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D Shugar

Publications and source records attributed to D Shugar.

At least 55 records · Page 3Linked to original sources
PubMed1993

2-Thio derivatives of dUrd and 5-fluoro-dUrd and their 5'-monophosphates: synthesis, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

A convenient synthesis of 5-fluoro-2-thiouracil (11) is based on hydrolytic deamination of 5-fluoro-2-thiocytosine (9). Lewis acid-catalyzed condensation of di-TMS-5-fluoro-2-thiouracil (13) or di-TMS-2-thiouracil (14) with 2-deoxy-3,5-di-O-p-toluyl-D-ribofuranosyl chloride (15) led to mixtures of the beta- and alpha-anomers of 3',5'-toluylated 2'-deoxy-5-fluoro-2-thiouridine (16 and 18) or 2'-deoxy-2-thiouridine (17 and 19), each of which was deblocked with MeOH-NH3 to give the desired free anomeric nucleoside pairs 1, 5 and 3, 7, respectively. These were selectively converted to the corresponding 5'-monophosphates 2, 6 and 4, 8, with the aid of the wheat shoot phosphotransferase system. Conformations of the nucleosides 1, 3, 5, 7 are deduced from 1H NMR spectra, and circular dichroism spectra for nucleotide anomeric pairs 2, 6 and 4, 8 are reported. Whereas beta-2-thio-dUMP (4) was a good substrate (Km approximately 10(-5) M), beta-5-fluoro-2-thio-dUMP (2) proved to be a potent competitive, slow-binding inhibitor (Ki approximately 10(-8) M) of the purified enzymes from Ehrlich ascites carcinoma and L1210 cells. The alpha-anomer 6 was a weak inhibitor, with Ki in the mM range, and its congener 8 hardly interacted with the enzyme. The beta-anomer 1 exhibited antitumor activity in a mouse leukemic cell line L5178Y (IC50 approximately 10(-6) M), hence 40-100-fold weaker than 5-fluoro-dUrd. Its alpha-anomer 5 was 10-fold less active, but exhibited at least 10-fold higher selectivity with respect to the tumor cells than the beta-anomer 1.

3T3 Cells↗
PubMed1993

Interaction of 2-thio-5-fluoro-dUMP and 4-thio-5-fluoro-dUMP with mammalian normal and tumour and helminthic thymidylate synthases: influence of C(4)-substituents on specificity for enzyme inactivation.

To determine how 5-fluoro-dUMP modifications may affect its specificity, 2-thio-5-fluoro-dUMP and 4-thio-5-fluoro-dUMP were compared as inhibitors of thymidylate synthases isolated from parental and FdUrd-resistant mouse leukemia L1210 cells, human and rat colon adenocarcinomas, regenerating rat liver and the tapeworm, Hymenolepis diminuta, differing in sensitivity to time- and N5,10-methylenetetrahydrofolate-dependent inactivation by 5-fluoro-dUMP (Ki values ranging from 10(-9) to 10(-7) M). Inactivation by 2-thio-5-fluoro-dUMP, relative to 5-fluoro-dUMP, was 5-20-fold weaker, with specificity for inactivation of different thymidylate synthases paralleling that of 5-fluoro-dUMP. By contrast, 4-thio-5-fluoro-dUMP showed very different specificity, being as potent an inactivator for some enzymes as 5-fluoro-dUMP, but 45-85-fold weaker for others. The results suggest that an interplay between substituents at C(4) and C(5) of the pyrimidine ring may affect the specificity of thymidylate synthase inactivation.

Animals↗
PubMed1993

5-Propyl-2-deoxyuridine induced interference with glycosylation in herpes simplex virus infected cells. Nature of PdU-induced modifications of N-linked glycans.

In herpes simplex virus-infected (HSV) cells, the antiviral nucleoside analogue 5-n-propyl-2'-deoxyuridine (PdU) may, under certain circumstances, induce a pattern of interference with late steps in formation of N-linked glycans, resulting in increased availability of viral glycoproteins for neutralizing antibodies. The PdU-induced changes in N-linked glycans, released by pronase digestion of the HSV-specified glycoprotein gC-1, were investigated by using lectin affinity chromatography and Bio-Gel P6 gel filtration of glycans, radiolabelled with [3H]galactose or [3H]glucosamine. PdU-treatment of HSV-infected cells totally inhibited addition of sialic acid and reduced the amount of galactose incorporated into N-linked glycans by 70%. In addition, the PDU-treatment caused a decrease in oligosaccharides with affinity for Phaseoulus vulgaris leuco-agglutinin and erythro-agglutinin, and an increase in Lens culinaris lectin (LCA)-binding oligosaccharides, suggesting a PdU-induced shift from multi-branched to moderately branched structures. This shift was also found in HSV-infected B16 mouse melanoma cells, where the large content of multi-branched oligosaccharides contributes to the metastatic potential. The LCA-binding glycans from PdU-treated cells were smaller and contained less galactose units than corresponding structures from untreated cells. In a cell-free system, PdU 5'-monophosphate inhibited the translocation of UDP-GlcNAc, and, to a smaller extent, also the translocation of UDP-galactose into Golgi vesicles, suggesting that nucleotide sugar translocation is one important target for the PdU-induced interference with glycosylation in HSV-infected cells.

Agglutinins↗
PubMed1993

Electrochemical reduction products of azido nucleosides, including zidovudine (AZT): mechanisms and relevance to their intracellular metabolism.

Previous studies on electrochemical reduction of the HIV reverse transcriptase inhibitor, 3'-azido-3'-deoxythymidine (Zidovudine, AZT) and several of its analogues, have been extended to 2'-AZdT and two of the intracellular metabolites of AZT, the 5'-O-glucuronide (GAZT) and the 5'-phosphate (AZTMP). Also investigated were azido nucleosides with aglycons susceptible to electrochemical reduction, cytosine and adenine. The surface activities of these compounds at the mercury electrode were examined. In all instances, reduction of the azido group was a two-electron process, with conversion to an amino group. For an azido adenine nucleoside, it proved possible to reduce the azido group without affecting the aglycon. Electrochemical reduction is shown to provide a simple one-step synthesis of amino nucleosides from the available azido nucleosides. The reduced compounds, several hitherto unknown, are useful reference standards for following intracellular metabolism of azido nucleosides, and may also prove of interest as new potential antimetabolites.

Antiviral Agents↗
PubMed1993

Cyclic phosphates of some antiviral acyclonucleosides: relationship between conformation and substrate/inhibitor properties in some enzyme systems.

Solution conformations, and substrate/inhibitor properties towards several phosphodiesterases and other nucleolytic enzymes, have been investigated for the cyclic phosphates of various acyclonucleosides, some with known antiviral activity, including 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG) and its carbocyclic congener, 9-(3,4-dihydroxybutyl)guanine (DHBG),9-[4-hydroxy-2-(hydroxymethyl)- butyl]-guanine (2HM-HBG), and 9-(2,3-dihydroxypropyl)guanine (HPG). Conformations of the cyclic phosphate rings were derived from analysis of the 1H-1H and 1H-31P vicinal coupling constants in the 1H n.m.r. spectra, subsequently optimalized by minimalization of the internal energy. The resulting structures were examined with respect to their ability to recognize various specific phosphodiesterases and nucleases, and some structural parameters were delineated for acyclonucleotide interactions. Qualitative data are presented for inhibitory properties of the acyclonucleoside cyclic phosphates, and, in those instances where they were substrates, kinetic constants were evaluated. An unusual finding was the apparent ability of nuclease P1 to hydrolyse a five-membered cyclic phosphate ring of an acyclonucleoside.

Antiviral Agents↗
PubMed1992

Formycins A and B and some analogues: selective inhibitors of bacterial (Escherichia coli) purine nucleoside phosphorylase.

Formycin B (FB), a moderate inhibitor (Ki approximately 100 microM) of mammalian purine nucleoside phosphorylase (PNP), and formycin A (FA), which is totally inactive vs. the mammalian enzyme, are both effective inhibitors of the bacterial (Escherichia coli) enzyme (Ki approximately 5 microM). Examination of a series of N-methyl analogues of FA and FB led to the finding that N(6)-methyl-FA, virtually inactive vs. the mammalian enzyme, is the most potent inhibitor of E. coli purine nucleoside phosphorylase (Ki approximately 0.3 uM) at neutral pH. Inhibition is competitive not only with respect to Ino, but also relative to 7-methyl-Guo and 7-methyl-Ado, as substrates. Both oxoformycins A and B are relatively poor inhibitors. For the most potent inhibitor, N(6)-methyl-FA, it was shown that the enzyme preferentially binds the neutral, and not the cationic, form. In accordance with this the neutral, but not the cationic form, of the structurally related N(1)-methyl-Ado was found to be an excellent substrate. Reported data on tautomerism of formycins were profited from, and extended, to infer which tautomeric species and ionic forms are the active inhibitors. A commercially available (Sigma) bacterial PNP, of unknown origin, was shown to differ from the E. coli enzyme by its inability to phosphorylase Ado; this enzyme was also resistant to FA and FB. These findings have been extended to provide a detailed comparison of the substrate/inhibitor properties of PNP from various microorganisms.

Bacillus cereus↗
PubMed1991

Benzimidazole nucleoside analogues as inhibitors of plant (maize seedling) casein kinases.

Halogeno benzimidazole and benzimidazole nucleoside analogues have been screened for inhibitory activity vs. purified plant (maize seedling) casein kinases I, IIA and IIB, and the results compared with those previously reported for some of the compounds as inhibitors of the corresponding mammalian CK-1 and CK-2 (Meggio et al. (1990) Eur. J. Biochem. 187, 89-94). One new analogue, the riboside of 5,7-dibromobenzimidazole, which is sterically constrained to the anti conformation about the glycosidic bond, and is a good inhibitor, exhibited appreciable (5-7-fold) discrimination between the type I and type II enzymes. An increase in the number of halogen substituents on the benzene ring of benzimidazole from two to three led to marked enhancement of inhibitory activity, particularly against the type II enzymes, with a decrease in Ki from 24 to 4 microM. The 2-aza analogue of 5,6-dichlorobenzimidazole, i.e. 5,6-dichlorobenzotriazole, as the free base, even more effectively discriminated between the two types of plant casein kinases, with Ki approximately 100 microM for CK-I, and Ki approximately 9 microM for CK-IIA and CK-IIB. Inhibition in all instances was competitive with respect to ATP (for CK-I), and ATP and GTP (for CK-IIA and CK-IIB). The results are compared with those for halogenated isoquinolinesulfonamide inhibitors reported by Chijiwa et al. (J. Biol. Chem. (1989) 264, 4924-4927), leading to proposals for the synthesis of potentially more effective and more discriminating inhibitors. Attention is drawn to the significant role of the halogen substituents in the mechanism(s) of action of the structurally related benzimidazole, benzotriazole and naphthalene and isoquinoline, inhibitors of protein kinases.

Adenosine Triphosphate↗
PubMed1991

Acyclonucleoside analogue inhibitors of mammalian purine nucleoside phosphorylase.

A series of about 60 purine acyclonucleosides, most with guanine as the aglycone and a 4-carbon chain as the acyclic moiety, was examined for ability to inhibit purine nucleoside phosphorylase from human erythrocytes and calf spleen. Compounds with shorter and longer acyclic chains were less effective inhibitors. Synthetic procedures are described. About 25 of the analogues were competitive inhibitors (relative to inosine or 7-methylguanosine as substrates) with Ki values in the range of 2 to 100 microM. The more potent ones (Ki 2-5 microM) included guanine as the aglycone, with various substituents at C(2') of the acyclic chain and hydroxyls at C(3') and C(4'). In one instance, 9-(2-fluoro-3,4-dihydroxybutyl)guanine, the (+)erythro enantiomer was 10-fold more effective than its (-) counterpart (2.5 microM vs 27 microM). Replacement of guanine by 8-bromo- or 8-aminoguanine enhanced affinity for the enzyme by an order of magnitude or more; 7-deazaacyclovir was also 10-fold more effective than acyclovir. With some of the inhibitors, Ki (human)/Ki (calf) varied over the range 0.4 to 4, reflecting differences between the two enzymes; nonetheless, the much more stable, and commercially available, calf spleen enzyme is recommended for preliminary screening of potential inhibitors of the human or other unstable enzymes. The overall results provide useful indications for the synthesis of potentially more potent inhibitors of the enzyme, by simultaneous modifications of the aglycone and the acyclic chains.

Acyclovir↗
PubMed1990

Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N4-hydroxy-dCMP, N4-hydroxy-5-fluoro-dCMP, and related analogues.

N4-Hydroxy-dCMP (N4-OH-dCMP), N4-methoxy-dCMP (N4-OMe-dCMP), and their 5-fluoro congeners (syntheses of which are described) were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N4-OH-dCMP was not a substrate (no dihydrofolate produced; no tritium released with 5-3H-labeled molecule), and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based, with arrest of a step posterior to addition of cofactor and blocking abstraction of the C(5) hydrogen. Ki values for N4-OH-dCMP and its 5-fluoro analogue were in the range 10(-7) - 10(-8) M, 2-3 orders of magnitude higher for the corresponding N4-OMe analogues. The 5-methyl analogue of N4-OH-dCMP was 10(4)-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N4-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N4-OH-dCMP, suggesting interaction of both N4-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N4-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Ribofuranosyl-benzimidazole derivatives as inhibitors of casein kinase-2 and casein kinase-1.

5,6-Dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DiCl-RB) is a powerful inhibitor of casein kinase-2 (CK-2) [Zandomeni, R. et al. (1986) J. Biol. Chem. 261, 3414-3420]. Here a series of 17 analogues of DiCl-RB has been employed for studying the specificity and the mode of action of this family of CK-2 inhibitors. The two halogen substituents on the benzene ring are shown to play a prominent role in inhibition, the 5,6-dibromo derivative (DiBr-RB) being fivefold more effective than DiCl-RB (Ki = 2 microM, with GTP as substrate), whereas the difluoro derivative (DiF-RB) is nearly as ineffective as unsubstituted 1-(beta-D-ribofuranosyl)benzimidazole. On the other hand, although some modifications of the ribose group significantly decrease the inhibitory efficiency, the sugar moiety is not strictly required, since dichlorobenzimidazole itself (DiCl-Bz) is an inhibitor almost as good as DiCl-RB. Inhibition of CK-2 by DiCl-RB and by its analogues, DiCl-Bz included, is of the competitive type with respect to the nucleotide substrate, the Ki values being lower with GTP than with ATP. The Ki values of the most potent inhibitor, DiBr-RB, with ATP and GTP, are 6 microM and 2 microM, respectively, denoting an affinity for the enzyme higher than that of the physiological substrates, ATP and GTP. DiBr-RB has been assayed for its inhibitory capacity toward several protein kinase other than CK-2. Protein kinase-C, cAMP-dependent protein kinase, the Ser/Thr protein kinase expressed by Pseudorabies virus, and four different tyrosine protein kinases from spleen, proved insensitive to DiBr-RB concentrations capable of almost entirely suppressing the activity of rat liver and maize seedling CK-2. Casein kinase-1 however is nearly as sensitive as CK-2 to DiBr-RB. Inhibition of CK-1 is also of the competitive type with respect to ATP (Ki = 14 microM). Although the inhibitory spectrum of CK-1 by the various analogues is reminiscent of that observed with CK-2, a remarkable difference is revealed by 5'-phosphorylation of ribose which increases the Ki with CK-2 while decreasing that with CK-1.

Animals↗
PubMed1990

Photochemical and enzymatic redox transformations of reduced forms of coenzyme NADP+.

The two reduced forms of NADP+, NADPH and its dimer (NADP)2, on irradiation in aqueous medium at 365 nm, are converted to enzymatically active NADP+, with accompanying formation of H2O2. The rate photooxidation of NADPH is strongly dependent on the presence of oxygen, but that of (NADP)2 is similar under aerobic and anaerobic conditions. In the presence of oxygen, but not in its absence, O2-. is an intermediate in the reaction. Generation of H2O2 under anaerobic conditions, confirmed by the fact that presence of peroxidase in irradiated solutions of (NADP)2 enhances photooxidation of the latter, is ascribed to attack on water of the excited dimer. Under anaerobic conditions at pH 9.5, Fe(EDTA)2+ and Fe(CN)4-(6) increase the rate of photooxidation of NADP dimer two-fold. gamma-Irradiation of (NADP)2 at pH 9.5 in the presence of N2O results in 80% conversion to enzymatically active NADP+. A mechanism for photooxidation of (NADP)2 under anaerobic conditions is suggested, and some relevant biological implications are presented.

Catalase↗
PubMed1988

Interaction of the 5'-phosphates of the anti-HIV agents, 3'-azido-3'-deoxythymidine and 3'-azido-2',3'-dideoxyuridine, with thymidylate synthase.

A study has been made of the interaction of 3'-azido-3'-deoxythymidine 5'-phosphate (AZTMP) and 3'-azido-2',3'-dideoxy-uridine 5'-phosphate (AZdUMP) with thymidylate synthase. With the enzyme from L1210 cells and the tapeworm Hymenolepis diminuta, AZTMP was a weak inhibitor competitive with respect to dUMP (Ki = 6.3 mM and 0.5 mM); hence cytotoxicity of AZT, in cells in which accumulation of AZTMP is not high, is not due to inhibition of cellular thymidylate synthase. AZdUMP, with the L1210 enzyme, was a weak substrate (competition with dUMP described by apparent Ki = 4.7 mM), excluding conversion of AZdUMP to AZTMP as a source of toxicity of 3'-azido-2',3'-dideoxyuridine. An efficient procedure is described for enzymatic phosphorylation on a preparative scale of dideoxynucleosides.

Animals↗
PubMed1988

Purine nucleoside phosphorylase. Structure-activity relationships for substrate and inhibitor properties of N-1-, N-7-, and C-8-substituted analogues; differentiation of mammalian and bacterial enzymes with N-1-methylinosine and guanosine.

The previous finding that 7-methylinosine (m7Ino) and 7-methylguanosine (m7Guo) are excellent, as well as fluorescent, substrates for calf spleen purine nucleoside phosphorylase has been extended to include a series of 7-alkylguanosines with higher alkyl groups (ethyl, propyl, butyl, isopropyl, isobutyl, benzyl). All of these are good substrates, with increases in Km compensated for by corresponding increases in Vmax, and excluding the ring N-7 as a binding site. Both m1Ino and m1Guo are neither substrates nor inhibitors of this enzyme, implicating the ring N-1 as a binding site. Included also are guanosine analogues with C-8 substituents (alpha-hydroxyisopropyl, methyl, bromo, chloro, amino) with known populations of syn and anti conformers about the glycosidic bond; while these did not unequivocally point to involvement of only one form, efficiency of phosphorolysis appeared to be correlated with a conformation in the anti region. The influence of various substituents is also considered in relation to steric and electronic effects. Kinetic parameters for all the foregoing have been determined fluorimetrically and/or spectrophotometrically from initial velocities and/or continuous monitoring of phosphorolysis including Ki values for inhibition by the base moieties. Furthermore, kinetic data demonstrated that, in striking contrast to the mammalian enzyme, both m1Ino and m1Guo are almost as good substrates as the parent nucleosides for a bacterial (Escherichia coli) enzyme, suggesting that the ring N-1 is not a binding site for the latter.

Animals↗
PubMed1988

Structure and conformation of a nucleoside analog 5-nitro-1-beta-D-arabinofuranosyluracil.

C9H11N3O8, Mr = 271, orthorhombic, P2(1)2(1)2(1), a = 9.241 (2), b = 20.518 (4), c = 6.187 (1) A, V = 1173.1 A3, Z = 4, D chi = 1.29 g cm-3, Mo K alpha, lambda = 0.7107 A, mu = 1.57 cm-1, F(000) = 600, T = 288 K, final R = 0.051 for 1078 observed reflections. Conformational features of the nucleoside include a glycosidic bond conformation in the anti range, a ribose moiety in the C(2')-endo (2E) form and the C(5')-(5') bond gauche to both C(4')-O(4') and C(4')-C(3').

Arabinofuranosyluracil↗
PubMed1987

Interaction of 5-fluoro-4-thio-2'-deoxyuridine 5'-phosphate with mammalian tumour thymidylate synthase: role of the pyrimidine N(3)-H dissociation.

The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.

Animals↗
PubMed1987

Inhibitor properties of some 5-substituted uracil acyclonucleosides, and 2,2'-anhydrouridines versus uridine phosphorylase from E. coli and mammalian sources.

Two series of 5-substituted uracil N(1)-acyclonucleosides, each with a different acyclic chain, were examined as inhibitors of uridine phosphorylase from rat intestinal mucosa, and several against the enzyme from Ehrlich ascites cells. In addition, several 5-substituted analogues of 2,2'-anhydrouridine were tested for their inhibitory effects vs a highly purified uridine phosphorylase from Escherichia coli. The results are compared with previously published data for inhibition of the E. coli enzyme by the acyclonucleosides, and of the rat enzyme by the anhydrouridines. In all instances, the inhibitors were active only vs the uridine, but not thymidine, phosphorylase from E. coli, and inhibition was competitive with respect to uridine as substrate. In general, with one or two exceptions, inhibitory effects were more pronounced against the enzyme from mammalian sources. Amongst the acyclonucleoside analogues, the most effective inhibitor of the enzyme from the rat and Ehrlich ascites cells exhibited a Ki = 0.1 microM, comparable to that reported with the Sarcoma-180 enzyme, whereas the Ki for inhibition of the E. coli enzyme was 0.7 microM. By contrast, another effective inhibitor of the bacterial enzyme was 7-fold less potent against the mammalian enzyme. The 2,2'-anhydrouridines were 10- to 30-fold more effective against the rat, as compared to the E. coli, enzyme. The overall quantitative data provide a reasonably good basis for the further design of potent inhibitors for possible use in chemotherapy.

Animals↗