Technical Highlight - January 2013
Short description: A cell-free binding assay is optimized step by step and applied in high throughput to screen for small molecule inhibitors of an influenza protein.
Fluorescence polarization (FP) is a robust approach for probing molecular interactions. It is essentially a measure of the tumbling rate of a fluorescently labeled molecule, which increases when the labeled probe binds a large ligand. The measurements are less variable than those based on fluorescence intensity, and the assay is cell-free, rapid and nonradioactive. As such, it has been employed to identify leads in inhibitor screens. The introduction of plate readers with the capacity for high-throughput FP analyses has enabled effective application of this technique in drug discovery.
Ellington, Cho and colleagues, in collaboration with Guy Montelione (PSI NESG), have now developed a FP-based screen to identify inhibitors of nonstructural protein 1 from influenza A virus (NS1A), a key player in viral infection. The N-terminal domain of NS1A nonspecifically binds double-stranded RNA (dsRNA), including viral dsRNAs, preventing their degradation by the human immune system. First, with a fluorescein-labeled dsRNA probe, the authors titrated in a number of NS1A constructs and control proteins, settling on glutathione S-transferase-tagged NS1A for the assay, which exhibited a dose-dependent increase in FP signal. tRNA was included to reduce background, reaction temperature and incubation time were optimized, and the effect of detergent was considered.
Following assay validation, 446 small molecules from the National Institutes of Health Clinical Collection were screened in two 384-well plates, in duplicate. The data were reproducible, with large signal-to-noise ratios. Subsequent stringent hit validation left just one compound, epigallocatechine gallate. This compound, which is found in green tea extract, has been shown to have anti-flu activity in several experiments, corroborating its identification in this screen. Since a structure of the dsRNA-biding site of NS1A is available, the authors suggest structure-based design to optimize the identified inhibitor molecule. As this FP-based assay was robust and reproducible, it should prove effective in the identification of other NS1A inhibitors.
E.J. Cho et al. Identification of influenza virus inhibitors targeting NS1A utilizing fluorescence polarization-based high-throughput assay.
J. Biomol. Screen. 17, 448-459 (2012). doi:10.1177/1087057111431488