Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
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Two isomers of methanocarba (MC) thymidine (T), one an effective antiherpes agent with the pseudosugar moiety locked in the North (N) hemisphere of the pseudorotational cycle (1a, N-MCT) and the other an inactive isomer locked in the antipodean South (S) conformation (1b, S-MCT) were used to determine whether kinases and polymerases discriminate between their substrates on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono-, di-, and triphosphate levels in control cells, cells infected with the Herpes simplex virus, or cells transfected with the corresponding viral kinase gene (HSV-tk), suggests that kinases prefer substrates that adopt the S sugar conformation. On the other hand, the cellular DNA polymerase(s) of a murine tumor cell line transfected with HSV-tk incorporated almost exclusively the triphosphate of the locked N conformer (N-MCTTP), notwithstanding the presence of higher triphosphate levels of the S-conformer (S-MCTTP).
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PMID:Experimental and structural evidence that herpes 1 kinase and cellular DNA polymerase(s) discriminate on the basis of sugar pucker. 1471 51

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS). Blocking lytic KSHV replication may hinder KS tumorigenesis. Here, we report potent in vitro anti-KSHV activity of 2'-exo-methanocarbathymidine [North-methanocarbathymidine (N-MCT)], a thymidine analog with a pseudosugar ring locked in the northern conformation, which has previously been shown to block the replication of herpes simplex virus types 1 and 2. N-MCT inhibited KSHV virion production in lytically induced KSHV-infected BCBL-1 cells with a substantially lower 50% inhibitory concentration (IC50) than those of cidofovir (CDV) and ganciclovir (GCV) (IC50, mean +/- standard deviation: 0.08 +/- 0.03, 0.42 +/- 0.07, and 0.96 +/- 0.49 microM for N-MCT, CDV, and GCV, respectively). The reduction in KSHV virion production was accompanied by a corresponding decrease in KSHV DNA levels in the N-MCT-treated BCBL-1 cells, indicating that the compound blocked lytic KSHV DNA replication. A time- and dose-dependent accumulation of N-MCT-triphosphate (TP) was demonstrated in lytically induced BCBL-1 cells, while uninfected cells showed virtually no accumulation. The levels of N-MCT-TP were significantly decreased in the presence of 5'-ethynylthymidine, a potent inhibitor of herpesvirus thymidine kinase, resulting in the abrogation of anti-KSHV activity of N-MCT. N-MCT-TP more effectively blocked in vitro DNA synthesis by KSHV DNA polymerase with an IC50 of 6.24 +/- 0.08 microM (mean +/- standard deviation) compared to CDV-diphosphate (14.70 +/-2.47 microM) or GCV-TP (24.59 +/- 5.60 microM). Taken together, N-MCT is a highly potent and target-specific anti-KSHV agent which inhibits lytic KSHV DNA synthesis through its triphosphate metabolite produced in KSHV-infected cells expressing a virally encoded thymidine kinase.
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PMID:Potent antiviral activity of north-methanocarbathymidine against Kaposi's sarcoma-associated herpesvirus. 1630 59

N-Methanocarbathymidine [(N)-MCT] is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. The antiviral activity of this molecule is dependent on the type I thymidine kinase (TK) in herpes simplex virus and also appears to be dependent on the type II TK expressed by cowpox and vaccinia viruses, suggesting that it is a substrate for both of these divergent forms of the enzyme. The drug is also a good inhibitor of viral DNA synthesis in both viruses and is consistent with inhibition of the viral DNA polymerase once it is activated by the viral TK homologs. This mechanism of action explains the rather unusual spectrum of activity, which is limited to orthopoxviruses, alphaherpesviruses, and Epstein-Barr virus, since these viruses express molecules with TK activity that can phosphorylate and thus activate the drug. The compound is also effective in vivo and reduces the mortality of mice infected with orthopoxviruses, as well as those infected with herpes simplex virus type 1 when treatment is initiated 24 h after infection. These results indicate that (N)-MCT is active in vitro and in vivo, and its mechanism of action suggests that the molecule may be an effective therapeutic for orthopoxvirus and herpesvirus infections, thus warranting further development.
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PMID:Activity and mechanism of action of N-methanocarbathymidine against herpesvirus and orthopoxvirus infections. 1656 49