A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

TitleA fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease
Publication TypeJournal Article
Year of Publication2009
AuthorsBodenreider C., Beer D., Keller T.H, Sonntag S., Wen D., Yap L., Yau Y.H, Shochat S.G, Huang D., Zhou T., Caflisch A., Su X.-C., Ozawa K., Otting G., Vasudevan S.G, Lescar J., Lim S.P
JournalAnalytical Biochemistry
Date Published2009 Dec 15
Type of ArticleResearch Article
KeywordsBinding Sites, Calorimetry, Computer Simulation, Dengue Virus, Fluorescent Dyes, Peptide Hydrolases, Protease Inhibitors, Serotyping, Spectrometry, Fluorescence, Structure-Activity Relationship, Surface Plasmon Resonance

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC50 values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC50 values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.



Alternate JournalAnal. Biochem.
PubMed ID19682971
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