Comparative inhibition of hepatitis B virus DNA polymerase and cellular DNA polymerases by triphosphates of sugar-modified 5-methyldeoxycytidines and of other nucleoside analogs

Matthes, E.; Reimer, K.; Janta-Lipinski, M. Von; Meisel, Helga; Lehmann, Christian

doi:10.1128/aac.35.6.1254

Cited by 17 publications

(17 citation statements)

References 25 publications

(30 reference statements)

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“…Table 1 summarizes these data and shows the percentages of the three phosphorylated species of nucleotides present before (0 min) and after (30 min) incubation in the presence of DHBV core preparation. However, for the subsequent experiments presented in this paper, dGTP and dATP were added at a concentration of 50 M, which is in great excess of the K m for the nucleotides (0.1 to 0.2 M) (14,21).…”

Section: Resultsmentioning

confidence: 99%

Mechanism of inhibition of duck hepatitis B virus polymerase by (-)-beta-L-2',3'-dideoxy-3'-thiacytidine

Severini

Liu

Wilson

et al. 1995

Antimicrob Agents Chemother

136

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We have used the endogenous reverse transcriptase reaction of viral core particles from duck liver to elucidate the mechanism of inhibition of duck hepatitis B virus (DHBV) replication by the nucleoside analog (؊)-␤-L-2,3-dideoxy-3-thiacytidine (3TC). As is the case in human immunodeficiency virus replication, 3TC-5-triphosphate (3TC-TP) acts as a chain terminator for the DNA polymerase activities. The results of several different experiments support this conclusion, which explains the potent activity of 3TC against the hepadnaviruses. In isolated DHBV core particles, 3TC-TP inhibited the reverse transcriptase in a manner that resembled competitive inhibition with respect to dCTP. However, the kinetics of inhibition was not linear on a double-reciprocal plot for the highest concentrations of 3TC-TP and the lowest concentration of dCTP. This anomaly would be expected if binding to the nucleotide site was followed by DNA chain termination. Calculations that used only the linear part of the curve yielded a K i of 0.78 ؎ 0.10 M 3TC-TP. The inhibition of core particles incubated in vitro with 3TC-TP was not reversed by removal of the free inhibitor. 3TC-TP inactivated the reverse transcriptase activity in a concentration-dependent manner. The K m of the chain termination reaction was calculated at 0.71 ؎ 0.05 M. Similar competitive kinetics and irreversible inhibition were also obtained on the endogenous DNA polymerase from viral particles from serum, suggesting that 3TC-TP also acts as a chain terminator of the DNA-directed DNA polymerase of DHBV replication. Core particles purified from DHBV-infected hepatocytes treated with 3TC also showed irreversible inhibition of the endogenous reverse transcriptase activity, indicating that chain termination is the mechanism of inhibition in the presence of the normal deoxynucleotide pools within cells. Endogenous sequencing of duck liver viral core particles with 3TC-TP as a chain terminator shows a pattern of bands similar to that obtained with ddCTP, and [ 32 P]3TC-TP labeled the growing DNA chain at the C position.) is a deoxycitidine analog in which the 3Ј carbon is replaced by a sulfur atom with loss of the 3Ј OH group necessary for the elongation of the DNA chain. 3TC was shown to be an effective inhibitor of human immunodeficiency virus (HIV) replication (7,13,28,33), and it was recently approved for compassionate use as an anti-HIV agent.Previous work showed that 3TC is also a very effective agent against human hepatitis B virus (5, 10, 32) and duck hepatitis B virus (DHBV [32]). In vivo and in infected hepatocytes, 3TC rapidly reduces the amount of viral DNA within days of the start of treatment, and it does not show significant toxicity (reference 5 and our unpublished results). 3TC is currently undergoing phase II clinical trials as an antiviral agent for human hepatitis B virus.For both HIV and hepadnaviruses, the mechanism of action of 3TC requires phosphorylation to 3TC-5Ј-triphosphate (3TC-TP), which in turn specifically inhibits the viral polymerases (3, 6, 15...

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Section: Resultsmentioning

confidence: 99%

Mechanism of inhibition of duck hepatitis B virus polymerase by (-)-beta-L-2',3'-dideoxy-3'-thiacytidine

Severini

Liu

Wilson

et al. 1995

Antimicrob Agents Chemother

136

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“…Modified dNTPs commonly exhibit a reduced level of uptake by DNA polymerases (10,(14)(15)(16)(17). Although certain modified dNTPs have been successfully used in PCR (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)21), accurate and comprehensive studies of their effects on rate and yield of DNA synthesis are rare.…”

Section: Substrate Properties Of Base-modified Dntps In Pcrmentioning

confidence: 99%

“…DNA polymerases are less able to accommodate modification of the sugar moiety in nucleotides (15). Many such modifications inhibit viral DNA polymerases, making these compounds interesting drug candidates for medicinal chemistry studies (16,17).…”

mentioning

confidence: 99%

Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

Kutyavin¹

2008

Biochemistry

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Several base-modified duplex-stabilizing deoxyribonucleoside 5′-triphosphates (dNTPs) have been evaluated as agents for enhancing the hybridization properties of primers and probes in real-time polymerase chain reaction (PCR). It was shown that pyrimidines substituted at the 5-position with bromine or iodine atoms and methyl or propynyl groups are incorporated into PCR amplicons by Taq DNA polymerase as efficiently as natural dNTPs. The dNTP of 2-aminoadenosine was incorporated somewhat less efficiently than dATP but still supported PCR. Incorporation of these modified nucleotides into the amplified DNA represents a simple and inexpensive way to stabilize duplexes of primers and probes and is particularly effective in improving the amplification and detection of A/T-rich sequences. This technology permits the use of higher PCR annealing temperatures or alternatively a reduction in the length of the oligonucleotide components. Examples of successful application in TaqMan and Scorpion real-time detection assays are provided. Limits of the approach are identified and discussed. For example, application of the 5-bromo and 5-iodo derivatives may be limited to relatively G/C-rich DNA targets and, in particular, to those lacking long runs of adenylate and/or thymidylate. Simultaneous use of base-modified analogues of dATP and dTTP should be avoided in PCR due to "overstabilization" of the amplicon.Structurally modified DNA and RNA have applications in bioengineering, nanotechnology, molecular biology, and medicine. Oftentimes, however, chemical synthesis of modified polynucleotides is difficult and inefficient. Moreover, certain modifications cannot be chemically introduced due to instability. Enzymatic synthesis is an alternative way to prepare DNA or RNA polymers. Relatively minor modifications at the R-phosphate moiety of deoxyribonucleoside 5′-triphosphates (dNTPs) are tolerated well by DNA polymerases (1). Examples include thio (2) and borano phosphate (3, 4) derivatives. DNA polymerases also tolerate certain base modifications in dNTPs as long as normal Watson-Crick base pairing is preserved.

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“…In view of the experience that some nucleosides with the unnatural ␤-L configuration were more powerful and selective antiviral agents than their corresponding ␤-D isomers, we synthesized and evaluated the corresponding opposite optical isomers of some of the ␤-D-MetCdR derivatives that we studied several years ago. These investigations have shown that the triphosphates of some of the ␤-D-MetCdR derivatives are extremely powerful inhibitors of HBV DNA polymerase in vitro (e.g., ␤-D-3Ј-FMetdCTP [IC 50 , 0.03 M]) (20), though they proved to be less active under cell culture and in vivo conditions (19,21). Now we found for the corresponding ␤-L analogues that ␤-L-FMetCdR, ␤-L-ddMetC, ␤-L-d4MetC, and ␤-L-araMetC as well as their triphosphates were unable to inhibit either HBV replication in HepG2.2.15 cells or cell-free HBV DNA polymerase activity (EC 50 , Ն50 M; IC 50 , 1.9 M to 10.8 M).…”

Section: Discussionmentioning

confidence: 99%

Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N ⁴ -Hydroxy- and 5-Methyl-β- l -Deoxycytidine Analogues

Matthes

Funk

Krahn

et al. 2007

Antimicrob Agents Chemother

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, and ␤-L-5-methyl-2-deoxycytidine (EC 50 , 0.9 M). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the ␤-L-cytidine derivatives was also assessed. In accordance with the cell culture data, ␤-L-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 M. The cytotoxicities of some of the 4-NHOH-modified ␤-L-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH 2 group. The 50% cytotoxic concentrations for ␤-L-Hyd4C in HepG2 and HL-60 cells were 2,500 M and 3,500 M, respectively. In summary, our results demonstrate that at least ␤-L-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.Complete and sustained suppression of viral replication remains the most important goal in the treatment of patients with chronic hepatitis B virus (HBV) infection. Nucleoside and nucleotide analogues have greatly improved the disease outcome for these patients and have also prevented hepatic decompensation or the development of hepatocellular carcinoma. While ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine (3TC; lamivudine) has been approved for the treatment of HBV infections, a series of further ␤-Lnucleosides are under clinical investigation as inhibitors of hepadnavirus infections. These include ␤-L-2Ј,3Ј-dideoxy-3Ј-The most important advantage associated with some, but not all, of these ␤-L-nucleosides seems to be a much higher selectivity for the viral target than for the cellular targets, resulting in reduced cytotoxicity. This quality of ␤-L-nucleosides stimulated the search for new ␤-L-nucleosides but also the synthesis of enantiomeric isomers of effective ␤-D-nucleosides (7).Some of the unmodified pyrimidine and purine ␤-L-nucleosides were synthesized for the first time more than 35 years ago (12, 28, 34) but were considered to be metabolized very poorly in mice (14). A systematic biochemical investigation of the ␤-L-enantiomers was initiated later and demonstrated the antihuman immunodeficiency virus (HIV) activity of ␤-L-2Ј,3Ј-dideoxycytidine (␤-L-ddC) in a cellular system (18). In addition, it was shown that ␤-L-dT might be phosphorylated by herpes simplex virus type 1 (HSV-1) thymidine kinase and further, by cellular enzymes, to ␤-L-dTTP (35).As a consequence, the effects of ␤-L-dTTP on various cellular DNA polymerases, TdT, the Klenow fragment of Escherichia coli DNA polymerase I, and viral DNA polymerases such as HSV-1 DNA polymerase and HIV reverse transcriptase were investigated (6,32,38). We showed that ␤-L-TTP does not influence the activity of any of the cellular DNA polymerases (␣, ␤, ␥, ␦, or ε) or of HIV reverse transcriptase, but we demonstrated for the first time a strong inhibition of human and duck HBV DNA polymerases by ␤-L-dTTP (concentrations resulting in 50% inhibition of HBV DNA polymerase activity [IC 50 ], 0.46 M and 1.0 M, respectively) (37). Investigations on the cellular level and in vivo experim...

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Comparative inhibition of hepatitis B virus DNA polymerase and cellular DNA polymerases by triphosphates of sugar-modified 5-methyldeoxycytidines and of other nucleoside analogs

Cited by 17 publications

References 25 publications

Mechanism of inhibition of duck hepatitis B virus polymerase by (-)-beta-L-2',3'-dideoxy-3'-thiacytidine

Mechanism of inhibition of duck hepatitis B virus polymerase by (-)-beta-L-2',3'-dideoxy-3'-thiacytidine

Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N ⁴ -Hydroxy- and 5-Methyl-β- l -Deoxycytidine Analogues

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Comparative inhibition of hepatitis B virus DNA polymerase and cellular DNA polymerases by triphosphates of sugar-modified 5-methyldeoxycytidines and of other nucleoside analogs

Cited by 17 publications

References 25 publications

Mechanism of inhibition of duck hepatitis B virus polymerase by (-)-beta-L-2',3'-dideoxy-3'-thiacytidine

Mechanism of inhibition of duck hepatitis B virus polymerase by (-)-beta-L-2',3'-dideoxy-3'-thiacytidine

Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N 4 -Hydroxy- and 5-Methyl-β- l -Deoxycytidine Analogues

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Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N ⁴ -Hydroxy- and 5-Methyl-β- l -Deoxycytidine Analogues