective cell lines indicate that these compounds may be an important new class of antifol.
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PMID:Folylpolyglutamate synthetase inhibition and cytotoxic effects of methotrexate analogs containing 2,omega-diaminoalkanoic acids. 242 84

We previously reported (J. Galivan et al., Proc. Natl. Acad. Sci. USA, 82: 2598-2602, 1985) the synthesis and characterization of DL-erythro,threo-gamma-fluoromethotrexate (FMTX). The individual diastereomers, DL-erythro-FMTX (eFMTX) and DL-threo-FMTX (tFMTX), and their radiolabeled counterparts have now been prepared and characterized. Transport of eFMTX (Km = 9.3 microM; Vmax = 7.5 pmol/min/10(7) cells) was similar to that of methotrexate (MTX: Km = 6.6-9.9 microM; Vmax = 11.4-14.2 pmol/min/10(7) cells), while tFMTX (Km = 65.1 microM; Vmax = 8.4 pmol/min/10(7) cells) was transported less efficiently. Both isomers were able to saturate intracellular dihydrofolate reductase and accumulate further as unbound intracellular drug. Based on competition experiments and studies with MTX transport-defective cell lines, both isomers utilized the reduced folate/MTX transport system. Efflux half-times for the isomers were similar to those of MTX. Each isomer was equivalent to MTX in its ability to inhibit dihydrofolate reductase activity and bind to intracellular dihydrofolate reductase when the intracellular drug concentration was limiting. Both isomers had drastically diminished capacity to be metabolized to poly(gamma-glutamyl) metabolites by isolated folylpolyglutamate synthetase and in whole cells; tFMTX was metabolized to a slightly lesser extent than eFMTX. Using the CCRF-CEM human leukemia and H35 rat hepatoma cell lines, the growth-inhibitory effects of eFMTX were almost the same as those of MTX during continuous exposure, while tFMTX was slightly less potent. This difference in growth-inhibitory potency of the two isomers correlated with their ability to inhibit de novo thymidylate synthesis in the H35 cell line. These results indicate that both diastereomers of FMTX are similar in their properties to MTX, except that both are incapable of being readily converted to polyglutamate derivatives. As a result of these properties, both isomers could be used under appropriate conditions in comparative studies with MTX to define the roles of MTX polyglutamates.
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PMID:Biochemical and growth inhibitory effects of the erythro and threo isomers of gamma-fluoromethotrexate, a methotrexate analogue defective in polyglutamylation. 247 80

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 novo purine 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 novo purine 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 novo purine synthesis and that the biochemical processes involved in their cytotoxicity display little stereochemical specificity.
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PMID:The 6S- and 6R-diastereomers of 5, 10-dideaza-5, 6, 7, 8-tetrahydrofolate are equiactive inhibitors of de novo purine synthesis. 259 65

Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.
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PMID:Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids. 289 31


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