3‘-Azido-3‘-deoxythymidine-(5‘)-tetraphospho-(5‘)-adenosine, the Product of ATP-Mediated Excision of Chain-Terminating AZTMP, Is a Potent Chain-Terminating Substrate for HIV-1 Reverse Transcriptase

Dharmasena, Sanjeewa; Pongracz, Zita; Arnold, Eddy; Sarafianos, Stefan G.; Parniak, Michael A.

doi:10.1021/bi061364s

Cited by 28 publications

(32 citation statements)

References 30 publications

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“…Single nucleotide incorporation measurements using Np 4 N as substrate at various concentrations could provide better estimates of k cat and K m for a more accurate value of k cat /K m . Nevertheless, current estimates were consistent to the observation with HIV RT showing that Np 4 N could be used as a substrate for DNA synthesis as efficiently as its corresponding nucleoside triphosphates (35,36).…”

Section: Discussionsupporting

confidence: 87%

NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

Zhang

Lévêque

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis C virus (HCV) RNA-dependent RNA polymerase replicates the viral genomic RNA and is a primary drug target for antiviral therapy. Previously, we described the purification of an active and stable polymerase-primer-template elongation complex. Here, we show that, unexpectedly, the polymerase elongation complex can use NTPs to excise the terminal nucleotide in nascent RNA. Mismatched ATP, UTP, or CTP could mediate excision of 3′-terminal CMP to generate the dinucleoside tetraphosphate products Ap 4 C, Up 4 C, and Cp 4 C, respectively. Pre-steady-state kinetic studies showed that the efficiency of NTP-mediated excision was highest with ATP. A chain-terminating inhibitor, 3′deoxy-CMP, could also be excised through this mechanism, suggesting important implications for nucleoside drug potency and resistance. The nucleotide excision reaction catalyzed by recombinant hepatitis C virus polymerase was 100-fold more efficient than the corresponding reaction observed with HIV reverse transcriptase.drug resistance | NS5B | nucleoside analog | pre-steady-state kinetics | pyrophosphorolysis C hronic infection with hepatitis C virus (HCV) is a leading cause of liver disease worldwide. Current treatment of the infection is based on the combination of pegylated IFN-α and ribavirin, which can lead to a sustained viral response (functional cure) in ∼50% of patients infected with HCV genotype 1 and in 80% of patients infected with HCV genotype 2 or 3. With the approval of the new HCV protease inhibitors, boceprevir and telaprevir, added to pegylated IFN and ribavirin, cure rates in treatment-naive patients infected with HCV genotype 1 have improved to ∼70%. However, these treatments are associated with significant side effects, and a large number of patients either do not respond or develop resistance to the current treatment options (1-3). New drugs in development include nucleoside analogs targeting the HCV polymerase (NS5B). These compounds, in contrast to protease inhibitors, are characterized by potency across all HCV genotypes and a higher barrier to resistance (4, 5).NS5B is a 68-kDa RNA-dependent RNA polymerase (RdRp) responsible for replication of the HCV genome. Its structure has the canonic right-hand polymerase structure with thumb, fingers, and palm domains, although extensions from the fingers domain enclose the active site and a β-loop originating from the thumb prevents the binding of duplex RNA (6, 7). HCV RNA replication initiates de novo without a primer in vivo, and RNA synthesis by NS5B proceeds through two very distinct mechanistic phases: initiation and elongation. Initiation of RNA synthesis is inefficient and associated with the production of a large amount of short, abortive RNA products, which are released into solution (8-10). Following initiation, RNA elongation is rapid and processive during the elongation phase (11). The different mechanistic and kinetic properties associated with the initiation and elongation phases suggest that different conformations of the NS5B protein may be required in th...

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Section: Discussionsupporting

confidence: 87%

NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

Zhang

Lévêque

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Excision of ACV-MP with ATP generates the ACV-(5Ј)-tetraphospho-(5Ј)-A (ACVp4A) that is eventually released from the complex. The tetraphosphate can be used as a substrate (16,57), resulting in re-incorporation of ACV-MP. At this level, the V75I mutant has a disadvantage over WT RT, which leads to an accumulation of the excised product.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase

Tchesnokov

Obikhod

Massud

et al. 2009

Journal of Biological Chemistry

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It has recently been demonstrated that the anti-herpetic drug acyclovir (ACV) also displays antiviral activity against the human immunodeficiency virus type 1 (HIV-1). The triphosphate form of ACV is accepted by HIV-1 reverse transcriptase (RT), and subsequent incorporation leads to classical chain termination. Like all approved nucleoside analogue RT inhibitors (NRTIs), the selective pressure of ACV is associated with the emergence of resistance. The V75I mutation in HIV-1 RT appears to be dominant in this regard. By itself, this mutation is usually not associated with resistance to currently approved NRTIs. Here we studied the underlying biochemical mechanism. We demonstrate that V75I is also selected under the selective pressure of a monophosphorylated prodrug that was designed to bypass the bottleneck in drug activation to the triphosphate form (ACV-TP). Pre-steady-state kinetics reveal that V75I discriminates against the inhibitor at the level of catalysis, whereas binding of the inhibitor remains largely unaffected. The incorporated ACV-monophosphate (ACV-MP) is vulnerable to excision in the presence of the pyrophosphate donor ATP. V75I compromises binding of the next nucleotide that can otherwise provide a certain degree of protection from excision. Collectively, the results of this study suggest that ACV is sensitive to two different resistance pathways, which warrants further investigation regarding the detailed resistance profile of ACV. Such studies will be crucial in assessing the potential clinical utility of ACV and its derivatives in combination with established NRTIs.

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“…Both pyrophosphate and ATP act as excision substrates in vitro, but the intracellular substrate used during replication of AZT-resistant HIV is still unknown. 4 The amount of azidothymidine-triphosphate (AZT-TP) was of the same order of magnitude as the intracellular concentrations of AZT-TP needed in blood cells for efficient treatment of HIV-infected patients treated with AZT. 5 There are some major problems associated with AZT chemotherapy, 6 and many H-phosphonates of AZT have also been shown to possess high anti-HIV activity, such as PZT (5 -hydrogenphosphate of AZT).…”

Section: Introductionmentioning

confidence: 99%

Synthesis of Novel Steroidal Bioconjugates of Phospholipid with AZT

Jin

et al. 2008

Phosphorus, Sulfur, and Silicon and the Related Elements

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3‘-Azido-3‘-deoxythymidine-(5‘)-tetraphospho-(5‘)-adenosine, the Product of ATP-Mediated Excision of Chain-Terminating AZTMP, Is a Potent Chain-Terminating Substrate for HIV-1 Reverse Transcriptase

Cited by 28 publications

References 30 publications

NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase

Synthesis of Novel Steroidal Bioconjugates of Phospholipid with AZT

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