Identification of Novel Inhibitors of the SARS Coronavirus Main Protease 3CLpro

Usman Bacha, Jennifer Barrila, Adrian Velazquez-Campoy, Stephanie A. Leavitt, Ernesto Freire

Research output: Contribution to journalArticle

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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 rnicrocalorimetric 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.

Original languageEnglish (US)
Pages (from-to)4906-4912
Number of pages7
JournalBiochemistry
Volume43
Issue number17
DOIs
StatePublished - May 4 2004
Externally publishedYes

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Severe Acute Respiratory Syndrome
Coronavirus
Catalytic Domain
Peptide Hydrolases
Boronic Acids
Differential Scanning Calorimetry
Differential scanning calorimetry
Titration
Viruses
Recombinant Proteins
Scaffolds
Pharmaceutical Preparations
Serine
Escherichia coli
Molecules
Substrates
Enzymes
Proteins
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of Novel Inhibitors of the SARS Coronavirus Main Protease 3CLpro . / Bacha, Usman; Barrila, Jennifer; Velazquez-Campoy, Adrian; Leavitt, Stephanie A.; Freire, Ernesto.

In: Biochemistry, Vol. 43, No. 17, 04.05.2004, p. 4906-4912.

Research output: Contribution to journalArticle

Bacha, U, Barrila, J, Velazquez-Campoy, A, Leavitt, SA & Freire, E 2004, 'Identification of Novel Inhibitors of the SARS Coronavirus Main Protease 3CLpro ', Biochemistry, vol. 43, no. 17, pp. 4906-4912. https://doi.org/10.1021/bi0361766
Bacha, Usman ; Barrila, Jennifer ; Velazquez-Campoy, Adrian ; Leavitt, Stephanie A. ; Freire, Ernesto. / Identification of Novel Inhibitors of the SARS Coronavirus Main Protease 3CLpro In: Biochemistry. 2004 ; Vol. 43, No. 17. pp. 4906-4912.
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