Identification of Efficiently Cleaved Substrates for HIV-1 Protease Using a Phage Display Library and Use in Inhibitor Development
Identifieur interne : 003737 ( Main/Exploration ); précédent : 003736; suivant : 003738Identification of Efficiently Cleaved Substrates for HIV-1 Protease Using a Phage Display Library and Use in Inhibitor Development
Auteurs : Zachary Q. Beck ; Laurence Hervio ; Philip E. Dawson ; John H. Elder ; Edwin L. MadisonSource :
- Virology [ 0042-6822 ] ; 2000.
English descriptors
- Teeft :
- Acid, Active site, Active site titration, Amide, Amide bond, Amide inhibitors, Amino, Amino acid, Amino acid analysis, Amino acid sequences, Amino acids, Analogue, Antibody epitope, Aspartic protease, Blot analysis, Boldface type, Broad range, Cleavage, Cleavage efficiency, Cleavage products, Cleavage site, Cleavage sites, Context dependence, Control phage, Corresponding substrates, Dixon plots, Enzyme concentration, Febs lett, First round, Fitting data, Fmoc chemistries, Fusion peptide, Glutamic, Glutamic acid, Hexamer, Hexamer library, Human immunodeficiency virus, Human immunodeficiency virus type, Immunodeficiency, Infective phage, Inhibitor, Larger variety, Last number, Mass spectrometry, Mass spectrometry analysis, Methods enzymol, Molecular biology, Morrison equation, Nacl, Natural substrates, Other inhibitors, Pansorbin cells, Peptide, Peptide bond, Peptide chain, Peptide sequences, Peptide substrate, Peptide substrates, Peptide synthesis, Phage, Phage fusion peptide, Phage selection, Phage substrates, Physiological conditions, Polgar, Polyproteins, Pore size, Position equivalent, Positive control phage, Primary sequences, Protease, Protease inhibitors, Protease specificity, Proteinase, Random hexamer region, Randomized, Randomized hexamer, Randomized hexamer region, Results show, Science park road, Scripps research institute, Solid phase synthesis, Standard deviation, Stephen kent, Stock solution, Subsite, Subsite preferences, Substrate, Substrate binding site, Substrate phage, Substrate specificity, Supernatant, Synthetic protease, Szeltner, Temporal order, Tight binding inhibitors, Torrey pines road, Total chemical synthesis, Tozser, Vascular biology.
Abstract
Abstract: The recognition sequences for substrate cleavage by aspartic protease of HIV-1 are diverse and cleavage specificities are controlled by complex interactions between at least six amino acids around the cleavage site. We have identified 45 efficiently cleaved peptide substrates of HIV-1 protease (PR) using substrate phage display, an approach that can elucidate both context-dependent and context-independent preferences at individual subsites of a protease substrate. Many of the selected peptides were cleaved more efficiently and had lower Km values than physiologically relevant substrates of HIV-1 PR. Therefore, mutations occurring in the cleavage sites of the Gag and Gag-pol polyproteins of HIV-1 could significantly lower the Km values to better compete against drugs for protease binding while maintaining cleavage rates necessary for viral replication. The most efficiently cleaved peptide substrate derived from these phage, Ac-GSGIF*LETSL-NH2, was cleaved 60 times more efficiently and had a Km approximately 260 times lower than a nine-amino-acid peptide based on the natural reverse transcriptase/integrase cleavage site when assayed at pH 5.6, 0.2 M NaCl. The peptide substrates selected served as frameworks for synthesis of tight binding reduced amide inhibitors of HIV-1 PR. The results show that the most efficiently cleaved substrates serve as the best templates for synthesis of the tightest binding inhibitors. Thus, defining changes in substrate preferences for drug-resistant proteases may aid in the development of more efficacious inhibitors.
Url:
DOI: 10.1006/viro.2000.0420
Affiliations:
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Madison</name><affiliation><wicri:noCountry code="subField">92121</wicri:noCountry></affiliation></author></analytic><monogr></monogr><series><title level="j">Virology</title><title level="j" type="abbrev">YVIRO</title><idno type="ISSN">0042-6822</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">274</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="391">391</biblScope><biblScope unit="page" to="401">401</biblScope></imprint><idno type="ISSN">0042-6822</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0042-6822</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acid</term><term>Active site</term><term>Active site titration</term><term>Amide</term><term>Amide bond</term><term>Amide inhibitors</term><term>Amino</term><term>Amino acid</term><term>Amino acid analysis</term><term>Amino acid sequences</term><term>Amino acids</term><term>Analogue</term><term>Antibody epitope</term><term>Aspartic protease</term><term>Blot analysis</term><term>Boldface type</term><term>Broad range</term><term>Cleavage</term><term>Cleavage efficiency</term><term>Cleavage products</term><term>Cleavage site</term><term>Cleavage sites</term><term>Context dependence</term><term>Control phage</term><term>Corresponding substrates</term><term>Dixon plots</term><term>Enzyme concentration</term><term>Febs lett</term><term>First round</term><term>Fitting data</term><term>Fmoc chemistries</term><term>Fusion peptide</term><term>Glutamic</term><term>Glutamic acid</term><term>Hexamer</term><term>Hexamer library</term><term>Human immunodeficiency virus</term><term>Human immunodeficiency virus type</term><term>Immunodeficiency</term><term>Infective phage</term><term>Inhibitor</term><term>Larger variety</term><term>Last number</term><term>Mass spectrometry</term><term>Mass spectrometry analysis</term><term>Methods enzymol</term><term>Molecular biology</term><term>Morrison equation</term><term>Nacl</term><term>Natural substrates</term><term>Other inhibitors</term><term>Pansorbin cells</term><term>Peptide</term><term>Peptide bond</term><term>Peptide chain</term><term>Peptide sequences</term><term>Peptide substrate</term><term>Peptide substrates</term><term>Peptide synthesis</term><term>Phage</term><term>Phage fusion peptide</term><term>Phage selection</term><term>Phage substrates</term><term>Physiological conditions</term><term>Polgar</term><term>Polyproteins</term><term>Pore size</term><term>Position equivalent</term><term>Positive control phage</term><term>Primary sequences</term><term>Protease</term><term>Protease inhibitors</term><term>Protease specificity</term><term>Proteinase</term><term>Random hexamer region</term><term>Randomized</term><term>Randomized hexamer</term><term>Randomized hexamer region</term><term>Results show</term><term>Science park road</term><term>Scripps research institute</term><term>Solid phase synthesis</term><term>Standard deviation</term><term>Stephen kent</term><term>Stock solution</term><term>Subsite</term><term>Subsite preferences</term><term>Substrate</term><term>Substrate binding site</term><term>Substrate phage</term><term>Substrate specificity</term><term>Supernatant</term><term>Synthetic protease</term><term>Szeltner</term><term>Temporal order</term><term>Tight binding inhibitors</term><term>Torrey pines road</term><term>Total chemical synthesis</term><term>Tozser</term><term>Vascular biology</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: The recognition sequences for substrate cleavage by aspartic protease of HIV-1 are diverse and cleavage specificities are controlled by complex interactions between at least six amino acids around the cleavage site. We have identified 45 efficiently cleaved peptide substrates of HIV-1 protease (PR) using substrate phage display, an approach that can elucidate both context-dependent and context-independent preferences at individual subsites of a protease substrate. Many of the selected peptides were cleaved more efficiently and had lower Km values than physiologically relevant substrates of HIV-1 PR. Therefore, mutations occurring in the cleavage sites of the Gag and Gag-pol polyproteins of HIV-1 could significantly lower the Km values to better compete against drugs for protease binding while maintaining cleavage rates necessary for viral replication. The most efficiently cleaved peptide substrate derived from these phage, Ac-GSGIF*LETSL-NH2, was cleaved 60 times more efficiently and had a Km approximately 260 times lower than a nine-amino-acid peptide based on the natural reverse transcriptase/integrase cleavage site when assayed at pH 5.6, 0.2 M NaCl. The peptide substrates selected served as frameworks for synthesis of tight binding reduced amide inhibitors of HIV-1 PR. The results show that the most efficiently cleaved substrates serve as the best templates for synthesis of the tightest binding inhibitors. Thus, defining changes in substrate preferences for drug-resistant proteases may aid in the development of more efficacious inhibitors.</div></front></TEI><affiliations><list></list><tree><noCountry><name sortKey="Beck, Zachary Q" sort="Beck, Zachary Q" uniqKey="Beck Z" first="Zachary Q." last="Beck">Zachary Q. Beck</name><name sortKey="Dawson, Philip E" sort="Dawson, Philip E" uniqKey="Dawson P" first="Philip E." last="Dawson">Philip E. Dawson</name><name sortKey="Elder, John H" sort="Elder, John H" uniqKey="Elder J" first="John H." last="Elder">John H. Elder</name><name sortKey="Hervio, Laurence" sort="Hervio, Laurence" uniqKey="Hervio L" first="Laurence" last="Hervio">Laurence Hervio</name><name sortKey="Madison, Edwin L" sort="Madison, Edwin L" uniqKey="Madison E" first="Edwin L." last="Madison">Edwin L. Madison</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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