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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytomegalovirus (CMV) DNA polymerase has immunologic specificity in relation to the virus specific polymerases of other herpesviruses. During the early phase of CMV infection in vitro, the virus DNA polymerase is rapidly induced. Due to the lack of virus specific thymidine kinase, cytomegalovirus is resistant to nucleosides which require herpesvirus thymidine kinases for activation. Cytomegalovirus DNA polymerase is therefore the known possible target for antiviral drugs. Several pyrophosphate analogs have been assayed for their inhibitory effects on this enzyme. The most active compound is phosphonoformate (PFA). PFA also effectively inhibits other partially purified herpesvirus DNA polymerases as well as the multiplication of all human herpesviruses, at concentrations which do not affect cellular DNA polymerases or normal cell growth. The mechanism of inhibition is a noncompetitive inhibition of CMV DNA polymerase activity with respect to the four deoxyribonucleoside triphosphates, and an uncompetitive inhibition with respect to the template used. In cell culture, PFA inhibits the formation of late CMV polypeptides, but not the synthesis of early CMV polypeptides. The CMV specific polymerase persists in the presence of PFA, as measured by immunological methods, and enzyme activity can be demonstrated after removal of PFA. The inhibition of CMV replication is reversible even after long exposure to PFA. Our interpretation is that the CMV genome is highly resistant to the cellular metabolism also in non-producing cells. A new rapid CMV neutralization test was established, based on the appearance of early CMV-induced antigens.
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PMID:Cytomegalovirus DNA polymerase inhibition and kinetics. 300 Jan 44

The antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine (iNDG) is phosphorylated by HSV1 thymidine kinase, and its phosphorylated products inhibit DNA polymerase activity. iNDG exists in two enantiomeric forms, each with a primary and a secondary hydroxyl; thus, a number of possibilities for preferential phosphorylation exist, which were explored in this study. HSV1 thymidine kinase phosphorylates the primary hydroxyl of both the R and the S isomers of iNDG. This was established by comparison with analogues in which either the primary or the secondary hydroxyl was replaced by fluorine or hydrogen and also by a study of the NMR spectrum of the monophosphate. GMP kinase phosphorylates the R and the S monophosphates to the respective diphosphates. Further phosphorylation, however, is much more efficient with the S than with the R isomer. Furthermore, (S)-iNDG triphosphate is a more potent inhibitor of HSV1 DNA polymerase than (R)-iNDG triphosphate. These differences in the biochemical specificities of the two isomers account for the observed higher antiviral potency of (S)-iNDG as compared to that of (R)-iNDG.
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PMID:Enzymatic phosphorylation of the antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine. 300 16

The activities of the purine acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) against two human and five animal strains of cytomegalovirus were compared with those of acyclovir. DHPG was significantly more active than acyclovir against all but one (mouse cytomegalovirus) of the strains tested, with 50% effective doses ranging from 5 to 13 microM, as determined by plaque reduction assays in human embryonic lung (MRC-5) and human embryonic tonsil cells. Both DHPG and acyclovir inhibited virus replication at concentrations considerably lower than those necessary to inhibit cell proliferation. In mode-of-action studies, the triphosphates of DHPG and acyclovir inhibited human cytomegalovirus DNA polymerase. DHPG phosphorylation to the active triphosphate was enhanced in infected cells; however, this enzymatic activity was unrelated to thymidine kinase. In animal studies, DHPG was slightly more effective than acyclovir in reducing mouse cytomegalovirus-induced mortality.
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PMID:Activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine compared with that of acyclovir against human, monkey, and rodent cytomegaloviruses. 301 Aug 40

Four kinds of 1-beta-D-arabinofuranosyl-5-halogenouracil were examined for inhibition of human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) replication. 1-beta-D-Arabinofuranosyl-5-fluorouracil (ara-FU) was the most effective against HCMV, whereas 1-beta-D-arabinofuranosyl-5-bromouracil was the most effective against HSV-1 and HSV-2. The mechanism of action of ara-FU on HCMV replication was also studied. The dTTP pool size in human embryonic fibroblasts was increased 33-fold by HCMV infection. However, treatment with ara-FU decreased the size of the dTTP pool by approximately 50%. On the other hand, 1-beta-D-arabinofuranosyl-5-fluorouracil-5'-triphosphate inhibited HCMV DNA polymerase activity competitively with dTTP. These results suggest that ara-FU acts as a bifunctional inhibitor of HCMV replication. Ara-FU is phosphorylated by cellular thymidine kinase to 1-beta-D-arabinofuranosyl-5-fluorouracil-5'-monophosphate, which inhibits cellular thymidylate synthetase, which in turn decreases the dTTP pool size in infected cells. As the dTTP pool size is reduced, inhibition of viral DNA polymerase by 1-beta-D-arabinofuranosyl-5-fluorouracil-5'-triphosphate becomes more efficient.
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PMID:Mechanism of selective inhibition of human cytomegalovirus replication by 1-beta-D-arabinofuranosyl-5-fluorouracil. 301 Aug 44

The properties of virus and host DNA polymerases are important factors in determining the selectivity of deoxynucleotide analogs used in antiviral chemotherapy. The high affinity of herpes DNA polymerase for nucleotide analogs may be particularly important in CMV and EBV-infected cells, since these viruses do not induce the synthesis of a virus-specified thymidine kinase. In general, the effect of nucleotide analog incorporation into DNA may be summarized as follows: analogs with modifications at the base moiety do not affect the rate of DNA chain elongation whereas those modified at the sugar moiety will inhibit the rate of chain elongation. ACGTP and DHPGTP competitively inhibit incorporation of dGTP into DNA; however, steric freedom of the acyclic phosphate may allow these nucleotides to bind virus enzyme in a conformation similar to that assumed by dGTP only at the transitional stage of the enzyme reaction. This may explain the high affinity of virus enzyme for these inhibitors. The interaction of aphidicolin with virus enzyme differs from that with host enzyme. These differences suggest new strategies for antiviral chemotherapy using aphidicolin derivatives.
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PMID:Interaction of DNA polymerase and nucleotide analog triphosphates. 301 71

Cross-resistance data for a group of nine acyclovir-resistant variants of herpes simplex virus type 1 are reported. These mutants, which express either altered thymidine kinase (TK) or DNA polymerase, were all derived from the same wild-type (wt) strain after exposure to acyclovir in tissue culture. Furthermore, all variants have pathogenic properties similar to the wt parental strain as assessed using mouse model systems (G. Darby, H.J. Field, and S.A. Salisbury, Nature (London) 289:81-83, 1981; B.A. Larder and G. Darby, Virology 146:262-271, 1985). Two groups of antiherpes compounds were used: those requiring activation by TK and those whose action is independent of that enzyme. The TK substrate-specificity mutants were generally resistant to the TK-activated drugs but showed wt susceptibility to phosphonoacetic acid, 9-beta-D-arabinofuranosyladenine, and aphidicolin. The DNA polymerase mutants were relatively susceptible to most TK-activated drugs, although two were resistant to 5-(trifluoromethyl)-2'-deoxyuridine. The polymerase mutants showed a more complex pattern of susceptibility, however, to those compounds whose mode of action is independent of TK. In general, these variants showed similar responses to phosphonoacetic acid, phosphonoformate, and 9-beta-D-arabinofuranosyladenine, a particular variant being either resistant, susceptible, or hypertensive to all three. The response of each variant to aphidicolin, however, appeared to be the inverse of its response to the other three drugs. The cross-resistance patterns are discussed, and their implications for combined or successive therapies are considered.
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PMID:Susceptibility to other antiherpes drugs of pathogenic variants of herpes simplex virus selected for resistance to acyclovir. 301 9

Sensitivity of Herpes Simplex Virus type I (HSV-I) mutants carrying genetic defect in the DNA polymerase and thymidine kinase genes to the action of some drugs was studied. TK- mutant of HSV-I was resistant to Ara-T and ACG and sensitive to PAA, Ara-A as well as to ribavirin and ADEA. PAAr mutant of HSV-I was resistant to PAA, Ara-A, ACG and sensitive to Ara-T, ribavirin and ADEA. A double mutant of HSV-I-TK-, PAAr was resistant to all drugs, except for ribavirin and ADEA. To inhibit reproduction of HSV with genetic defect, it is important using drugs of independent mode of action on the function of defective viral gene.
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PMID:[Inhibition of the reproduction of a herpes simplex I virus carrying mutations in the thymidine kinase and DNA polymerase genes]. 301 23

The acyclovir resistant mutant of varicella-zoster virus ACV-R (A 8) induced the same level of thymidine kinase activity in infected cells as the parent Kawaguchi strain. However, it induced less deoxycytidine kinase activity and did not induce phosphorylating activity for the nucleotide analogue, 9-(2 hydroxy-ethoxymethyl)-guanine-(acyclovir). Another acyclovir resistant mutant, ACV-R (A 4), which is cross-resistant to phosphonoacetate and is thought to be a viral DNA polymerase mutant, induced the same level of phosphorylating activities for thymidine, deoxycytidine and acyclovir as the parent strain. The altered substrate specificity of thymidine kinase induced by ACV-R (A 8) is concluded to confer resistance to acyclovir on ACV-R (A 8).
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PMID:Thymidine kinase with altered substrate specificity of acyclovir resistant varicella-zoster virus. 302 11

Replication of equine herpesvirus type 1 (EHV-1) was sensitive to 9-(1,3-dihydroxy-2-propoxymethyl)guanine(DHPG) but relatively resistant to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). Likewise, plaque formation by EHV-1 was inhibited by DHPG, but not by BVDU. Plaque formation by a thymidine kinase-negative (tk-) mutant of EHV-1 was not inhibited by DHPG. In order to investigate biochemical mechanisms determining the differential sensitivity of EHV-1 to these drugs, the EHV-1-encoded thymidine kinase enzyme activity (TK)1 was partially purified from EHV-1-infected cells and analyzed. The EHV-1-induced enzyme utilized both ATP and CTP as phosphate donors and differed in relative electrophoretic mobility from the TKs of mock-infected and HSV-1-infected cells. Phosphorylation of 3H-dThd by the EHV-1 TK was inhibited by AraT, IdUrd, BVDU, and DHPG. The EHV-1 TK phosphorylated 125I-dCyd and 3H-ACV. The results indicate that EHV-1 encodes a pyrimidine deoxyribonucleoside kinase with broad nucleoside substrate specificity. These observations suggest that the failure of BVDU to inhibit EHV-1 replication is not attributable to an inability of the EHV-1 TK to phosphorylate BVDU, but may result from the incapacity of the viral TK to convert BVDU monophosphate to the triphosphate or from lack of inhibitory effect of BVDU triphosphate on viral DNA polymerase reactions.
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PMID:Phosphorylation of nucleoside analogs by equine herpesvirus type 1 pyrimidine deoxyribonucleoside kinase. 302 47

The antiviral compound 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (2'-nor-2'-deoxyguanosine, 2'-NDG) is phosphorylated by the HSV-1-induced thymidine kinase to the monophosphate (2'-NDG-MP) and this is further phosphorylated by cellular kinases to the triphosphate (2'-NDG-TP) which is a potent inhibitor of DNA polymerases. Since phosphorylation of 2'-NDG creates a chiral center in the molecule, it was of interest to examine whether both monophosphate enantiomers were produced by the viral thymidine kinase, whether they both could be further phosphorylated by cellular kinases and, if so, whether the respective triphosphates were equally inhibitory to the DNA polymerases. The time course of the phosphorylation by GMP kinase of a chemically synthesized, racemic 2'-NDG-MP was compared to that of a 2'-NDG-MP preparation obtained by enzymatic phosphorylation of 2'-NDG with HSV-1 thymidine kinase. The results indicated that the two enantiomeric monophosphates were phosphorylated by GMP kinase with different rates and that phosphorylation of 2'-NDG by HSV-1 thymidine kinase gave only one of the isomers, whose structure was determined to be S. Both enantiomeric diphosphates were further phosphorylated to the respective triphosphates and it was shown that, in contrast to the triphosphate obtained from the 2'-NDG-MP prepared by viral thymidine kinase which was a potent inhibitor of HSV-1 DNA polymerase, the triphosphate obtained from the slow-reacting R isomer had little or no inhibitory activity against this enzyme.
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PMID:Stereochemical considerations in the enzymatic phosphorylation and antiviral activity of acyclonucleosides. I. Phosphorylation of 2'-nor-2'-deoxyguanosine. 302 84


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