Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amino-terminal presequences of rat peroxisomal 3-ketoacyl-CoA thiolase precursors (types A and B) were reported to be cleavable signal peptides for peroxisomal protein translocation. In the present study, this was proven by immunoelectron microscopy of the cultured Chinese hamster ovary cells stably expressing fusion proteins of the amino-terminal sequences of the thiolase precursor and Escherichia coli dihydrofolate reductase. The fusion proteins were processed into mature forms of the apparently correct sizes. Site-directed mutagenesis studies of the charged residues in the B-type presequence (26 amino acid residues) revealed that arginine at position -24 and histidine at position -17 were both indispensable. Even replacement of these residues with other basic amino acids abolished the import activity. Both Arg-24 and His-17 were also required in a longer presequence (36 amino acid residues) of the thiolase A, thereby suggesting that the signal can function in an internal position. When glutamic acid at position -11 was changed to amino acids other than aspartic acid, the signal peptide became apparently effective in both peroxisomal and mitochondrial targeting. All of these data indicate that the thiolase signal peptide is a newly defined type of peroxisomal targeting signal recognized by a mechanism presumably different from that for a known peroxisomal signal, the carboxy-terminal Ser-Lys-Leu-COOH motif.
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PMID:Characterization of the signal peptide at the amino terminus of the rat peroxisomal 3-ketoacyl-CoA thiolase precursor. 811 46

Classical antifolate analogues containing a novel furo[2,3-d]pyrimidine ring system which include N-[4-[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5- yl)methyl]amino]benzoyl]-L-glutamic acid (1) and its N-9 methyl analogue 2 were synthesized as potential dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Four nonclassical antifolates, 2,4-diamino-5-(anilinomethyl)furo[2,3-d]pyrimidines 3-6 with 3,4,5-trimethoxy, 3,4,5-trichloro, 3,4-dichloro, and 2,5-dimethoxy substituents, respectively, in the phenyl ring, were also synthesized as potential inhibitors of DHFRs including those from Pneumocystis carinii and Toxoplasma gondii, which are organisms responsible for opportunistic infections in AIDS patients. The classical and nonclassical analogues were obtained via nucleophilic displacements of the key intermediate 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with the appropriate (p-aminobenzoyl)-L-glutamate or substituted aniline. The key intermediate was in turn synthesized from 2,4-diamino-6-hydroxypyrimidine and 1,3-dichloroacetone. The final compounds were tested in vitro against rat liver, (recombinant) human, P. carinii, T. gondii, and Lactobacillus casei DHFRs. The classical analogues showed moderate to good DHFR inhibitory activity (IC50 10(-6)-10(-8) M) with the N-CH3 analogue 2 about twice as potent as 1. The nonclassical analogues were inactive with IC50S > 3 x 10(-5) M. The classical analogues were also evaluated as inhibitors of TS (L. casei, (recombinant) human and human CCRF-CEM), glycinamide ribonucleotide formyltransferase, and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and were found to be inactive against these enzymes. The classical analogues (particularly 2) were significantly cytotoxic toward a variety of tumor cell lines in culture. The nonclassical analogues were marginally active. Both classical compounds were good substrates for human folylpolyglutamate synthetase. Further evaluation of the cytotoxicity of 1 and 2 in CCRF-CEM cells and its sublines, having defined mechanisms of methotrexate (MTX) resistance, demonstrated that the analogues utilize the reduced folate/MTX-transport system and primarily inhibit DHFR and that poly-gamma-glutamylation was crucial to their mechanism of action. Protection studies in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin or the combination of thymidine plus hypoxanthine. Furthermore, for compounds 1 and 2, in contrast to MTX, the FaDu cells were better protected by thymidine alone than hypoxanthine alone, suggesting a predominantly antithymidylate effect.
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PMID:Classical and nonclassical furo[2,3-d]pyrimidines as novel antifolates: synthesis and biological activities. 816 59

Analogues of classical antifolates with the 4-aminobenzoyl group replaced by 4-amino-1-naphthoyl were synthesized for study after molecular modeling indicated ample spatial accommodation for the naphthalene ring and even larger groups in models based on reported X-ray crystallographic data describing the binding of methotrexate to human dihydrofolate reductase (DHFR). The side-chain precursors, N-(4-amino- and 4-(methylamino)-1-naphthoyl)-L-glutamic acid diethyl esters, were synthesized, and the 2,4-diamino-substituted heterocyclic groups were attached using several methods. Target compounds included naphthoyl analogues of aminopterin (AMT), methotrexate (MTX), 5-deazaAMT, 5-deazaMTX, 5-methyl-5-deazaAMT, 5-methyl-5-deazaMTX, and 5,8-dideazaAMT. A 5,6,7,8-tetrahydronaphthoyl analogue of 5-deazaAMT was also prepared. None of the naphthoyl analogues showed loss in binding to DHFR compared with the corresponding antifolate bearing the benzoyl group, thus confirming the anticipated bulk tolerance. Only the 5,6,7,8-tetrahydronaphthoyl analogue displayed reduced antifolate effects. Substrate activity toward folylpolyglutamate synthetase was, however, severely compromised. The naphthoyl compounds were transported into L1210 cells 3-6 times more readily than MTX, and despite apparently low levels of intracellular polyglutamylation, each compound was found to be significantly more potent than MTX in inhibiting tumor cell growth in vitro in three lines (L1210, HL60, and S180). The MTX, 5-methyl-5-deazaAMT, and 5-methyl-5-deazaMTX analogues were evaluated in vivo alongside MTX against E0771 mammary adenocarcinoma in mice. All three proved more effective than MTX in retarding the tumor growth. The naphthoyl analogue of 5-deazaAMT strongly inhibited DHFR from Pneumocystis carinii, Toxoplasma gondii, and rat liver giving IC50 (pM) values of 0.53, 2.1, and 1.6 respectively, but this compound did not inhibit in vitro growth of T. gondii, thus indicating lack of transport.
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PMID:Studies on analogues of classical antifolates bearing the naphthoyl group in place of benzoyl in the side chain. 827 97

gamma-Glutamyl hydrolase has been partially purified and characterized from conditioned culture medium of H35 hepatoma cells. Evidence for heterogeneity of the enzyme is derived from its elution as three distinct peaks of enzymatic activity when the enzyme is purified by TSK-butyl-Sepharose column chromatography. These three enzyme fractions appear to have identical catalytic properties but, as yet, the basis for their resolution is not understood. A rapid, sensitive and simple assay based on reverse-phase HPLC fluorescent detection with pre-column derivatization using o-phthalaldehyde (OPA) was developed to separate OPA-derivatives of poly-gamma-glutamates and glutamic acid. Using this assay and the standard HPLC assay for pteroylpolyglutamates, the enzyme appears to be an endopeptidase with respect to pteroylpenta-gamma-glutamate (PteGlu5), methotrexate penta-gamma-glutamate (4-NH2-10-CH3PteGlu5) and p-aminobenzoyl-penta-gamma-glutamate (pABAGlu5). The initial products are PteGlu1 (or 4-NH2-10-CH3PteGlu1 or pABAGlu1) and intact tetra-gamma-glutamate, which is subsequently degraded to glutamic acid. When penta-gamma-glutamate is the substrate, the cleavage of the gamma-bonds by the enzyme is less ordered, with the early appearance of mono-, di-, tri- and tetraglutamate. Poly-alpha-glutamate is not a substrate nor are pABA-gamma-Glu5 or penta-gamma-glutamate covalently linked to albumin. 4-NH2-10-CH3PteGlu2 or Glu5 bound to dihydrofolate reductase is not a substrate for the enzyme, offering further evidence that protein-associated poly-gamma-glutamates are poor substrates for gamma-glutamyl hydrolase from H35 hepatoma cells.
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PMID:The properties of the secreted gamma-glutamyl hydrolases from H35 hepatoma cells. 834 22

The crystal structure of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) as a binary complex with folinic acid (5-formyl-5,6,7,8-tetrahydrofolate; also called leucovorin or citrovorum factor) has been solved in two space groups, P6(1) and P6(5), with, respectively, two molecules and one molecule per asymmetric unit. The crystal structures have been refined to an R-factor of 14.2% at resolutions of 2.0 and 1.9 A. The P6(1) structure is isomorphous with several previously reported ecDHFR binary complexes [Bolin, J.T., Filman, D.J., Matthews, D.A., Hamlin, R.C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662; Reyes, V.M., Sawaya, M.R., Brown, K.A., & Kraut, J. (1995) Biochemistry 34, 2710-2723]; enzyme and ligand conformations are very similar to the P6(1) 5,10-dideazatetrahydrofolate complex. While the two enzyme subdomains of the P6(1) structure are nearly in the closed conformation, exemplified by the methotrexate P6(1) binary complex, in the P6(5) structure they are in an intermediate conformation, halfway between the closed and the fully open conformation of the apoenzyme [Bystroff, C., Oatley, S.J., & Kraut, J. (1990) Biochemistry 29, 3263-3277]. Thus crystal packing strongly influences this aspect of the enzyme structure. In contrast to the P6(1) structure, in which the Met-20 loop (residues 9-23) is turned away from the substrate binding pocket, in the P6(5) structure the Met-20 loop blocks the pocket and protrudes into the cofactor binding site. In this respect, the P6(5) structure is unique. Additionally, positioning of a Ca2+ ion (a component of the crystallization medium) is different in the two crystal packings: in the P6(1) structure it lies at the boundary between the two molecules of the asymmetric unit, while in P6(5) it coordinates two water molecules, the hydroxyl group of an ethanol molecule, and the backbone carbonyl oxygens of Glu-17, Asn-18, and Met-20. The Ca2+ ion thus stabilizes a single turn of 3(10) helix (residues 16-18 in the Met-20 loop), a second unique feature of the P6(5) crystal structure. The disposition of the N5-formyl group in these structures indicates formation, at least half of the time, of an intramolecular hydrogen bond between the formyl oxygen and O4 of the tetrahydropterin ring. This observation is consistent with the existence of an enol-keto equilibrium in which the enolic tautomer is favored when a hydrogen-bond acceptor is present between O4 and N5. Such would be the case whenever a water molecule occupies that site as part of a hypothetical proton-relay mechanism. Two arginine side chains, Arg-52 in the P6(5) structure and Arg-44 in molecule A of the P6(1) structure, are turned away drastically from the ligand (p-aminobenzoyl)glutamic acid moiety as compared with previously reported DHFR binary complex structures. As in the ecDHFR dideazatetrahydrofolate complex, in both the P6(1) and P6(5) structures a water molecule bridges pteridine O4 and Trp-22(N epsilon 1) with ideal geometry for hydrogen bonding, perhaps contributing to the slow release of 5,6,7,8-tetrahydrofolate from the enzyme-product complex. When either the P6(1) or the P6(5) structures are superimposed with the NADPH holoenzyme [Sawaya, M. R. (1994) Ph.D. Dissertation, University of California, San Diego], we find that the distances between the nicotinamide C4 and pteridine C6 and C7 are very short, 2.1 and 1.7 A in the P6(1) case and 2.0 and 1.4 A in the P6(5) case, perhaps in part explaining the more rapid release of tetrahydrofolate from the enzyme-product complex when NADPH is bound.
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PMID:Crystal structures of Escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups: evidence for enolization of pteridine O4. 867 26

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

Six novel 2-amino-4-oxo-5-[(substituted phenyl)sulfanyl]pyrrolo[2,3-d]pyrimidines 7-12 were synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor and/or antibacterial agents. The analogues contain a 5-thio substituent with a phenyl, 4'-chlorophenyl, 3',4'-dichlorophenyl, 4'-nitrophenyl, 3',4'-dimethoxyphenyl, and 2'-naphthyl on the sulfur, and were synthesized from the key intermediate 2-(pivaloylamino)-4-oxo-6-methylpyrrolo[2,3-d]-pyrimidine, 17. Appropriately substituted aryl thiols were appended to the 5-position of 17 via an oxidative addition reaction using iodine, ethanol, and water under conditions which also resulted in the deprotection of the 2-amino group. The compounds were evaluated against human, Lactobacillus casei, Escherichia coli, Streptococcus faecium, and Pneumocystis carinii (pc) TSs and against human, rat liver (rl), pc, and Toxoplasma gondii (tg) DHFRs. The nonclassical analogues with the 3',4'-dichloro and the 4'-nitro substituents in the side chain (9 and 10) were more potent than N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop- 2-ynylamino]benzoyl]-L-glutamic acid (PDDF, 1) and N-[5-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N- methylamino]-2-thenoyl]-L-glutamic acid (ZD1694,2) against human TS. Analogues with the 4'-chloro, 3',4'-dimethoxy, and naphthyl side chains (8, 11 and 12) were more potent than the unsubstituted phenyl analogue (7) but less than 2, 9, and 10 by 1 order of magnitude. They were all poor inhibitors of human, rl, and pc DHFRs (IC50 = 10(-5) M) but moderate inhibitors (IC50 = 10(-6) M) of tg DHFR. The 4-nitro analogue, 10 (EC50 1.5 microM), was comparable to PDDF in its potency as an inhibitor of the growth of the FaDu human squamous cell carcinoma cell line.
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PMID:2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as nonclassical antifolate inhibitors of thymidylate synthase. 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

The synthesis of seven 2,4-diamino-5,6,7,8-tetrahydro-7-substituted pyrido[4',3':4,5]furo[2,3-d]pyrimidines 1-6 are reported as nonclassical antifolate inhibitors of dihydrofolate reductase (DHFR) and compound 7 as a classical antifolate inhibitor of tumor cells in culture. The compounds were designed as conformationally restricted analogues of trimetrexate. The synthesis was accomplished from the cyclocondensation of 3-bromo-4-piperidone with 2, 4-diamino-6-hydroxypyrimidine to afford regiospecifically 2, 4-diamino-5,6,7,8-tetrahydropyrido[4',3':4,5]furo[2, 3-d]pyrimidine-7-hydrobromide (16). This in turn was alkylated with the appropriate benzyl halide to afford the target compounds 1-6. The classical antifolate 7 utilized 4-(chloromethyl)benzoyl-l-glutamic acid diethyl ester (17) instead of the benzyl halide for alkylation, followed by saponification to afford 7. Compounds 1-6 showed moderate inhibitory potency against DHFR from Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and rat liver. The classical analogue 7 was 88-fold more potent against M. avium DHFR than against rat liver DHFR. The classical analogue was also inhibitory against the growth of tumor cells, CCRF-CEM, and FaDu, in culture.
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PMID:Synthesis and biological activities of tricyclic conformationally restricted tetrahydropyrido annulated furo[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases. 955 74


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