Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate.
Identifieur interne : 000816 ( Ncbi/Curation ); précédent : 000815; suivant : 000817Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate.
Auteurs : Chih-Jung Kuo [République populaire de Chine] ; Ya-Hui Chi ; John T-A Hsu ; Po-Huang LiangSource :
- Biochemical and biophysical research communications [ 0006-291X ] ; 2004.
Descripteurs français
- KwdFr :
- Cinétique, Colorants fluorescents (métabolisme), Dimérisation, Endopeptidases (), Endopeptidases (génétique), Endopeptidases (métabolisme), Escherichia coli (génétique), Escherichia coli (métabolisme), Inhibiteurs de protéases (pharmacologie), N,N-Diméthyl-4-phényldiazényl-aniline (analogues et dérivés), N,N-Diméthyl-4-phényldiazényl-aniline (métabolisme), Naphtalènesulfonates (métabolisme), Oligopeptides (métabolisme), Protéines recombinantes (), Protéines recombinantes (antagonistes et inhibiteurs), Protéines recombinantes (génétique), Protéines recombinantes (métabolisme), Protéines virales (), Protéines virales (antagonistes et inhibiteurs), Protéines virales (génétique), Protéines virales (métabolisme), Spécificité du substrat, Séquence d'acides aminés, Transfert d'énergie par résonance de fluorescence, Virus du SRAS (enzymologie).
- MESH :
- analogues et dérivés : N,N-Diméthyl-4-phényldiazényl-aniline.
- antagonistes et inhibiteurs : Protéines recombinantes, Protéines virales.
- enzymologie : Virus du SRAS.
- génétique : Endopeptidases, Escherichia coli, Protéines recombinantes, Protéines virales.
- métabolisme : Colorants fluorescents, Endopeptidases, Escherichia coli, N,N-Diméthyl-4-phényldiazényl-aniline, Naphtalènesulfonates, Oligopeptides, Protéines recombinantes, Protéines virales.
- pharmacologie : Inhibiteurs de protéases.
- Cinétique, Dimérisation, Endopeptidases, Protéines recombinantes, Protéines virales, Spécificité du substrat, Séquence d'acides aminés, Transfert d'énergie par résonance de fluorescence.
English descriptors
- KwdEn :
- Amino Acid Sequence, Dimerization, Endopeptidases (chemistry), Endopeptidases (genetics), Endopeptidases (metabolism), Escherichia coli (genetics), Escherichia coli (metabolism), Fluorescence Resonance Energy Transfer, Fluorescent Dyes (metabolism), Kinetics, Naphthalenesulfonates (metabolism), Oligopeptides (metabolism), Protease Inhibitors (pharmacology), Recombinant Proteins (antagonists & inhibitors), Recombinant Proteins (chemistry), Recombinant Proteins (genetics), Recombinant Proteins (metabolism), SARS Virus (enzymology), Substrate Specificity, Viral Proteins (antagonists & inhibitors), Viral Proteins (chemistry), Viral Proteins (genetics), Viral Proteins (metabolism), p-Dimethylaminoazobenzene (analogs & derivatives), p-Dimethylaminoazobenzene (metabolism).
- MESH :
- chemical , analogs & derivatives : p-Dimethylaminoazobenzene.
- chemical , antagonists & inhibitors : Recombinant Proteins, Viral Proteins.
- chemical , chemistry : Endopeptidases, Recombinant Proteins, Viral Proteins.
- chemical , genetics : Endopeptidases, Recombinant Proteins, Viral Proteins.
- chemical , metabolism : Endopeptidases, Fluorescent Dyes, Naphthalenesulfonates, Oligopeptides, Recombinant Proteins, Viral Proteins, p-Dimethylaminoazobenzene.
- enzymology : SARS Virus.
- genetics : Escherichia coli.
- metabolism : Escherichia coli.
- chemical , pharmacology : Protease Inhibitors.
- Amino Acid Sequence, Dimerization, Fluorescence Resonance Energy Transfer, Kinetics, Substrate Specificity.
Abstract
SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.
DOI: 10.1016/j.bbrc.2004.04.098
PubMed: 15147951
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pubmed:15147951Le document en format XML
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<author><name sortKey="Kuo, Chih Jung" sort="Kuo, Chih Jung" uniqKey="Kuo C" first="Chih-Jung" last="Kuo">Chih-Jung Kuo</name>
<affiliation wicri:level="1"><nlm:affiliation>Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, ROC.</nlm:affiliation>
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<author><name sortKey="Chi, Ya Hui" sort="Chi, Ya Hui" uniqKey="Chi Y" first="Ya-Hui" last="Chi">Ya-Hui Chi</name>
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<author><name sortKey="Hsu, John T A" sort="Hsu, John T A" uniqKey="Hsu J" first="John T-A" last="Hsu">John T-A Hsu</name>
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<author><name sortKey="Liang, Po Huang" sort="Liang, Po Huang" uniqKey="Liang P" first="Po-Huang" last="Liang">Po-Huang Liang</name>
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<series><title level="j">Biochemical and biophysical research communications</title>
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<term>Dimerization</term>
<term>Endopeptidases (chemistry)</term>
<term>Endopeptidases (genetics)</term>
<term>Endopeptidases (metabolism)</term>
<term>Escherichia coli (genetics)</term>
<term>Escherichia coli (metabolism)</term>
<term>Fluorescence Resonance Energy Transfer</term>
<term>Fluorescent Dyes (metabolism)</term>
<term>Kinetics</term>
<term>Naphthalenesulfonates (metabolism)</term>
<term>Oligopeptides (metabolism)</term>
<term>Protease Inhibitors (pharmacology)</term>
<term>Recombinant Proteins (antagonists & inhibitors)</term>
<term>Recombinant Proteins (chemistry)</term>
<term>Recombinant Proteins (genetics)</term>
<term>Recombinant Proteins (metabolism)</term>
<term>SARS Virus (enzymology)</term>
<term>Substrate Specificity</term>
<term>Viral Proteins (antagonists & inhibitors)</term>
<term>Viral Proteins (chemistry)</term>
<term>Viral Proteins (genetics)</term>
<term>Viral Proteins (metabolism)</term>
<term>p-Dimethylaminoazobenzene (analogs & derivatives)</term>
<term>p-Dimethylaminoazobenzene (metabolism)</term>
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<keywords scheme="KwdFr" xml:lang="fr"><term>Cinétique</term>
<term>Colorants fluorescents (métabolisme)</term>
<term>Dimérisation</term>
<term>Endopeptidases ()</term>
<term>Endopeptidases (génétique)</term>
<term>Endopeptidases (métabolisme)</term>
<term>Escherichia coli (génétique)</term>
<term>Escherichia coli (métabolisme)</term>
<term>Inhibiteurs de protéases (pharmacologie)</term>
<term>N,N-Diméthyl-4-phényldiazényl-aniline (analogues et dérivés)</term>
<term>N,N-Diméthyl-4-phényldiazényl-aniline (métabolisme)</term>
<term>Naphtalènesulfonates (métabolisme)</term>
<term>Oligopeptides (métabolisme)</term>
<term>Protéines recombinantes ()</term>
<term>Protéines recombinantes (antagonistes et inhibiteurs)</term>
<term>Protéines recombinantes (génétique)</term>
<term>Protéines recombinantes (métabolisme)</term>
<term>Protéines virales ()</term>
<term>Protéines virales (antagonistes et inhibiteurs)</term>
<term>Protéines virales (génétique)</term>
<term>Protéines virales (métabolisme)</term>
<term>Spécificité du substrat</term>
<term>Séquence d'acides aminés</term>
<term>Transfert d'énergie par résonance de fluorescence</term>
<term>Virus du SRAS (enzymologie)</term>
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<keywords scheme="MESH" type="chemical" qualifier="analogs & derivatives" xml:lang="en"><term>p-Dimethylaminoazobenzene</term>
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<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="chemistry" xml:lang="en"><term>Endopeptidases</term>
<term>Recombinant Proteins</term>
<term>Viral Proteins</term>
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<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Endopeptidases</term>
<term>Recombinant Proteins</term>
<term>Viral Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Endopeptidases</term>
<term>Fluorescent Dyes</term>
<term>Naphthalenesulfonates</term>
<term>Oligopeptides</term>
<term>Recombinant Proteins</term>
<term>Viral Proteins</term>
<term>p-Dimethylaminoazobenzene</term>
</keywords>
<keywords scheme="MESH" qualifier="analogues et dérivés" xml:lang="fr"><term>N,N-Diméthyl-4-phényldiazényl-aniline</term>
</keywords>
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<term>Protéines virales</term>
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<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Endopeptidases</term>
<term>Escherichia coli</term>
<term>Protéines recombinantes</term>
<term>Protéines virales</term>
</keywords>
<keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term>
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<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Colorants fluorescents</term>
<term>Endopeptidases</term>
<term>Escherichia coli</term>
<term>N,N-Diméthyl-4-phényldiazényl-aniline</term>
<term>Naphtalènesulfonates</term>
<term>Oligopeptides</term>
<term>Protéines recombinantes</term>
<term>Protéines virales</term>
</keywords>
<keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Inhibiteurs de protéases</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Protease Inhibitors</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Dimerization</term>
<term>Fluorescence Resonance Energy Transfer</term>
<term>Kinetics</term>
<term>Substrate Specificity</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>Cinétique</term>
<term>Dimérisation</term>
<term>Endopeptidases</term>
<term>Protéines recombinantes</term>
<term>Protéines virales</term>
<term>Spécificité du substrat</term>
<term>Séquence d'acides aminés</term>
<term>Transfert d'énergie par résonance de fluorescence</term>
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<front><div type="abstract" xml:lang="en">SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.</div>
</front>
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