Biomedical subjects
Publications and source records attributed to W Rode.
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Explore the source record for details and available documents.
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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)
The outer part of Hymenolepis diminuta tegument was extracted with 3 M KCl. The antigen was adsorbed from the extract on an affinity column containing H. diminuta-infected rat immunoglobulin immobilized on Sepharose 4B and could be eluted with 2.5 M urea at pH 2.8. Analysis of polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of both the crude extract and material eluted from the column showed that the latter was markedly purified. Double diffusion and immunoelectrophoresis revealed 11 and 4 antigenic components in the crude extract and purified preparation, respectively.
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.
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.
The role of the phosphate moiety of dUMP, and some analogues, in their interaction with mammalian thymidylate synthase, has been investigated. Substrate and inhibitor activities, and the pH-dependence of these activities, of dUMP and 5-FdUMP, as well as analogues with modified phosphate groups, were compared. The methyl ester of dUMP was neither a substrate nor an inhibitor. By contrast, the methyl ester of 5-FdUMP was a slow-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells, with Ki values in the micromolar range. Both 5-FdUrd and the newly synthesized 5'-methylphosphonate of 5-FdUrd were also slow-binding inhibitors of the Ehrlich carcinoma enzyme, but with Ki values in the millimolar range. The interaction of dUMP, 5-FdUMP, and the methyl ester of the latter decreased with increase in pH, whereas that of the 5'-methyl-phosphonate of 5-FdUrd remained unchanged. The results are discussed in relation to the role of the phosphate hydroxyls of dUMP in binding to the enzyme. 5-FdUMP and its analogues exhibited differing interactions with two binding sites on the enzyme molecule, consistent with cooperativity of binding. A convenient procedure is described for the synthesis of 5-fluoro-2'-deoxyuridine-5'-methylphosphonate, applicable also to the preparation of other 5'-methylphosphonate analogues.
Extracts of the tapeworm, Hymenolepis diminuta, catalyse N5,10-methylene-tetrahydrofolate-dependent release of tritium from [5-3H]dUMP, indicating the presence of thymidylate synthase. The enzyme activity was found in immature, mature and gravid proglottids, as well as in immature and mature oncospheres. The reaction showed pH optimum at 7.5. Its Michaelis constants were approximately 2 and 15 microM for dUMP and (+/-), L-N5,10-methylenetetrahydrofolate, respectively. Incubation of the tapeworm extracts with 5-F-[3H]dUMP and N5,10-methylenetetrahydrofolate resulted in formation of a labelled complex, separable under conditions of SDS polyacrylamide electrophoresis (mol. wt. of approx. 34,000), corresponding to thymidylate synthase subunit. Results of gel filtration of the above complex, under nondenaturing conditions, pointed to a dimeric structure of the enzyme.
Reaction of the reagent of Lawesson, 2,4-bis(p-methoxyphenyl)-1,3,4-dithiadiphosphatane-2,4-disulfide, with blocked uracil nucleosides in dioxane leads to quantitative thionation at C(4). With the bases, thionation occurs at C(4) and, with two equivalents of the reagent, at C(2) and C(4). Enzymatic phosphorylation of 4-thio-FdUrd gave the 5'-monophosphate, which was further converted with NH2OH to N4-hydroxy-FdCMP. Both nucleotides were examined as potential inhibitors of thymidylate synthase, and 4-thio-FdUrd for cytotoxic activities vs monkey and human leukemic cell lines.
Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells. The enzyme is a dimer composed of 35,000 mol. wt monomers. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34 degrees C, reflecting a change of rate-limiting step in dTMP formation. MgATP at millimolar concentrations inhibits mouse thymus enzyme.
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This paper presents improved synthetic methods for the modification of 2'-deoxyuridine-5'-monophosphate and its 5-fluoro derivative, using trimethylphosphate in aqueous medium at pH 10. These modifications include methylation of the pyrimidine ring N(3) and/or esterification of the phosphate group. The 5'-methyl ester of dUMP was neither a substrate nor an inhibitor of Ehrlich ascites carcinoma thymidylate synthetase. By contrast, the corresponding methyl ester of FdUMP was a tight-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells. 3-Methyl-dUMP, fixed in the 4-keto form, exhibited only very weak substrate activity with the Ehrlich ascites carcinoma enzyme. The dUMP analogues 5-ethyl-dUMP and 5-propyl-dUMP were found to be competitive inhibitors of thymidylate synthetase from L1210, Ehrlich ascites carcinoma and HeLa cells, the former being the more potent inhibitor. Both analogues were shown to bind cooperatively to each of the mouse tumour enzymes. Two molecules of inhibitor interacted with a single enzyme molecule, reflected by the parabolic character of the replots of the slope versus inhibitor concentration. The parent dTMP was a stronger inhibitor of the mouse tumour enzymes than its higher alkyl homologues.
Activity of thymidylate synthase was measured in situ in leukemia cells by tritium release from [5-3H]dUrd. Aphidicolin, an inhibitor of DNA polymerase alpha, but not thymidylate synthase, caused a time dependent inhibition of the enzyme when added to the cells after [5-3H]dUrd. Cells treated with hydroxyurea and aphidicolin in sequence before addition of [5-3H]dUrd had a high initial thymidylate synthase activity that decreased with time. This pattern indicates that thymidylate synthase activity is linked to DNA synthesis; however, its inhibition by drugs that inhibit DNA synthesis may be due to accumulation of thymidine nucleotide(s), rather than to an allosteric interaction in the replitase complex.
Methionine synthase reaction may be coupled with thymidylate synthase-catalysed tritium release from [5-3H]dUMP via non-enzymatic reaction of formaldehyde with tetrahydrofolate. A convenient and sensitive assay of methionine synthase activity, based on this principle, is described.
Improved syntheses, based on Lewis acid-catalyzed nucleosidation, are described for the preparation of 5-alkyl-2'-deoxyuridines. These were converted to their 5'-phosphates with the use of wheat shoot phosphotransferase. The dUMP analogues, 5-ethyl-dUMP and 5-propyl-dUMP, were competitive vs dUMP inhibitors of thymidylate synthetase purified from mouse L1210, Ehrlich ascites and HeLa cells, the former being the stronger inhibitor. Both analogues were shown to bind cooperatively to each of the mouse tumour enzymes, two molecules of inhibitor interacting with a single enzyme molecule, as reflected by the parabolic character of the replots of the slope vs inhibitor concentrations. dTMP was a stronger inhibitor of the mouse tumour enzymes than its higher alkyl homologues.
Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.
Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.