Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities
Identifieur interne : 001A54 ( Istex/Curation ); précédent : 001A53; suivant : 001A55Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities
Auteurs : Cathryn A. Shaw-Reid [États-Unis] ; Bradley Feuston [États-Unis] ; Vandna Munshi [États-Unis] ; Krista Getty [États-Unis] ; Julie Krueger [États-Unis] ; Daria J. Hazuda [États-Unis] ; Michael A. Parniak [États-Unis] ; Michael D. Miller [États-Unis] ; Dale Lewis [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 2005.
Abstract
Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3‘-azido-3‘-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.
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DOI: 10.1021/bi0486740
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<sourceDesc><biblStruct><analytic><title level="a" type="main">Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor
Combinations on HIV-1 Reverse-Transcriptase Activities</title>
<author><name sortKey="Shaw Reid, Cathryn A" sort="Shaw Reid, Cathryn A" uniqKey="Shaw Reid C" first="Cathryn A." last="Shaw-Reid">Cathryn A. Shaw-Reid</name>
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</affiliation>
<affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation>
</affiliation>
<affiliation wicri:level="2"><mods:affiliation> Present address: Amgen, Inc., One Amgen Center Drive, ThousandOaks, CA 91320-1799.</mods:affiliation>
<country xml:lang="fr">États-Unis</country>
<placeName><region type="state">Californie</region>
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<author><name sortKey="Feuston, Bradley" sort="Feuston, Bradley" uniqKey="Feuston B" first="Bradley" last="Feuston">Bradley Feuston</name>
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<country xml:lang="fr">États-Unis</country>
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<wicri:cityArea>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh</wicri:cityArea>
</affiliation>
<affiliation><mods:affiliation> Department of Molecular Systems, Merck Research Laboratories.</mods:affiliation>
</affiliation>
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<author><name sortKey="Munshi, Vandna" sort="Munshi, Vandna" uniqKey="Munshi V" first="Vandna" last="Munshi">Vandna Munshi</name>
<affiliation wicri:level="2"><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation>
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</affiliation>
<affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation>
</affiliation>
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<author><name sortKey="Getty, Krista" sort="Getty, Krista" uniqKey="Getty K" first="Krista" last="Getty">Krista Getty</name>
<affiliation wicri:level="2"><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation>
<country xml:lang="fr">États-Unis</country>
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<wicri:cityArea>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh</wicri:cityArea>
</affiliation>
<affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Krueger, Julie" sort="Krueger, Julie" uniqKey="Krueger J" first="Julie" last="Krueger">Julie Krueger</name>
<affiliation wicri:level="2"><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation>
<country xml:lang="fr">États-Unis</country>
<placeName><region type="state">Pennsylvanie</region>
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<wicri:cityArea>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh</wicri:cityArea>
</affiliation>
<affiliation><mods:affiliation> Department of Cardiovascular Diseases, Merck Research Laboratories.</mods:affiliation>
</affiliation>
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<author><name sortKey="Hazuda, Daria J" sort="Hazuda, Daria J" uniqKey="Hazuda D" first="Daria J." last="Hazuda">Daria J. Hazuda</name>
<affiliation wicri:level="2"><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation>
<country xml:lang="fr">États-Unis</country>
<placeName><region type="state">Pennsylvanie</region>
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<wicri:cityArea>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh</wicri:cityArea>
</affiliation>
<affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation>
</affiliation>
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<author><name sortKey="Parniak, Michael A" sort="Parniak, Michael A" uniqKey="Parniak M" first="Michael A." last="Parniak">Michael A. Parniak</name>
<affiliation wicri:level="2"><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation>
<country xml:lang="fr">États-Unis</country>
<placeName><region type="state">Pennsylvanie</region>
</placeName>
<wicri:cityArea>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh</wicri:cityArea>
</affiliation>
<affiliation><mods:affiliation> Department of Medicine/Division of Infectious Diseases, Universityof Pittsburgh.</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Miller, Michael D" sort="Miller, Michael D" uniqKey="Miller M" first="Michael D." last="Miller">Michael D. Miller</name>
<affiliation wicri:level="2"><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation>
<country xml:lang="fr">États-Unis</country>
<placeName><region type="state">Pennsylvanie</region>
</placeName>
<wicri:cityArea>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh</wicri:cityArea>
</affiliation>
<affiliation><mods:affiliation> Department of Antiviral Research, Merck Research Laboratories.</mods:affiliation>
</affiliation>
<affiliation wicri:level="2"><mods:affiliation> To whom correspondence should be addressed: Department ofAntiviral Research, Merck and Co., Inc., P.O. Box 4, WP42-209, WestPoint, PA 19486-0004. Telephone: 215-652-0480. Fax: 215-993-5798.E-mail: michael_miller1@merck.com.</mods:affiliation>
<country xml:lang="fr">États-Unis</country>
<placeName><region type="state">Pennsylvanie</region>
</placeName>
<wicri:cityArea> To whom correspondence should be addressed: Department ofAntiviral Research, Merck and Co., Inc., P.O. Box 4, WP42-209, WestPoint</wicri:cityArea>
</affiliation>
</author>
<author><name sortKey="Lewis, Dale" sort="Lewis, Dale" uniqKey="Lewis D" first="Dale" last="Lewis">Dale Lewis</name>
<affiliation wicri:level="2"><mods:affiliation>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh, Pennsylvania 15261</mods:affiliation>
<country xml:lang="fr">États-Unis</country>
<placeName><region type="state">Pennsylvanie</region>
</placeName>
<wicri:cityArea>Department of Antiviral Research, Department of Molecular Systems, Department of Cardiovascular Diseases, Merck ResearchLaboratories, West Point, Pennsylvania 19486-0004, and Department of Medicine/Division of Infectious Diseases, University ofPittsburgh, Pittsburgh</wicri:cityArea>
</affiliation>
<affiliation><mods:affiliation> Department of Cardiovascular Diseases, Merck Research Laboratories.</mods:affiliation>
</affiliation>
</author>
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<series><title level="j" type="main">Biochemistry</title>
<title level="j" type="abbrev">Biochemistry</title>
<idno type="ISSN">0006-2960</idno>
<idno type="eISSN">1520-4995</idno>
<imprint><publisher>American Chemical Society</publisher>
<date type="e-published">2005</date>
<date type="published">2005</date>
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<front><div type="abstract">Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3‘-azido-3‘-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.</div>
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