A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease
| Autor: | Lijian Yap, Julien Lescar, Xun-Cheng Su, Danzhi Huang, Subhash G. Vasudevan, Siew Pheng Lim, Amedeo Caflisch, Gottfried Otting, Kiyoshi Ozawa, David Beer, Thomas H. Keller, Yin Hoe Yau, Daying Wen, Christophe Bodenreider, Sebastian Sonntag, Ting Zhou, Susana Geifman Shochat |
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| Přispěvatelé: | University of Zurich, Lim, S P |
| Rok vydání: | 2009 |
| Předmět: |
1303 Biochemistry
medicine.medical_treatment Biophysics Dengue virus Calorimetry medicine.disease_cause Biochemistry 1307 Cell Biology Structure-Activity Relationship medicine 10019 Department of Biochemistry 1312 Molecular Biology Computer Simulation Protease Inhibitors Serotyping Molecular Biology Fluorescent Dyes Protease Quenching (fluorescence) Binding Sites Chemistry Ligand binding assay Isothermal titration calorimetry Cell Biology Dengue Virus Surface Plasmon Resonance Molecular biology Protease inhibitor (biology) Enzyme binding Förster resonance energy transfer Spectrometry Fluorescence 570 Life sciences biology medicine.drug 1304 Biophysics Peptide Hydrolases |
| Zdroj: | Analytical biochemistry. 395(2) |
| ISSN: | 1096-0309 |
| Popis: | 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 IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) 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. |
| Databáze: | OpenAIRE |
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