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)

A series of eight previously undescribed 2,4-diaminothieno[2,3-d]pyrimidine analogues of the potent dihydrofolate reductase (DHFR) inhibitors trimetrexate (TMQ) and piritrexim (PTX) were synthesized as potential drugs against Pneumocystis carinii and Toxoplasma gondii, which are major causes of severe opportunistic infections in AIDS patients. 2,4-Diamino-5-methyl-6-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 3,4,5-trimethoxy or 2,5-dimethoxy substitution in the aryl/aralkyl moiety and 2,4-diamino-5-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 2,5-dimethoxy substitution in the aryl/aralkyl moiety were obtained by reaction of the corresponding 2-amino-3-cyanothiophenes with chloroformamidine hydrochloride. The aryl group in the 5,6-disubstituted analogues was either attached directly to the hetero ring or was separated from it by one or two carbons, whereas the aryl group in the 5-monosubstituted analogues was separated from the hetero ring by two or three carbons. 2-Amino-3-cyano-5-methyl-6-(aryl/alkyl)thiophene intermediates for the preparation of the 5,6-disubstituted analogues were prepared from omega-aryl-2-alkylidene-malononitriles and sulfur in the presence of a secondary amine, and 2-amino-3-cyano-4-(aryl/aralkyl)thiophene intermediates for the preparation of the 5-monosubstituted analogues were obtained from omega-aryl-1-chloro-2-alkylidenemalononitriles and sodium hydrosulfide. Synthetic routes to the heretofore unknown ylidenemalononitriles, and the ketone precursors thereof, were developed. The final products were tested in vitro as inhibitors of DHFR from Pneumocystis carinii, Toxoplasma gondii, rat liver, beef liver, and Lactobacillus casei. A selected number of previously known 2,4-diaminothieno[2,3-d]pyrimidines lacking the 3,4,5-trimethoxyphenyl and 2,5-dimethoxyphenyl substitution pattern of TMQ and PTX, respectively, were also tested for comparison. None of the compounds was as potent as TMQ or PTX, and while some of them showed some selectivity in their binding to Pneumocystis carinii and Toxoplasma gondii versus rat liver DHFR, this effect was not deemed large enough to warrant further preclinical evaluation.
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PMID:2,4-Diaminothieno[2,3-d]pyrimidine analogues of trimetrexate and piritrexim as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. 823 96

These structural studies reveal unusual intermolecular interactions for the binding of inhibitors and cofactor in ternary complexes with both wild type and F31 mutant recombinant human DHFR and show that these inhibitors have flexibility in occupying the active site. These studies also possibly indicate the first structural data for a ternary complex with a folate inhibitor and a polyglutamate side chain. However, further refinement of this data is necessary before this can be confirmed. In contrast to the ternary complexes of folate and MTX, the lipophilic antifolate PTX binds with its methoxybenzoyl ring oriented toward the cofactor nicotinamide ring, while that of TMQ it is bound closer to the Phe-31 position. Furthermore, the nicotinamide ring makes a close contact to the N10 amine of TMQ, significantly different from its binding site interactions in MTX complexes. These data also reveal that the conserved contacts between the cofactor carboxyamide with the enzyme backbone residues Ala-9 and Ile-16 are dictated by the enzyme and that changes in the orientation of the structural elements requires only subtle changes in the secondary structural units in which they are contained. Therefore, only by careful analysis of a series of enzyme complexes can the mechanisms of binding action be delineated.
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PMID:Conformational analysis of human dihydrofolate reductase inhibitor complexes: crystal structure determination of wild type and F31 mutant binary and ternary inhibitor complexes. 830 63

Chinese hamster ovary cells with a single allele for dihydrofolate reductase were used as a model system to study the effect of exposure to an alkylating agent, ethylmethanesulfonate, on rates and types of mutations at the dihydrofolate reductase locus leading to antifolate resistance. After overnight exposure to 400 micrograms/ml ethylmethanesulfonate, cells were allowed to recover for 3 days, and resistant colonies were selected in 8 x 10(-8) M trimetrexate. Trimetrexate, rather than methotrexate, was used as the selecting agent to increase the probability of obtaining mutations in dihydrofolate reductase, rather than in the reduced folate transport carrier protein. Seven of several hundred surviving colonies were selected at random, and cell lines were established. Cell lines 1-3 were maintained in culture in the presence of 8 x 10(-8) M trimetrexate and were 66-170-fold resistant to the drug. Cell lines 4-7 were initially expanded in 8 x 10(-8) M trimetrexate but were then maintained in the absence of the drug. These cell lines were 4.4-26-fold resistant to the drug, compared with the parental cell line. Cell line 1 was found to have an increase in dihydrofolate reductase activity, a corresponding increase in mRNA for dihydrofolate reductase, and amplification of this gene. Cell lines 2 and 6 had a mutated dihydrofolate reductase with altered trimetrexate- and methotrexate-binding properties. Cell line 3 had a 3-fold increase in dihydrofolate reductase activity. In cell lines 4, 5, and 7 the mechanisms of resistance to trimetrexate remain unknown.
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PMID:Mutations leading to antifolate resistance in Chinese hamster ovary cells after exposure to the alkylating agent ethylmethanesulfonate. 834 Dec 68

Three methods for analyzing the products of polymerase chain reaction were applied to detect complex alterations of dihydrofolate reductase (DHFR) gene, in order to assess their value in detection of folate-resistance in leukemia cells. A single point mutation in the second position of codon 31, a T-to-C transition, in trimetrexate (TMQ) resistant MOLT-3/TMQ200 cells was detected by either allele-specific oligonucleotide hybridization or restriction pattern of the PCR product. These two analyses allowed us to detect not only the presence of the mutation, but also the amplification of the mutated gene in TMQ-methotrexate (MTX) doubly-resistant MOLT-3/TMQ200-MTX500 cells. The base change was confirmed by direct sequencing method of the PCR product. Using these analyses of the PCR product, the complex alterations of DHFR gene are to be examined in leukemic patient cells.
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PMID:[Detection of alterations of dihydrofolate reductase gene in folate-resistant leukemia cells by in vitro enzymatic amplification]. 836 Oct 48

In order to clarify a molecular mechanism of folate resistance in leukemia cells, we studied alterations of the dihydrofolate reductase (DHFR) gene in a human leukemia cell line, MOLT-3, and its sublines made resistant to methotrexate (MTX), trimetrexate (TMQ) and N10-propargyl-5,8-dideazafolic acid (CB3717), alone or in combination. Major alterations of the DHFR gene were examined by Southern analysis of high-molecular-weight DNA. The presence of a base change (T-->C) at nucleotide position 91 of the DHFR gene, which is reported to be responsible for the reduced affinity of the enzyme for MTX in an MTX-resistant human colon carcinoma cell, was examined by allele-specific oligonucleotide hybridization. In a 10,000-fold MTX-resistant subline (MOLT-3/MTX10,000), the normal allele of DHFR gene had been amplified. In contrast, a 200-fold TMQ-resistant subline (MOLT-3/TMQ200) and a 30-fold CB3717-resistant subline selected from MOLT-3/TMQ200 (MOLT-3/TMQ200-CB-3717(30)) were shown to have the mutant allele. Furthermore, the mutant allele had been amplified in a 500-fold MTX-resistant subline, which was established by the continuous exposure of the MOLT-3/TMQ200 cells to stepwise increases of drug concentration and designated as MOLT-3/TMQ200-MTX500. On the other hand, a 40-fold-resistant subline to CB3717 alone (MOLT-3/CB3717(40)) showed the normal allele without amplification. These data suggest that complex alterations of the DHFR gene are involved in the molecular mechanisms of folate resistance that can be differentially introduced into leukemia cells by exposure to various folate analogues, alone or in combination.
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PMID:Differential alterations of dihydrofolate reductase gene in human leukemia cell lines made resistant to various folate analogues. 844 31

The clinical efficacy of trimetrexate, a dihydrofolate reductase inhibitor with potent in vitro antitoxoplasma activity, was assessed in 9 sulfonamide-intolerant patients with AIDS and biopsy-proven cerebral toxoplasmosis. The 9 patients were treated for 28-149 days with trimetrexate (30-280 mg/m2/day) plus leucovorin (20-90 mg/m2 every 6 h). Radiographic responses were documented in 8 patients, and clinical responses in 5 patients. Despite continued therapy, all patients deteriorated clinically and radiographically within 13-109 days of their initial improvement. Trimetrexate at very high doses for extended periods was not associated with serious toxicity. Trimetrexate alone had dramatic but transient activity in sulfonamide-intolerant patients and thus is not adequate as single-agent therapy for AIDS-associated toxoplasmosis.
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PMID:Salvage trial of trimetrexate-leucovorin for the treatment of cerebral toxoplasmosis in patients with AIDS. 850 35

Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y-containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers.
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PMID:A gene transfer strategy for making bone marrow cells resistant to trimetrexate. 863 Apr 26

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

Nine novel 2,4-diamino-5-methyl-6-substituted-pyrido[2,3-d]pyrimidines, 2-10, were synthesized as potential inhibitors of Pneumocystis carinii dihydrofolate reductase (pcDHFR) and Toxoplasma gondii dihydrofolate reductase (tgDHFR). Compounds 2-5 were designed as conformationally restricted analogues of trimetrexate (TMQ), in which rotation around tau 3 was constrained by incorporation of the side chain nitrogen as part of an indoline or an indole ring. Analogue 6, which has an extra atom between the side chain nitrogen and the phenyl ring, has its nitrogen as part of a tetrahydroisoquinoline ring. Analogues 7-9 are epiroprim (Ro 11-8958) analogues and contain a pyrrole ring as part of the side chain substitution on the phenyl ring similar to epiroprim. These analogues were designed to investigate the role of the pyrrole substitution on the phenyl ring of 2,4-diamino-5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines. Molecular modeling indicated that a pyrrole substituent in the ortho position of the side chain phenyl ring was most likely to interact with pcDHFR in a manner similar to the pyrrole moiety of epiroprim. Analogue 10, in which a phenyl ring replaced a methoxy group, was synthesized to determine the contribution of a phenyl ring on selectivity, lipophilicity, and cell penetration. The synthesis of analogues 2-4 was achieved via reductive amination of 2,4-diamino-5-methyl 6-carboxaldehyde with the appropriately substituted indolines. The indolines were obtained from the corresponding indoles via NaCNBH3 reductions. Analogues 5-10 were synthesized by nucleophilic displacement of 2,4-diamino-5-methyl-6-(bromomethyl)-pyrido[2,3-d]pyrimidine with the 5-methoxyindolyl anion, 6,7-dimethoxytetrahydroisoquinoline, the appropriately substituted pyrroloaniline or 2-methoxy-5-phenylaniline. The pyrroloanilines were synthesized in two steps by treating the substituted nitroanilines with 2,5-dimethoxy-tetrahydrofuran to afford the nitropyrrole intermediates, followed by reduction of the nitro group with Raney Ni. The analogues were more potent than trimethoprim and epiroprim and more selective than TMQ and piritrexim against pcDHFR and tgDHFR. Compounds 5 and 10 had IC50 values of 1 and 0.64 microM, respectively, for the inhibition of the growth of T. gondii cells in culture, and showed excellent culture IC50/enzyme IC50 ratios, which were correlated with their calculated log P values, indicating a direct relationship between calculated lipophilicity and cell penetration.
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PMID:Synthesis and biological evaluation of nonclassical 2,4-diamino-5-methylpyrido[2,3-d]pyrimidines with novel side chain substituents as potential inhibitors of dihydrofolate reductases. 904 38

Seven novel tricyclic pyrimido[4,5-c][2,7]naphthyridones 5-8 and the corresponding naphthyridines 9-11 were synthesized as conformationally restricted inhibitors of dihydrofolate reductase (DHFR) and as antitumor and/or antiinfectious agents. The analogues were designed to orient the side chain trimethoxyphenyl group in different conformationally defined positions in order to explore the effect of the side chain orientation on binding affinity and selectivity for DHFR from various species. The semirigid orientations were achieved by bridging the C5 and N10 of compound 12 with a N-ethyl bridge and by variation of the position of double bonds in rings B and C as well as substitution at the 2',6'-positions of the phenyl ring. The synthesis of compounds 5-11 were accomplished by cyclocondensation of the appropriate keto ester (as the biselectrophile) with 2,4,6-triaminopyrimidine to afford the lactam 5. The dehydrolactams 6 and 7 were prepared by air oxidation and PtO2-catalyzed dehydrogenation of 7, respectively. The dichloro dehydro lactam 8 was obtained by refluxing lactam 5 and/or 6 in POCl3 or a mixture of POCl3/PCl5. Compounds 9-11 were obtained by two methods, direct borane reduction of lactam 5 or 6 or thiation of the dipivoylated lactam 15 followed by reductive dethiation. Compounds 9-11 were interconverted by air oxidation or PtO2-catalyzed reduction/oxidation, respectively. The compounds were evaluated as inhibitors of DHFR from Pneumocystis carinii (pc) and Toxoplasma gondii (tg) with rat liver (rl) serving as the reference mammalian enzyme. In the lactam series 5-8, the most unsaturated analogue 7 showed an IC50 of 86 nM against rlDHFR, almost 100-fold more active than 5 and 3-fold more active than 6. The 2',6'-dichloro dehydro lactam 8 was less active than the corresponding dehydro lactam 6 against rlDHFR. In the naphthyridine series 9-11, the dehydro analogue 10 was more active than 9 against rlDHFR. The fully reduced analogue 11 (as a mixture of cis and trans isomers) was the most active in the naphthyridine series. The analogues were, in general, more inhibitory against rlDHFR than against pcDHFR, or tgDHFR, and thus lacked selectivity. In addition, they were less potent than the bicyclic compounds trimetrexate 3 (TMQ) and piritrixim 4 (PTX).
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PMID:Synthesis and biological activities of conformationally restricted, tricyclic nonclassical antifolates as inhibitors of dihydrofolate reductases. 919 71


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