A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease.
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.
Bodenreider
Christophe
C
Novartis Institute for Tropical Diseases, Chromos 138670, Singapore.
Beer
David
D
Keller
Thomas H
TH
Sonntag
Sebastian
S
Wen
Daying
D
Yap
Lijian
L
Yau
Yin Hoe
YH
Shochat
Susana Geifman
SG
Huang
Danzhi
D
Zhou
Ting
T
Caflisch
Amedeo
A
Su
Xun-Cheng
XC
Ozawa
Kiyoshi
K
Otting
Gottfried
G
Vasudevan
Subhash G
SG
Lescar
Julien
J
Lim
Siew Pheng
SP
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*
D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D010447:Peptide Hydrolases