Evaluating the 3C-like protease activity of SARS-Coronavirus: recommendations for standardized assays for drug discovery.
Identifieur interne : 003115 ( Main/Exploration ); précédent : 003114; suivant : 003116Evaluating the 3C-like protease activity of SARS-Coronavirus: recommendations for standardized assays for drug discovery.
Auteurs : Valerie Grum-Tokars [États-Unis] ; Kiira Ratia ; Adrian Begaye ; Susan C. Baker ; Andrew D. MesecarSource :
- Virus research [ 0168-1702 ] ; 2008.
Descripteurs français
- KwdFr :
- Antienzymes (pharmacologie), Cinétique, Colorants fluorescents (métabolisme), Cristallisation, Cysteine endopeptidases (analyse), Cysteine endopeptidases (génétique), Cysteine endopeptidases (métabolisme), Histidine (génétique), Histidine (métabolisme), Humains, Oligopeptides (génétique), Oligopeptides (métabolisme), Protéines de fusion recombinantes (analyse), Protéines de fusion recombinantes (antagonistes et inhibiteurs), Protéines de fusion recombinantes (génétique), Protéines de fusion recombinantes (métabolisme), Spécificité du substrat, Séquence d'acides aminés, Transfert d'énergie par résonance de fluorescence, Virus du SRAS (), Virus du SRAS (enzymologie), Évaluation préclinique de médicament.
- MESH :
- analyse : Cysteine endopeptidases, Protéines de fusion recombinantes.
- antagonistes et inhibiteurs : Protéines de fusion recombinantes.
- enzymologie : Virus du SRAS.
- génétique : Cysteine endopeptidases, Histidine, Oligopeptides, Protéines de fusion recombinantes.
- métabolisme : Colorants fluorescents, Cysteine endopeptidases, Histidine, Oligopeptides, Protéines de fusion recombinantes.
- pharmacologie : Antienzymes.
- Cinétique, Cristallisation, Humains, Spécificité du substrat, Séquence d'acides aminés, Transfert d'énergie par résonance de fluorescence, Virus du SRAS, Évaluation préclinique de médicament.
English descriptors
- KwdEn :
- Amino Acid Sequence, Crystallization, Cysteine Endopeptidases (analysis), Cysteine Endopeptidases (genetics), Cysteine Endopeptidases (metabolism), Drug Evaluation, Preclinical, Enzyme Inhibitors (pharmacology), Fluorescence Resonance Energy Transfer, Fluorescent Dyes (metabolism), Histidine (genetics), Histidine (metabolism), Humans, Kinetics, Oligopeptides (genetics), Oligopeptides (metabolism), Recombinant Fusion Proteins (analysis), Recombinant Fusion Proteins (antagonists & inhibitors), Recombinant Fusion Proteins (genetics), Recombinant Fusion Proteins (metabolism), SARS Virus (drug effects), SARS Virus (enzymology), Substrate Specificity.
- MESH :
- chemical , analysis : Cysteine Endopeptidases, Recombinant Fusion Proteins.
- chemical , antagonists & inhibitors : Recombinant Fusion Proteins.
- chemical , genetics : Cysteine Endopeptidases, Histidine, Oligopeptides, Recombinant Fusion Proteins.
- chemical , metabolism : Cysteine Endopeptidases, Fluorescent Dyes, Histidine, Oligopeptides, Recombinant Fusion Proteins.
- chemical , pharmacology : Enzyme Inhibitors.
- drug effects : SARS Virus.
- enzymology : SARS Virus.
- Amino Acid Sequence, Crystallization, Drug Evaluation, Preclinical, Fluorescence Resonance Energy Transfer, Humans, Kinetics, Substrate Specificity.
Abstract
Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. One potential target for SARS-CoV antiviral drug development is the 3C-like protease (3CLpro). This enzyme is an attractive target since it is essential for viral replication, and since there are now a number of high resolution X-ray structures of SARS-CoV 3CLpro available making structure-based drug-design possible. As a result, SARS-CoV 3CLpro has become the focus of numerous drug discovery efforts worldwide, but as a consequence, a variety of different 3CLpro expression constructs and kinetic assays have been independently developed making evaluation and comparison between potential inhibitors problematic. Here, we review the literature focusing on different SARS-CoV 3CLpro expression constructs and assays used to measure enzymatic activity. Moreover, we provide experimental evidence showing that the activity of 3CLpro enzymatic is significantly reduced when non-native sequences or affinity-tags are added to the N- or C-termini of the enzyme, or when the enzyme used in assays is at concentrations below the equilibrium dissociation constant of the 3CLpro dimer. We demonstrate for the first time the utility of a highly sensitive and novel Alexa488-QSY7 FRET-based peptide substrate designed for routine analysis and high-throughput screening, and show that kinetic constants determined from FRET-based assays that are uncorrected for inner-filter effects can lead to artifacts. Finally, we evaluated the effects of common assay components including DTT, NaCl, EDTA and DMSO on enzymatic activity, and we recommend standardized assay conditions and constructs for routine SARS-CoV 3CLpro assays to facilitate direct comparisons between SARS-CoV 3CLpro inhibitors under development worldwide.
DOI: 10.1016/j.virusres.2007.02.015
PubMed: 17397958
Affiliations:
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Le document en format XML
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Proteins (genetics)</term><term>Recombinant Fusion Proteins (metabolism)</term><term>SARS Virus (drug effects)</term><term>SARS Virus (enzymology)</term><term>Substrate Specificity</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Antienzymes (pharmacologie)</term><term>Cinétique</term><term>Colorants fluorescents (métabolisme)</term><term>Cristallisation</term><term>Cysteine endopeptidases (analyse)</term><term>Cysteine endopeptidases (génétique)</term><term>Cysteine endopeptidases (métabolisme)</term><term>Histidine (génétique)</term><term>Histidine (métabolisme)</term><term>Humains</term><term>Oligopeptides (génétique)</term><term>Oligopeptides (métabolisme)</term><term>Protéines de fusion recombinantes (analyse)</term><term>Protéines de fusion recombinantes (antagonistes et inhibiteurs)</term><term>Protéines de fusion recombinantes (génétique)</term><term>Protéines de fusion recombinantes (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 ()</term><term>Virus du SRAS (enzymologie)</term><term>Évaluation préclinique de médicament</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Recombinant Fusion Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="antagonists & inhibitors" xml:lang="en"><term>Recombinant Fusion Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Histidine</term><term>Oligopeptides</term><term>Recombinant Fusion Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Fluorescent Dyes</term><term>Histidine</term><term>Oligopeptides</term><term>Recombinant Fusion Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Enzyme Inhibitors</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>Cysteine endopeptidases</term><term>Protéines de fusion recombinantes</term></keywords><keywords scheme="MESH" qualifier="antagonistes et inhibiteurs" xml:lang="fr"><term>Protéines de fusion recombinantes</term></keywords><keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Cysteine endopeptidases</term><term>Histidine</term><term>Oligopeptides</term><term>Protéines de fusion recombinantes</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Colorants fluorescents</term><term>Cysteine endopeptidases</term><term>Histidine</term><term>Oligopeptides</term><term>Protéines de fusion recombinantes</term></keywords><keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Antienzymes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Crystallization</term><term>Drug Evaluation, Preclinical</term><term>Fluorescence Resonance Energy Transfer</term><term>Humans</term><term>Kinetics</term><term>Substrate Specificity</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cinétique</term><term>Cristallisation</term><term>Humains</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</term><term>Évaluation préclinique de médicament</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. One potential target for SARS-CoV antiviral drug development is the 3C-like protease (3CLpro). This enzyme is an attractive target since it is essential for viral replication, and since there are now a number of high resolution X-ray structures of SARS-CoV 3CLpro available making structure-based drug-design possible. As a result, SARS-CoV 3CLpro has become the focus of numerous drug discovery efforts worldwide, but as a consequence, a variety of different 3CLpro expression constructs and kinetic assays have been independently developed making evaluation and comparison between potential inhibitors problematic. Here, we review the literature focusing on different SARS-CoV 3CLpro expression constructs and assays used to measure enzymatic activity. Moreover, we provide experimental evidence showing that the activity of 3CLpro enzymatic is significantly reduced when non-native sequences or affinity-tags are added to the N- or C-termini of the enzyme, or when the enzyme used in assays is at concentrations below the equilibrium dissociation constant of the 3CLpro dimer. We demonstrate for the first time the utility of a highly sensitive and novel Alexa488-QSY7 FRET-based peptide substrate designed for routine analysis and high-throughput screening, and show that kinetic constants determined from FRET-based assays that are uncorrected for inner-filter effects can lead to artifacts. Finally, we evaluated the effects of common assay components including DTT, NaCl, EDTA and DMSO on enzymatic activity, and we recommend standardized assay conditions and constructs for routine SARS-CoV 3CLpro assays to facilitate direct comparisons between SARS-CoV 3CLpro inhibitors under development worldwide.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Illinois</li></region></list><tree><noCountry><name sortKey="Baker, Susan C" sort="Baker, Susan C" uniqKey="Baker S" first="Susan C" last="Baker">Susan C. Baker</name><name sortKey="Begaye, Adrian" sort="Begaye, Adrian" uniqKey="Begaye A" first="Adrian" last="Begaye">Adrian Begaye</name><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D" last="Mesecar">Andrew D. Mesecar</name><name sortKey="Ratia, Kiira" sort="Ratia, Kiira" uniqKey="Ratia K" first="Kiira" last="Ratia">Kiira Ratia</name></noCountry><country name="États-Unis"><region name="Illinois"><name sortKey="Grum Tokars, Valerie" sort="Grum Tokars, Valerie" uniqKey="Grum Tokars V" first="Valerie" last="Grum-Tokars">Valerie Grum-Tokars</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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