Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay.
Identifieur interne : 005432 ( Main/Exploration ); précédent : 005431; suivant : 005433Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay.
Auteurs : Richard Y. Kao [République populaire de Chine] ; Amanda P C. To ; Louisa W Y. Ng ; Wayne H W. Tsui ; Terri S W. Lee ; Hoi-Wah Tsoi ; Kwok-Yung YuenSource :
- FEBS letters [ 0014-5793 ] ; 2004.
Descripteurs français
- KwdFr :
- Amorces ADN, Chromatographie en phase liquide à haute performance, Cinétique, Clonage moléculaire, Cysteine endopeptidases, Endopeptidases (métabolisme), Escherichia coli (enzymologie), Escherichia coli (génétique), Protéines recombinantes (antagonistes et inhibiteurs), Protéines recombinantes (métabolisme), Protéines virales (antagonistes et inhibiteurs), Protéines virales (métabolisme), Spectrométrie de fluorescence (), Séquence nucléotidique, Virus du SRAS (enzymologie).
- MESH :
- antagonistes et inhibiteurs : Protéines recombinantes, Protéines virales.
- enzymologie : Escherichia coli, Virus du SRAS.
- génétique : Escherichia coli.
- métabolisme : Endopeptidases, Protéines recombinantes, Protéines virales.
- Amorces ADN, Chromatographie en phase liquide à haute performance, Cinétique, Clonage moléculaire, Cysteine endopeptidases, Spectrométrie de fluorescence, Séquence nucléotidique.
English descriptors
- KwdEn :
- Base Sequence, Chromatography, High Pressure Liquid, Cloning, Molecular, Cysteine Endopeptidases, DNA Primers, Endopeptidases (metabolism), Escherichia coli (enzymology), Escherichia coli (genetics), Kinetics, Recombinant Proteins (antagonists & inhibitors), Recombinant Proteins (metabolism), SARS Virus (enzymology), Spectrometry, Fluorescence (methods), Viral Proteins (antagonists & inhibitors), Viral Proteins (metabolism).
- MESH :
- chemical , antagonists & inhibitors : Recombinant Proteins, Viral Proteins.
- chemical , metabolism : Endopeptidases, Recombinant Proteins, Viral Proteins.
- chemical : Cysteine Endopeptidases, DNA Primers.
- enzymology : Escherichia coli, SARS Virus.
- genetics : Escherichia coli.
- methods : Spectrometry, Fluorescence.
- Base Sequence, Chromatography, High Pressure Liquid, Cloning, Molecular, Kinetics.
Abstract
Severe acute respiratory syndrome associated coronavirus main protease (SARS-CoV Mpro) has been proposed as a prime target for anti-SARS drug development. We have cloned and overexpressed the SARS-CoV Mpro in Escherichia coli, and purified the recombinant Mpro to homogeneity. The kinetic parameters of the recombinant SARS-CoV Mpro were characterized by high performance liquid chromatography-based assay and continuous fluorescence-based assay. Two novel small molecule inhibitors of the SARS-CoV Mpro were identified by high-throughput screening using an internally quenched fluorogenic substrate. The identified inhibitors have Ki values at low microM range with comparable anti-SARS-CoV activity in cell-based assays.
DOI: 10.1016/j.febslet.2004.09.026
PubMed: 15498556
Affiliations:
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Le document en format XML
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Kao</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, China. rytkao@hkucc.hku.hk</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong</wicri:regionArea><wicri:noRegion>Hong Kong</wicri:noRegion></affiliation></author><author><name sortKey="To, Amanda P C" sort="To, Amanda P C" uniqKey="To A" first="Amanda P C" last="To">Amanda P C. To</name></author><author><name sortKey="Ng, Louisa W Y" sort="Ng, Louisa W Y" uniqKey="Ng L" first="Louisa W Y" last="Ng">Louisa W Y. Ng</name></author><author><name sortKey="Tsui, Wayne H W" sort="Tsui, Wayne H W" uniqKey="Tsui W" first="Wayne H W" last="Tsui">Wayne H W. Tsui</name></author><author><name sortKey="Lee, Terri S W" sort="Lee, Terri S W" uniqKey="Lee T" first="Terri S W" last="Lee">Terri S W. Lee</name></author><author><name sortKey="Tsoi, Hoi Wah" sort="Tsoi, Hoi Wah" uniqKey="Tsoi H" first="Hoi-Wah" last="Tsoi">Hoi-Wah Tsoi</name></author><author><name sortKey="Yuen, Kwok Yung" sort="Yuen, Kwok Yung" uniqKey="Yuen K" first="Kwok-Yung" last="Yuen">Kwok-Yung Yuen</name></author></analytic><series><title level="j">FEBS letters</title><idno type="ISSN">0014-5793</idno><imprint><date when="2004" type="published">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Sequence</term><term>Chromatography, High Pressure Liquid</term><term>Cloning, Molecular</term><term>Cysteine Endopeptidases</term><term>DNA Primers</term><term>Endopeptidases (metabolism)</term><term>Escherichia coli (enzymology)</term><term>Escherichia coli (genetics)</term><term>Kinetics</term><term>Recombinant Proteins (antagonists & inhibitors)</term><term>Recombinant Proteins (metabolism)</term><term>SARS Virus (enzymology)</term><term>Spectrometry, Fluorescence (methods)</term><term>Viral Proteins (antagonists & inhibitors)</term><term>Viral Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Amorces ADN</term><term>Chromatographie en phase liquide à haute performance</term><term>Cinétique</term><term>Clonage moléculaire</term><term>Cysteine endopeptidases</term><term>Endopeptidases (métabolisme)</term><term>Escherichia coli (enzymologie)</term><term>Escherichia coli (génétique)</term><term>Protéines recombinantes (antagonistes et inhibiteurs)</term><term>Protéines recombinantes (métabolisme)</term><term>Protéines virales (antagonistes et inhibiteurs)</term><term>Protéines virales (métabolisme)</term><term>Spectrométrie de fluorescence ()</term><term>Séquence nucléotidique</term><term>Virus du SRAS (enzymologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="antagonists & inhibitors" xml:lang="en"><term>Recombinant Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Endopeptidases</term><term>Recombinant Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Cysteine Endopeptidases</term><term>DNA Primers</term></keywords><keywords scheme="MESH" qualifier="antagonistes et inhibiteurs" xml:lang="fr"><term>Protéines recombinantes</term><term>Protéines virales</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Escherichia coli</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Escherichia coli</term><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Spectrometry, Fluorescence</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Endopeptidases</term><term>Protéines recombinantes</term><term>Protéines virales</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Base Sequence</term><term>Chromatography, High Pressure Liquid</term><term>Cloning, Molecular</term><term>Kinetics</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Amorces ADN</term><term>Chromatographie en phase liquide à haute performance</term><term>Cinétique</term><term>Clonage moléculaire</term><term>Cysteine endopeptidases</term><term>Spectrométrie de fluorescence</term><term>Séquence nucléotidique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Severe acute respiratory syndrome associated coronavirus main protease (SARS-CoV Mpro) has been proposed as a prime target for anti-SARS drug development. We have cloned and overexpressed the SARS-CoV Mpro in Escherichia coli, and purified the recombinant Mpro to homogeneity. The kinetic parameters of the recombinant SARS-CoV Mpro were characterized by high performance liquid chromatography-based assay and continuous fluorescence-based assay. Two novel small molecule inhibitors of the SARS-CoV Mpro were identified by high-throughput screening using an internally quenched fluorogenic substrate. The identified inhibitors have Ki values at low microM range with comparable anti-SARS-CoV activity in cell-based assays.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><noCountry><name sortKey="Lee, Terri S W" sort="Lee, Terri S W" uniqKey="Lee T" first="Terri S W" last="Lee">Terri S W. Lee</name><name sortKey="Ng, Louisa W Y" sort="Ng, Louisa W Y" uniqKey="Ng L" first="Louisa W Y" last="Ng">Louisa W Y. Ng</name><name sortKey="To, Amanda P C" sort="To, Amanda P C" uniqKey="To A" first="Amanda P C" last="To">Amanda P C. To</name><name sortKey="Tsoi, Hoi Wah" sort="Tsoi, Hoi Wah" uniqKey="Tsoi H" first="Hoi-Wah" last="Tsoi">Hoi-Wah Tsoi</name><name sortKey="Tsui, Wayne H W" sort="Tsui, Wayne H W" uniqKey="Tsui W" first="Wayne H W" last="Tsui">Wayne H W. Tsui</name><name sortKey="Yuen, Kwok Yung" sort="Yuen, Kwok Yung" uniqKey="Yuen K" first="Kwok-Yung" last="Yuen">Kwok-Yung Yuen</name></noCountry><country name="République populaire de Chine"><noRegion><name sortKey="Kao, Richard Y" sort="Kao, Richard Y" uniqKey="Kao R" first="Richard Y" last="Kao">Richard Y. Kao</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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