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Journal of Biomolecular Screening
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Optimization of Assay Conditions fo r Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents

Christian Steuer

Institut für Pharmazie und Molekulare Biotechnologie, Universität Heidelberg, Heidelberg, Germany

Karl H. Heinonen

Institut für Pharmazie und Molekulare Biotechnologie, Universität Heidelberg, Heidelberg, Germany

Lars Kattner

Endotherm Life Science Molecules, Saarbrücken, Germany

Christian D. Klein

Institut für Pharmazie und Molekulare Biotechnologie, Universität Heidelberg, Heidelberg, Germany, c.klein{at}uni-heidelberg.de

The aim of this work was to perform a systematic study of the effect of nonionic detergents on the activity of the dengue virus NS2B-NS3 protease. To ensure a high activity of the protease, the assay procedures for the dengue virus and other flaviviral proteases published to date are performed in the presence of up to 35% glycerol, which does not represent the cellular physicochemical environment. In addition, the high viscosity of glycerol-containing solutions leads to various experimental problems in miniaturized assays. Using an internally quenched peptide substrate, the authors show that glycerol is not essential for enzymatic activity if certain nonionic detergents are added to the assay buffer. In addition, nonionic detergents may help to avoid false-positive screening results caused by "promiscuous" inhibitors. Other polyalcohols can substitute glycerol and have less effect on the viscosity of the assay buffer. The assay was used to screen a compound library and allowed the identification of small-molecular nonpeptidic inhibitors of dengue NS3 protease. Finally, the authors discuss the mode of action of nonionic detergents and the influence that they may have on the conformational properties of the NS2B-NS3 protease. (Journal of Biomolecular Screening 2009:1102-1108)

Key Words: dengue virus • protease • high-throughput screening • promiscuous inhibitors • nonionic detergents

This version was published on October 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 9, 1102-1108 (2009)
DOI: 10.1177/1087057109344115
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