Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1 Reverse Transcriptase Using a Transient Kinetic Analysis†
Identifieur interne : 003240 ( Main/Exploration ); précédent : 003239; suivant : 003241Probing the Molecular Mechanisms of AZT Drug Resistance Mediated by HIV-1 Reverse Transcriptase Using a Transient Kinetic Analysis†
Auteurs : Adrian S. Ray [États-Unis] ; Eisuke Murakami [États-Unis] ; Aravind Basavapathruni [États-Unis] ; Joseph A. Vaccaro [États-Unis] ; Dagny Ulrich [États-Unis] ; Chung K. Chu [États-Unis] ; Raymond F. Schinazi [États-Unis] ; Karen S. Anderson [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 2003.
Abstract
Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase (RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision, suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain terminator from an RNA−RNA primer−template may show how slight changes in selectivity against AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance. Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations. Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.
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DOI: 10.1021/bi034435l
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Reverse Transcriptase Using a Transient Kinetic Analysis<ref type="bib" target="#bi034435lAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Ray, Adrian S" sort="Ray, Adrian S" uniqKey="Ray A" first="Adrian S." last="Ray">Adrian S. Ray</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur</wicri:cityArea></affiliation><affiliation></affiliation></author><author><name sortKey="Murakami, Eisuke" sort="Murakami, Eisuke" uniqKey="Murakami E" first="Eisuke" last="Murakami">Eisuke Murakami</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur</wicri:cityArea></affiliation><affiliation></affiliation></author><author><name sortKey="Basavapathruni, Aravind" sort="Basavapathruni, Aravind" uniqKey="Basavapathruni A" first="Aravind" last="Basavapathruni">Aravind Basavapathruni</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur</wicri:cityArea></affiliation><affiliation></affiliation></author><author><name sortKey="Vaccaro, Joseph A" sort="Vaccaro, Joseph A" uniqKey="Vaccaro J" first="Joseph A." last="Vaccaro">Joseph A. Vaccaro</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur</wicri:cityArea></affiliation><affiliation></affiliation><affiliation></affiliation></author><author><name sortKey="Ulrich, Dagny" sort="Ulrich, Dagny" uniqKey="Ulrich D" first="Dagny" last="Ulrich">Dagny Ulrich</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur</wicri:cityArea></affiliation><affiliation></affiliation></author><author><name sortKey="Chu, Chung K" sort="Chu, Chung K" uniqKey="Chu C" first="Chung K." last="Chu">Chung K. Chu</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur</wicri:cityArea></affiliation><affiliation></affiliation></author><author><name sortKey="Schinazi, Raymond F" sort="Schinazi, Raymond F" uniqKey="Schinazi R" first="Raymond F." last="Schinazi">Raymond F. Schinazi</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur</wicri:cityArea></affiliation><affiliation></affiliation></author><author><name sortKey="Anderson, Karen S" sort="Anderson, Karen S" uniqKey="Anderson K" first="Karen S." last="Anderson">Karen S. Anderson</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520,Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,Athens, Georgia 30602-2352, and Laboratory of Biochemical Pharmacology, Department of Pediatrics,Emory University School of Medicine/Veterans Affairs Medical Center, Decatur</wicri:cityArea></affiliation><affiliation></affiliation><affiliation></affiliation></author></analytic><monogr></monogr><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">2003</date><date type="published">2003</date><biblScope unit="vol">42</biblScope><biblScope unit="issue">29</biblScope><biblScope unit="page" from="8831">8831</biblScope><biblScope unit="page" to="8841">8841</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase (RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision, suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain terminator from an RNA−RNA primer−template may show how slight changes in selectivity against AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance. Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations. Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Géorgie (États-Unis)</li></region></list><tree><country name="États-Unis"><region name="Géorgie (États-Unis)"><name sortKey="Ray, Adrian S" sort="Ray, Adrian S" uniqKey="Ray A" first="Adrian S." last="Ray">Adrian S. Ray</name></region><name sortKey="Anderson, Karen S" sort="Anderson, Karen S" uniqKey="Anderson K" first="Karen S." last="Anderson">Karen S. Anderson</name><name sortKey="Basavapathruni, Aravind" sort="Basavapathruni, Aravind" uniqKey="Basavapathruni A" first="Aravind" last="Basavapathruni">Aravind Basavapathruni</name><name sortKey="Chu, Chung K" sort="Chu, Chung K" uniqKey="Chu C" first="Chung K." last="Chu">Chung K. Chu</name><name sortKey="Murakami, Eisuke" sort="Murakami, Eisuke" uniqKey="Murakami E" first="Eisuke" last="Murakami">Eisuke Murakami</name><name sortKey="Schinazi, Raymond F" sort="Schinazi, Raymond F" uniqKey="Schinazi R" first="Raymond F." last="Schinazi">Raymond F. Schinazi</name><name sortKey="Ulrich, Dagny" sort="Ulrich, Dagny" uniqKey="Ulrich D" first="Dagny" last="Ulrich">Dagny Ulrich</name><name sortKey="Vaccaro, Joseph A" sort="Vaccaro, Joseph A" uniqKey="Vaccaro J" first="Joseph A." last="Vaccaro">Joseph A. Vaccaro</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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