Reducing agents affect inhibitory activities of compounds: results from multiple drug targets.
Identifieur interne : 001C75 ( Main/Exploration ); précédent : 001C74; suivant : 001C76Reducing agents affect inhibitory activities of compounds: results from multiple drug targets.
Auteurs : Hyun Lee [États-Unis] ; Jaime Torres ; Lena Truong ; Rima Chaudhuri ; Anuradha Mittal ; Michael E. JohnsonSource :
- Analytical biochemistry [ 1096-0309 ] ; 2012.
Descripteurs français
- KwdFr :
- 2-Sulfanyl-éthanol (), Cinétique, Dithiothréitol (), Glutathion (), Hepacivirus (enzymologie), Inhibiteurs de protéases (), Peptide hydrolases (), Peptide hydrolases (métabolisme), Phosphines (), Protéines (antagonistes et inhibiteurs), Protéines (métabolisme), Protéines virales (antagonistes et inhibiteurs), Protéines virales (métabolisme), Protéines virales non structurales (antagonistes et inhibiteurs), Protéines virales non structurales (métabolisme), Tests de criblage à haut débit, Virus du SRAS (enzymologie).
- MESH :
- antagonistes et inhibiteurs : Protéines, Protéines virales, Protéines virales non structurales.
- enzymologie : Hepacivirus, Virus du SRAS.
- métabolisme : Peptide hydrolases, Protéines, Protéines virales, Protéines virales non structurales.
- 2-Sulfanyl-éthanol, Cinétique, Dithiothréitol, Glutathion, Inhibiteurs de protéases, Peptide hydrolases, Phosphines, Tests de criblage à haut débit.
English descriptors
- KwdEn :
- Dithiothreitol (chemistry), Glutathione (chemistry), Hepacivirus (enzymology), High-Throughput Screening Assays, Kinetics, Mercaptoethanol (chemistry), Peptide Hydrolases (chemistry), Peptide Hydrolases (metabolism), Phosphines (chemistry), Protease Inhibitors (chemistry), Proteins (antagonists & inhibitors), Proteins (metabolism), SARS Virus (enzymology), Viral Nonstructural Proteins (antagonists & inhibitors), Viral Nonstructural Proteins (metabolism), Viral Proteins (antagonists & inhibitors), Viral Proteins (metabolism).
- MESH :
- chemical , antagonists & inhibitors : Proteins, Viral Nonstructural Proteins, Viral Proteins.
- chemical , chemistry : Dithiothreitol, Glutathione, Mercaptoethanol, Peptide Hydrolases, Phosphines, Protease Inhibitors.
- enzymology : Hepacivirus, SARS Virus.
- chemical , metabolism : Peptide Hydrolases, Proteins, Viral Nonstructural Proteins, Viral Proteins.
- High-Throughput Screening Assays, Kinetics.
Abstract
High-throughput screening (HTS) of large compound libraries has become a commonly used method for the identification of drug leads, and nonphysiological reducing agents have been widely used for HTS. However, a comparison of the difference in the HTS results based on the choice of reducing agent used and potency comparisons of selected inhibitors has not been done with the physiological reducing agent reduced glutathione (GSH). Here, we compared the effects of three reducing agents-dithiothreitol (DTT), β-mercaptoethanol (β-MCE), and tris(2-carboxyethyl)phosphine (TCEP)-as well as GSH against three drug target proteins. Approximately 100,000 compounds were computationally screened for each target protein, and experimental testing of high-scoring compounds (~560 compounds) with the four reducing agents surprisingly produced many nonoverlapping hits. More importantly, we found that various reducing agents altered inhibitor potency (IC(50)) from approximately 10 μM with one reducing agent to complete loss (IC(50)>200 μM) of inhibitory activity with another reducing agent. Therefore, the choice of reducing agent in an HTS is critical because this may lead to the pursuit of falsely identified active compounds or failure to identify the true active compounds. We demonstrate the feasibility of using GSH for in vitro HTS assays with these three target enzymes.
DOI: 10.1016/j.ab.2012.01.006
PubMed: 22310499
Affiliations:
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Le document en format XML
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Johnson</name></author></analytic><series><title level="j">Analytical biochemistry</title><idno type="eISSN">1096-0309</idno><imprint><date when="2012" type="published">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Dithiothreitol (chemistry)</term><term>Glutathione (chemistry)</term><term>Hepacivirus (enzymology)</term><term>High-Throughput Screening Assays</term><term>Kinetics</term><term>Mercaptoethanol (chemistry)</term><term>Peptide Hydrolases (chemistry)</term><term>Peptide Hydrolases (metabolism)</term><term>Phosphines (chemistry)</term><term>Protease Inhibitors (chemistry)</term><term>Proteins (antagonists & inhibitors)</term><term>Proteins (metabolism)</term><term>SARS Virus (enzymology)</term><term>Viral Nonstructural Proteins (antagonists & inhibitors)</term><term>Viral Nonstructural Proteins (metabolism)</term><term>Viral Proteins (antagonists & inhibitors)</term><term>Viral Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>2-Sulfanyl-éthanol ()</term><term>Cinétique</term><term>Dithiothréitol ()</term><term>Glutathion ()</term><term>Hepacivirus (enzymologie)</term><term>Inhibiteurs de protéases ()</term><term>Peptide hydrolases ()</term><term>Peptide hydrolases (métabolisme)</term><term>Phosphines ()</term><term>Protéines (antagonistes et inhibiteurs)</term><term>Protéines (métabolisme)</term><term>Protéines virales (antagonistes et inhibiteurs)</term><term>Protéines virales (métabolisme)</term><term>Protéines virales non structurales (antagonistes et inhibiteurs)</term><term>Protéines virales non structurales (métabolisme)</term><term>Tests de criblage à haut débit</term><term>Virus du SRAS (enzymologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="antagonists & inhibitors" xml:lang="en"><term>Proteins</term><term>Viral Nonstructural Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Dithiothreitol</term><term>Glutathione</term><term>Mercaptoethanol</term><term>Peptide Hydrolases</term><term>Phosphines</term><term>Protease Inhibitors</term></keywords><keywords scheme="MESH" qualifier="antagonistes et inhibiteurs" xml:lang="fr"><term>Protéines</term><term>Protéines virales</term><term>Protéines virales non structurales</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Hepacivirus</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Hepacivirus</term><term>SARS Virus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Peptide Hydrolases</term><term>Proteins</term><term>Viral Nonstructural Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Peptide hydrolases</term><term>Protéines</term><term>Protéines virales</term><term>Protéines virales non structurales</term></keywords><keywords scheme="MESH" xml:lang="en"><term>High-Throughput Screening Assays</term><term>Kinetics</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>2-Sulfanyl-éthanol</term><term>Cinétique</term><term>Dithiothréitol</term><term>Glutathion</term><term>Inhibiteurs de protéases</term><term>Peptide hydrolases</term><term>Phosphines</term><term>Tests de criblage à haut débit</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">High-throughput screening (HTS) of large compound libraries has become a commonly used method for the identification of drug leads, and nonphysiological reducing agents have been widely used for HTS. However, a comparison of the difference in the HTS results based on the choice of reducing agent used and potency comparisons of selected inhibitors has not been done with the physiological reducing agent reduced glutathione (GSH). Here, we compared the effects of three reducing agents-dithiothreitol (DTT), β-mercaptoethanol (β-MCE), and tris(2-carboxyethyl)phosphine (TCEP)-as well as GSH against three drug target proteins. Approximately 100,000 compounds were computationally screened for each target protein, and experimental testing of high-scoring compounds (~560 compounds) with the four reducing agents surprisingly produced many nonoverlapping hits. More importantly, we found that various reducing agents altered inhibitor potency (IC(50)) from approximately 10 μM with one reducing agent to complete loss (IC(50)>200 μM) of inhibitory activity with another reducing agent. Therefore, the choice of reducing agent in an HTS is critical because this may lead to the pursuit of falsely identified active compounds or failure to identify the true active compounds. We demonstrate the feasibility of using GSH for in vitro HTS assays with these three target enzymes.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Illinois</li></region><settlement><li>Chicago</li></settlement><orgName><li>Université de l'Illinois à Chicago</li></orgName></list><tree><noCountry><name sortKey="Chaudhuri, Rima" sort="Chaudhuri, Rima" uniqKey="Chaudhuri R" first="Rima" last="Chaudhuri">Rima Chaudhuri</name><name sortKey="Johnson, Michael E" sort="Johnson, Michael E" uniqKey="Johnson M" first="Michael E" last="Johnson">Michael E. Johnson</name><name sortKey="Mittal, Anuradha" sort="Mittal, Anuradha" uniqKey="Mittal A" first="Anuradha" last="Mittal">Anuradha Mittal</name><name sortKey="Torres, Jaime" sort="Torres, Jaime" uniqKey="Torres J" first="Jaime" last="Torres">Jaime Torres</name><name sortKey="Truong, Lena" sort="Truong, Lena" uniqKey="Truong L" first="Lena" last="Truong">Lena Truong</name></noCountry><country name="États-Unis"><region name="Illinois"><name sortKey="Lee, Hyun" sort="Lee, Hyun" uniqKey="Lee H" first="Hyun" last="Lee">Hyun Lee</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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