title: A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease.
abstract: 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.
authors.affiliation: Novartis Institute for Tropical Diseases, Chromos 138670, Singapore.
authors.forename: Christophe
authors.initials: C
authors.lastname: Bodenreider
authors.forename: David
authors.initials: D
authors.lastname: Beer
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Keller
authors.forename: Sebastian
authors.initials: S
authors.lastname: Sonntag
authors.forename: Daying
authors.initials: D
authors.lastname: Wen
authors.forename: Lijian
authors.initials: L
authors.lastname: Yap
authors.forename: Yin Hoe
authors.initials: YH
authors.lastname: Yau
authors.forename: Susana Geifman
authors.initials: SG
authors.lastname: Shochat
authors.forename: Danzhi
authors.initials: D
authors.lastname: Huang
authors.forename: Ting
authors.initials: T
authors.lastname: Zhou
authors.forename: Amedeo
authors.initials: A
authors.lastname: Caflisch
authors.forename: Xun-Cheng
authors.initials: XC
authors.lastname: Su
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ozawa
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
authors.forename: Subhash G
authors.initials: SG
authors.lastname: Vasudevan
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
mesh_terms: D001665:Binding Sites; D002151:Calorimetry / Q000379:methods*; D003198:Computer Simulation; D003716:Dengue Virus / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D012703:Serotyping; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D010447:Peptide Hydrolases