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)

5-Deaza-10-propargylfolic acid (4), an analogue of the thymidylate synthase (TS) inhibitor 10-propargyl-5,8-dideazafolic acid (PDDF, 1), was prepared via alkylation of diethyl N-[4-(propargylamino)benzoyl]-L-glutamate (7) by 2-amino-6-(bromomethyl)-4(3H)-pyrido[2,3-d]pyrimidinone (15). Bromomethyl intermediate 15 was prepared from the corresponding hydroxymethyl precursor 14 by treatment with 48% HBr. Hydroxymethyl compound 14 was obtained by deamination of reported 2,4-diaminopyrido[2,3-d]pyrimidine-6-methanol (12a) in refluxing 1 N NaOH. Both 12a and its 5-methyl-substituted analogue 12b were converted to versatile 6-bromomethyl intermediates 13a and 13b from which important antifolates may be readily derived. Alkylation of 7 by 13a,b led to 10-propargyl-5-deazaaminopterin (5) and 5-methyl-10-propargyl-5-deazaaminopterin (6). As an inhibitor of TS from H35F/F cells, 4 gave an IC50 value showing it to be approximately 6-fold less inhibitory than PDDF (90 nM for 4 vs 14 nM for PDDF). In in vitro studies, IC50 (microM) values obtained for 4 vs L1210 and S180 of 1.50 and 2.35, respectively, were similar to those obtained for PDDF (2.61 and 1.97). Against HL60 cells, 4 was about 7-fold more cytotoxic than PDDF (IC50 values 0.72 and 5.29 microM). Inclusion of thymidine did not establish TS as the site of cytotoxic action for either 4 or PDDF in the cell lines used. In in vivo tests against L1210 in mice, 4 failed to show therapeutic effect. The 2,4-diamino compounds 5 and 6 were as potent inhibitors of DHFR from L1210 cells as MTX and 7- and 35-fold, respectively, more inhibitory than MTX toward L1210 cell growth. In mediated influx into L1210 cells, 5 and 6 were transported 2.7- and 8.5-fold, respectively, more readily than MTX. Against the EO771 mammary adenocarcinoma in mice, 6 produced greater antitumor effect than MTX. A dose of 36 mg/kg per day for 5 days caused no toxic deaths while the average tumor volume among 10 mice was reduced to 8-9% of that of the control, and 20% of the test animals were rendered tumor free.
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PMID:Synthesis and antifolate evaluation of the 10-propargyl derivatives of 5-deazafolic acid, 5-deazaaminopterin, and 5-methyl-5-deazaaminopterin. 173 51

Rat surfactant protein A (SP-A) was expressed in a Chinese hamster ovary (CHO-K1) cell line and characterized for biologic activity using assays for receptor binding and modulation of phospholipid secretion from isolated type II cells. The CHO-K1 cell line was cotransfected with separate plasmids encoding for the rat SP-A, dihydrofolate reductase and neomycin phosphotransferase, respectively. Antibiotic (Geneticin-G418)-resistant transformants were screened by ELISA for the secretion of recombinant SP-A into the media. Northern analysis of the transfected cell lines demonstrated the expression of both 1.6 kb and 0.9 kb mRNA species for SP-A, consistent with the proposed differential polyadenylation of the primary transcript. Amplification with methotrexate resulted in a dose-dependent increase in mRNA for SP-A and a 20-fold increase in the production of recombinant SP-A relative to untreated cells. Maximum production of SP-A was 370 micrograms of SP-A/l of media in a 4-day incubation. Recombinant SP-A was purified from the serum-free media of large scale cultures of transfected, amplified CHO cells by affinity chromatography on mannose-Sepharose. The recombinant SP-A migrated similarly to native SP-A by NaDodSO4-PAGE analysis under reducing and nonreducing conditions and under reducing conditions after digestion with N-glycanase. Recombinant SP-A effectively competed with 125I-native SP-A for binding to the high affinity receptor for SP-A on isolated plasma membranes from rat alveolar type II cells. The recombinant SP-A was as effective as native SP-A in the inhibition of secretion of phospholipid from isolated type II cells. We conclude that recombinant rat SP-A produced in Chinese hamster ovary cells is physically and functionally similar to native rat SP-A.
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PMID:Expression and characterization of rat surfactant protein A synthesized in Chinese hamster ovary cells. 217 80

Two analogues of dihydrofolic acid possessing a 7,8-dihydro-8-oxapterin ring system have been synthesized and evaluated for their antifolate activities. These compounds, N-[(2-amino-4-hydroxy-7,8-dihydro-8-oxa-6-pteridinyl)benzoyl]-L-glutamic acid (3) and N-[[(2-amino-4-hydroxy-7,8-dihydro-8-oxa-6-pteridinyl) methyl]benzoyl]-L-glutamic acid (4), were synthesized by reacting the appropriately substituted alpha-halo ketones with 2,5-diamino-4,6-dihydroxypyrimidine (2). Elaboration of p-carbomethoxybenzaldehyde (5) to p-carbomethoxyphenacyl bromide (7) was accomplished by its oxidation with Jones reagent and the successive treatment of the oxidation product with SOCl2, CH2N2, and HBr. Commercially available p-vinylbenzoic acid (11) was converted to its glutamate conjugate 12 and was further converted to the bromo ketone, diethyl N-[p-(1-bromo-2-oxopropyl)benzoyl]-L-glutamate (17), by a series of reactions involving epoxidation, oxirane ring opening with HBr, Jones oxidation, Zn/HOAc reduction, and successive treatment of the reduction product 16 with SOCl2, CH2N2, and HBr. These bromo ketones, 7 and 17, upon reaction with pyrimidine 2, gave the diethyl esters of the target compounds, which were hydrolyzed to 3 and 4 with NaOH. Compound 4 underwent an interesting acid-catalyzed isomerization where the double bond of 4 was shifted from the 5,6-position to the 6,9-position to give the isomer 19. Both compounds 3 and 4 were inactive against Lactobacillus casei (ATCC 7469) and did not serve as synthetic substrates of L. casei dihydrofolate reductase. Compound 4 showed activity against Streptococcus faecium (ATCC 8043), but 3 was inactive against this organism.
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PMID:Folate analogues. 22. Synthesis and biological evaluation of two analogues of dihydrofolic acid possessing a 7,8-dihydro-8-oxapterin ring system. 641 65

Six previously unknown 2,4-diamino-6-(anilinomethyl)- and 2,4-diamino-6-[(N-methylanilino)-methyl]pyrido[3,2-d]pyrimidines (5-10) were synthesized from 2,4-diamino-6-(bromomethyl)-pyrido[3,2-d]pyrimidine hydrobromide (11.HBr) by treatment with the appropriate aniline or N-methylaniline in dimethylformamide at room temperature, with or without NaHCO3 present. Compounds 5-10 were tested as inhibitors of dihydrofolate reductase from Pneumocystis carinii, Toxoplasma gondii, and rat liver as a part of a larger effort directed toward the discovery of lipophilic nonclassical antifolates combining high enzyme selectivity and high potency. Of the six analogues tested, the most potent and selective against T. gondii DHFR was 2,4-diamino-6-[(3',4',5'-trimethoxy-N-methylanilono)methyl]pyrido[ 3,2-d d pyrimidine (7), which had an IC50 of 0.0047 microM against this enzyme as compared with 0.026 microM against the rat liver enzyme. The potency of 7 against T. gondii DHFR was similar to that of trimetrexate (TMQ, 1) and piritrexim (PTX, 2) but was > 500-fold greater than that of trimethoprim (TMP, 3). However, while 7 was more selective than either TMQ (19x) or PTX (63x) against this enzyme, its selectivity in comparison with TMP was 8-fold lower. 2,4-Diamino-6-[3',4',5'-trimethoxyanilino)methyl]pyrido[3,2-d]pyri midin e (6) was 17-fold less active than 7 and was also less selective. 2,4-Diamino-6-[(3',4'-dichloro-N-methylanilino)methyl]pyrido[3, 2-d]pyrimidine (10) had an IC50 of 0.022 microM against P. carinii DHFR and was comparable in potency to TMQ and PTX. The species selectivity of 10 for P. carinii versus rat liver DHFR was greater than that of either TMQ (21-fold) or PTX (31-fold). On the other hand, even though 10 was slightly more active than TMQ against the P. carinii enzyme, its selectivity was 7-fold lower than that of TMP. Thus, the goal of combining high enzyme binding activity, which is characteristic of the fused-ring compounds TMQ and PTX, with high selectivity for T. gondii and P. carinii DHFR versus rat liver DHFR, which is characteristic of the monocyclic compound TMP, remained unmet in this limited series.
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PMID:2,4-Diaminopyrido[3,2-d]pyrimidine inhibitors of dihydrofolate reductase from Pneumocystis carinii and Toxoplasma gondii. 762 1

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.
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PMID:High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease. 855 76

A stable transfection system for the lower trypanosomatid Leptomonas collosoma was established using the Leishmania expression vector pX that was derived from an extrachromosomal amplified DNA carrying the dihydrofolate reductase-thymidylate synthase gene. Transformants harboring the pX vector were selected on Geneticin, and cell lines harboring as many as 200 copies per cell were obtained by increasing the drug concentration. The system was utilized to examine the expression of the SL RNA genes of Trypanosoma brucei and Leishmania mexicana amazonensis. Despite the high copy number of the foreign genes, no heterologous SL RNA was detected in steady-state RNA populations or by nascent transcription of cells made permeable by lysolecithin, suggesting the existence of a transcription barrier for this gene among the trypanosomatids. Such a barrier does not exist for the T. brucei 5S rRNA gene, since transcription of this gene was detected in permeable cells carrying the heterologous gene and in steady-state RNA population. To overcome the transcription barrier, the authentic regulatory region of the L. collosoma SL gene was identified. Chimeric constructs carrying 50 or 415 nt of the L. collosoma SL upstream sequence and 24 nt of the L. collosoma exon portion were fused to the T. brucei SL RNA gene at the SL portion. Expression of a chimeric SL RNA of 150 nt, composed of 24 nt L. collosoma RNA and 126 nt T. brucei RNA, was observed only in cell lines carrying the 415-nt upstream sequence. The efficient expression of the chimeric SL RNA, using the L. collosoma SL gene regulatory regions, may facilitate a structure-function analysis of chimeric and site-directed mutated SL RNA in trans-splicing.
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PMID:Stable transfection in the monogenetic trypanosomatid Leptomonas collosoma--transcription barrier of heterologous trypanosomatid SL RNA genes and expression of a chimeric SL RNA molecule. 888 31