Identification of Novel Inhibitors of the SARS Coronavirus Main Protease 3CLpro†
Identifieur interne : 000021 ( Istex/Curation ); précédent : 000020; suivant : 000022Identification of Novel Inhibitors of the SARS Coronavirus Main Protease 3CLpro†
Auteurs : Usman Bacha [États-Unis] ; Jennifer Barrila [États-Unis] ; Adrian Velazquez-Campoy [États-Unis] ; Stephanie A. Leavitt [États-Unis] ; Ernesto Freire [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 2004.
Abstract
SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CLpro (also termed SARS-CoV 3CLpro). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CLpro as well as a truncated form containing only the catalytic domains. The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the α-helical domain. Differential scanning calorimetry indicates that the α-helical domain does not contribute to the structural stability of the catalytic domains. Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid. This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM. Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CLpro in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.
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DOI: 10.1021/bi0361766
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A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CLpro (also termed SARS-CoV 3CLpro). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CLpro as well as a truncated form containing only the catalytic domains. The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the α-helical domain. Differential scanning calorimetry indicates that the α-helical domain does not contribute to the structural stability of the catalytic domains. Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid. This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM. Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CLpro in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.</div></front></TEI></record>Pour manipuler ce document sous Unix (Dilib)
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