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)

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.
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PMID:Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells. 241 21

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 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

9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (2'-nor-2'-deoxyguanosine; 2'NDG) selectively inhibits the replication of herpes group viruses. In cell culture studies 2'NDG was at least 10-fold more potent than acyclovir (ACV) in inhibition of human cytomegalovirus replication and Epstein-Barr virus-induced lymphocyte transformation and was about as effective as ACV in inhibition of herpes simplex viruses 1 and 2 and varicella zoster virus. Orally administered 2'NDG was 6- to 50-fold more efficacious than ACV in treating systemic or local HSV-1 infection or HSV-2 intravaginal infection in mice. The mode of action of 2'NDG appears to involve phosphorylation by herpes simplex virus thymidine kinase and subsequent phosphorylations by cellular kinases to produce 2'NDG triphosphate, which is a potent inhibitor of herpes virus DNA polymerase. Compared to ACV, 2'NDG was a more efficient substrate for HSV-1 thymidine kinase (Vmax/Km for 2'NDG 30-fold higher than that of ACV), whereas 2'NDG monophosphate is a more efficient substrate for GMP kinase (Vmax/Km for 2'NDG monophosphate 492-fold higher than that for ACV monophosphate). The combined effect is more rapid production of the inhibitory triphosphate from 2'NDG than from ACV.
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PMID:9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication. 630 64

We characterized the cross-talk between activators of protein kinase A (PKA) and thyroid hormone (T3) in T3 receptor (TR)-mediated transcription. U937 cells were cotransfected with a plasmid expressing the TR and a reporter plasmid containing a T3 response element (TRE) oriented either as a direct repeat or as a palindrome upstream of the thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene. T3 activated transcription by 10-fold. T3 response was potentiated 2.5-3-fold by activators of PKA, but an activator of protein kinase C or of guanylate kinase was ineffective. In the absence of T3, activators of PKA had no effect on transcription. TR heterodimerization with the retinoid X receptor may facilitate T3/PKA cross-talk because coexpression of the retinoid X receptor potentiated cross-talk. Synergy was not observed in JEG-3, F9, CV-1, HeLa, L929, and HTC cells, indicating that it may require cell-specific factors. Synergy required the DNA- and ligand-binding domains, but not the amino-terminal domain, indicating that T3- and TRE-induced conformational changes on the TR are essential for cross-talk. PKA phosphorylated the TR in vitro, suggesting that, like other nuclear receptors, the TR is a target for PKA. These results imply that PKA cross-talks with T3 at the level of the TRE-bound TR, enhancing its transcriptional activity in a cell-specific manner.
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PMID:Thyroid hormone activation of transcription is potentiated by activators of cAMP-dependent protein kinase. 870

Herpes simplex virus-thymidine kinase (HSV-TK) phosphorylates the prodrugs ganciclovir (GCV) and acyclovir (ACV), leading to disruption of DNA synthesis and inhibition of cell proliferation. HSV-TK vectors have been successfully employed in cardiovascular and cancer gene therapy. Activation of GCV and ACV, after an initial phosphorylation step by the viral thymidine kinase, is carried out by guanylate kinase. We reasoned that coexpression of guanylate kinase (GK) with HSV-TK would augment phosphorylation of GCV or ACV, leading to increased cell killing. To test this hypothesis, a vector expressing TK with GK (TKciteGK) was developed and tested on vascular smooth muscle cells (vsmcs) in vitro and in vivo. Compared to HSV-TK vectors, killing of vascular cells transduced with TKciteGK and exposed to GCV was significantly increased (P = 0.03). The TKciteGK construct was evaluated with three promoters: CMV, EF1alpha, and SM22alpha. TKciteGK expression driven by a CMV promoter induced cell killing more effectively than SM22alpha or EF1alpha promoters in primary vsmcs. Based upon these in vitro findings, TKciteGK vectors with a CMV promoter were tested in two animal models of cardiovascular disease: balloon angioplasty and stent deployment in pig arteries. Following vascular injury, expression of CMV-TKciteGK with GCV significantly reduced vsmc proliferation and intimal lesion formation compared to control vectors with GCV. In the angioplasty model, there was an 80% reduction in intima-to-media area ratio (P = 0.0002). These findings were paralleled in a stent model with 66% reduction in intimal lesions (P = 0.006). Coexpression of GK with TK increases cell killing and permits administration of GCV at lower doses. These modifications in TKciteGK vectors and GCV showed enhanced efficacy at lower prodrug doses, leading to improved safety for cardiovascular gene therapy.
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PMID:Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo. 1135 82

Herpes simplex virus thymidine kinase (HSVTK) with the guanosine analog ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system for gene therapy of cancer. Despite the broad application of the HSVTK/GCV approach, phosphorylation of GCV to its active state is inefficient such that high, myelosuppressive doses of GCV are needed to observe an antitumor effect. One strategy used to overcome the poor substrate specificity of HSVTK towards GCV (Km = 45 microM) has been to create novel forms of TK with altered substrate preferences. Such mutant TKs have shown benefit and are currently in clinical use. We describe here a second strategy to increase the amount of intracellular triphosphorylated GCV by involving the second enzyme in the GCV activation pathway, guanylate kinase (GMK). As a means to overcome the bottleneck of prodrug activation from the monophosphate to the diphosphate, we sought to combine both the critical HSVTK and GMK activities together. In this report we describe the construction of a fusion or chimeric protein of HSVTK and guanylate kinase, show data that demonstrate it confers a approximately 175-fold decrease in IC50 compared to HSVTK alone in response to ganciclovir treatment in stably transfected C6 glioma cells and finally, we present biochemical evidence of a kinetic basis for this improved cell killing.
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PMID:A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing. 1681 Jan 97

Herpes simplex virus thymidine kinase (HSVTK) with ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system in cancer gene therapy. A major limitation in this therapy is the inefficient activation of GCV by HSVTK to its active antimetabolites. We described earlier two strategies to overcome this limitation: (1) generation of HSVTK mutants with improved GCV activation potential and (2) construction of a fusion protein encoding HSVTK and mouse guanylate kinase (MGMK), the second enzyme in the GCV activation pathway. As a means to further enhance GCV activation, two MGMK/HSVTK constructs containing the HSVTK mutants, mutant 30 and SR39, were generated and evaluated for their tumor and bystander killing effects in vitro and in vivo. One fusion mutant, MGMK/30, shows significant reduction in IC(50) values of approximately 12 500-fold, 100-fold, and 125-fold compared with HSVTK, mutant 30 or MGMK/HSVTK, respectively. In vitro bystander analyses show that 5% of MGMK/30-expressing cells are sufficient to induce 75% of tumor cell killing. In an xenograft tumor model, MGMK/30 displays the greatest inhibition of tumor growth at a GCV concentration (1 mg kg(-1)) that has no effect on wild-type HSVTK-, MGMK/HSVTK-, or mutant 30-transfected cells. Another fusion construct, MGMK/SR39, sensitizes rat C6 glioma cells to GCV by 2500-fold or 25-fold compared with HSVTK or MGMK/HSVTK, respectively. In vitro analyses show similar IC(50) values between cells harboring SR39 and MGMK/SR39, although MGMK/SR39 seems to elicit stronger bystander killing effects in which 1% of MGMK/SR39-transfected cells result in 60% cell death. In a xenograft tumor model, despite observable tumor growth inhibition, no statistical significance in tumor volume was detected between mice harboring SR39- and MGMK/SR39-transfected cells when dosed with 1 mg kg(-1) GCV. However, at a lower dose of GCV (0.1 mg kg(-1)), MGMK/SR39 seems to have slightly greater tumor growth inhibition properties compared with SR39 (P< or =0.05). In vivo studies indicate that both mutant fusion proteins display substantial improvements in bystander killing in the presence of 1 mg kg(-1) GCV, even when only 5% of the tumor cells are transfected. Such fusion mutants with exceptional prodrug converting properties will allow administration of lower and non-myelosuppressive doses of GCV concomitant with improved tumor killing and as such are promising candidates for translational gene therapy studies.
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PMID:Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo. 1976 47

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