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)

BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 x 10(-5) and 1 x 10(-5), respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and surveys on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 micro M hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.
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PMID:Alteration of human breast tumor cell membrane functions by chromosome-mediated gene transfer. 23 36

In approximately one-third of primary hyperoxaluria type 1 patients, disease is associated with a unique protein sorting defect in which hepatic L-alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), which is normally peroxisomal, is mistargeted to mitochondria. In all such patients analyzed to date, the gene encoding the aberrantly targeted AGT carries three point mutations, each of which specifies an amino acid substitution. In this paper we show that one of these substitutions, a proline-to-leucine at residue 11, is necessary and sufficient for the generation of a mitochondrial targeting sequence in the AGT protein. AGT with this substitution appears to interact specifically with the mitochondrial protein import machinery, via a discrete N-terminal domain of the AGT protein. The N-terminal 19 amino acids of AGT with this substitution are sufficient to direct mouse cytosolic dihydrofolate reductase to mitochondria, and a synthetic peptide corresponding to this same 19-amino acid region reversibly inhibits mitochondrial protein import, not only of AGT but also of ornithine transcarbamoylase, a genuine cytoplasmically synthesized mitochondrial protein. We have extended these studies to analyze a region of normal human AGT cDNA directly upstream of the coding region. This sequence appears to correspond to an ancestral mitochondrial targeting sequence deleted from the human coding region by point mutation at the initiation codon. We show that reestablishment of this initiation codon produces an active mitochondrial targeting sequence that is different to that found in the hyperoxaluria patients. These results are discussed with reference to the AGT targeting defect in primary hyperoxaluria and also in relation to the highly unusual species specificity of subcellular distribution of AGT among mammals.
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PMID:Mistargeting of peroxisomal L-alanine:glyoxylate aminotransferase to mitochondria in primary hyperoxaluria patients depends upon activation of a cryptic mitochondrial targeting sequence by a point mutation. 196 59

The heretofore unknown gamma-(m-carboxyanilide) and gamma-(m-boronoanillide) derivatives of methotrexate (MTX) and the gamma-(m-carboxyanilide) derivatives of aminopterin (AMT) were prepared and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of cell growth in culture with the aim of comparing their activity with that of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, a potent antifolate whose side chain likewise contains a hydrophobic aromatic ring with an acid group on the ring. All three anilides were potent DHFR inhibitors, with activity comparable to MTX and AMT. The gamma-(m-boronoanilide) displayed growth inhibitory potency similar to that of the hemiphthaloylornithine analogue, with an IC50 of only 0.7 nM. This compound, which is the most potent of the gamma-amides of MTX tested to date, is also the first reported example of an antifolate with a B(OH)2 group in the side chain and is especially novel because of its potential to form a stable tetrahedral boronate complex by reaction with electron rich OH or NH2 groups in the active site of DHFR or other folate enzymes. In antitumor assays against L1210 leukemia in mice, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine gave a T/C of greater than 263% at 20 mg/kg (qdx9) and 300% at 16 mg/kg (bidx10), whereas maximally tolerated doses of MTX of 8 mg/kg (qdx9) and 1 mg/kg (bidx10) gave T/C values of 213 and 188%, respectively. MTX gamma-(m-boronoanilide) was also active, with a T/C of 175% at 32 mg/kg (qdx9), the highest dose tested.
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PMID:Synthesis and biological activity of methotrexate analogues with two acid groups and a hydrophobic aromatic ring in the side chain. 199 80

The 5-deaza and 5,8-dideaza analogues of N alpha-pteroyl-L-ornithine (Pter-Orn), the 5-deaza, 8-deaza, and 5,8-dideaza analogues of N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-Orn), and the N delta-carboxymethyl derivative of N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-ornithine (mAPA-Orn) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of tumor cell growth in culture. Reductive amination of 2-acetamido-6-formylpyrido[2,3-d]pyrimidine-4(3H)-one with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate followed by removal of the blocking groups afforded the 5-deaza analogue of Pter-Orn, whereas N-alkylation of methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate with 2-amino-6-(bromomethyl)quinazolin-4(3H)-one and deprotection gave the corresponding 5,8-dideaza analogue. Reductive coupling of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile and 4-aminobenzoic acid followed by reaction with 95-97% formic acid yielded 4-amino-4-deoxy-5-deaza-N10-formylpteroic acid, which on condensation with methyl N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection gave the 5-deaza analogue of APA-Orn. A similar sequence starting from 2,4-diamino-quinazoline-6-carbonitrile led to the corresponding 5,8-dideaza compound, whereas treatment of 2,4-diamino-pyrido[3,2-d]pyrimidine-6-methanol with phosphorus tribromide followed by condensation with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection afforded the 8-deaza analogue. For the preparation of the N delta-carboxymethyl derivative of mAPA-Orn, N alpha-(benzyloxycarbonyl)-L-ornithine was subjected to N delta-monoalkylation with glyoxylic acid and sodium cyanoborohydride, followed by N delta-acylation with ethyl trifluoroacetate, N alpha-deprotection by hydrogenolysis, condensation with 4-amino-4-deoxy-N10-methylpteroic acid, and N delta-deprotection by gentle treatment with ammonia. The 2,4-diamino derivatives all inhibited the growth of tumor cells in culture, with IC50 values of 0.2-2 microM, and inhibited purified DHFR with IC50 values of 0.02-0.08 microM. Deletion of ring nitrogens and N delta-carboxymethylation both increased potency in the cell growth assay; however, the ornithine derivatives were less potent than aminopterin or methotrexate.
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PMID:Synthesis and in vitro biological activity of new deaza analogues of folic acid, aminopterin, and methotrexate with an L-ornithine side chain. 201 22

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

Six new 5,8-dideaza analogues of folic acid and aminopterin containing a terminal L-ornithine residue were prepared by using multistep synthetic sequences. Each was evaluated as an inhibitor of hog liver folylpolyglutamate synthetase and human dihydrofolate reductase. Structural modifications at positions 2, 4, 5, and 10 were included to help define structure-activity relationships for compounds of this type. The compound N alpha-(4-amino-4-deoxy-5-chloro-5,8-dideazapteroyl)-L-ornithine (3f) was identified as the most potent inhibitor of mammalian folylpolyglutamate synthetase reported thus far (Ki congruent to 2 nM). Its 4-oxy counterpart, N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, was only 5-fold less inhibitory than 3f toward folylpolyglutamate synthetase but was found to be a much weaker inhibitor of dihydrofolate reductase than 3f.
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PMID:Inhibition of mammalian folylpolyglutamate synthetase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin bearing a terminal L-ornithine. 273 91

Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.
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PMID:Methotrexate analogues. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogues containing a basic amino acid side chain. 287 Nov 91

Analogues of methotrexate (MTX) with strong alkylating activity were prepared by replacing the L-glutamate side chain with N omega-haloacetyl derivatives of L-lysine and L-ornithine. Haloacetylation was accomplished in 30-40% yield by reaction of the preformed L-lysine and L-ornithine analogues of MTX with p-nitrophenyl bromoacetate or chloroacetate in aqueous sodium bicarbonate at room temperature. All four haloacetamides were potent inhibitors in spectrophotometric assays measuring noncovalent binding to purified dihydrofolate reductase (DHFR) from L1210 cells. In experiments designed to measure time-dependent inactivation of DHFR from L1210 cells and Candida albicans, the N epsilon-(bromoacetyl)-L-lysine and N delta-(bromoacetyl)-L-ornithine analogues gave results consistent with covalent binding, whereas N epsilon- and N delta-chloroacetyl analogues did not. The N delta-(bromoacetyl)-L-ornithine analogue appeared to be the more reactive one toward both enzymes. Amino acid analysis of acid hydrolysates of the L1210 enzyme following incubation with the bromoacetamides failed to demonstrate the presence of a carboxymethylated residue, suggesting that alkylation had perhaps formed an acid-labile bond. In growth inhibition assays with L1210 cultured murine leukemia cells, the four haloacetamides were all more potent than their nonacylated precursors but less potent than MTX. The greater than 40,000-fold MTX-resistant mutant cell line L1210/R81 was only partly cross-resistant to the haloacetamides. An analogue of MTX with acivicin replacing glutamate was a potent inhibitor of DHFR from chicken liver and L1210 cells but was 200 times less potent than MTX against L1210 cells in culture.
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PMID:Methotrexate analogues. 30. Dihydrofolate reductase inhibition and in vitro tumor cell growth inhibition by N epsilon-(haloacetyl)-L-lysine and N delta-(haloacetyl)-L-ornithine analogues and an acivicin analogue of methotrexate. 311 97

A series of folate analogs containing ornithine instead of glutamate was synthesized and tested for inhibition of folylpolyglutamate synthetase (FPGS) and other folate-dependent enzymes of human leukemia cell lines. Reduced derivatives of 2-amino-4-oxo-10-methyl-pteroyl-ornithine had dramatically increased inhibitory potency against FPGS compared to the oxidized parent. The amino-pterin analog (2,4-diamino-pteroylornithine) was a potent inhibitor of both dihydrofolate reductase and FPGS. It was a much more potent linear competitive inhibitor of human FPGS than the corresponding methotrexate derivative previously described (Ki = 0.15-0.26 and 3 microM respectively). A quinazoline folate analog, 2-amino-4-oxo-5,8-dideazapteroyl-ornithine, was a relatively poor inhibitor of isolated dihydrofolate reductase and thymidylate synthase; however, it is the most potent human FPGS inhibitor identified to date (Ki = 100-150 nM). Because of the lack of appreciable interaction with other folate-dependent enzymes, structures incorporating the 2-amino-4-oxo-5,8-dideazapteroate nucleus may thus lead to selective inhibition of FPGS. Substitution of ornithine for glutamate caused a profound decrease in cytotoxic potency for these analogs; this was apparently the result of poor transport. Together with earlier studies, these data indicate that the potency of FPGS inhibition by an analog containing ornithine closely parallels the relative substrate activity of its glutamate-containing counterpart. The substitution of ornithine apparently does not perturb the pterin specificity of FPGS. The close parallel between substrate and inhibitor specificity may thus allow the use of currently available structure-activity studies on FPGS to design more potent and more selective inhibitors of FPGS.
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PMID:Structural specificity of inhibition of human folylpolyglutamate synthetase by ornithine-containing folate analogs. 319 Jul 39

N delta-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base. The N delta-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N10-formylpteroic acid by reaction with persilylated N delta-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N delta-phthaloyl moiety with base. The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC50's ranging from 0.027 and 0.052 microM as compared with 0.072 microM for APA-L-Orn. Several of the N delta-acyl-N10-formyl intermediates also proved to be good DHFR inhibitors. One of them, N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-N delta-(4-chlorobenzoyl)-L- ornithine, had a 2-fold lower IC50 than its deformylated product, confirming that the N10-formyl group is well tolerated for DHFR binding. While N delta-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS. N delta-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells. However, N delta-acyl derivatives also containing an N10-formyl substituent were less cytotoxic than the corresponding N10-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N10-formylation may be unfavorable for transport. Two compounds, the N delta-benzoyl and N delta-hemiphthaloyl derivatives of APA-L-Orn, with IC50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system. These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture. Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10-20-fold MTX resistance, the N delta-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Methotrexate analogues. 33. N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity. 338 30


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