| Assay Method Information | |
| | Endonuclease Inhibition Assay |
| Description: | Compounds were dissolved and serially diluted in 100% DMSO then 0.5 μl was transferred to 384-well plates. 50 nM truncated Influenza A/victoria/75 PA(1-209) was prepared in assay buffer (20 mM Tris, pH=7.5, 10 mM MgCl2, 0.01% Tween 20, 100 mM NaCl and 1 mM DTT) and 20 ul was added to each well of assay plate with compounds, centrifuged for 1 min at 1000 rpm and incubated for 30 min at room temperature. 20 μl of 20 nM fluorescein-labeled probe [5-(4-(3-carboxy-3-oxopropanoyl)-4-(4-chlorobenzyl)piperidine-1-carbonyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid] in assay buffer was then added, centrifuged for 1 min at 1000 rpm and incubated for 60 min at room temperature.Fluorescence polarization was measured on Perkin Elmer Envision plate reader with excitation at 480 nm and emission at 535 nm and reported in millipolarization units (mP). IC50 values were determined relative to wells containing 125 uM of a compound known to bind to the active site of PA and uninhibited wells containing 1.25% DMSO. |
| Affinity data for this assay | |
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