| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | Inhibition of PDE4 is measured using a luminescence-coupled assay system developed by Cambrex. This assay system couples the formation of AMP, derived from PDE4-catalyzyed hydrolysis of cAMP, to the formation of ATP. The ATP is then used as a substrate for Luciferase and results in light as a signal output. When PDE is inhibited or inactive, no AMP is produced, the luciferase is inactive, and no light signal is produced. This assay is used in a quenched assay format, where PDE4 enzyme and cAMP substrate are added sequentially to a 384 well assay plate (Greiner784075) pre-stamped with compound at the desired concentration. The reaction is incubated at room temperature for 1 h, and then is quenched by the addition of enzyme stop solution and then the light signal is generated by the addition of detection reagent. The luminescence is then measured on a Viewlux imager (Perkin Elmer) using emission filters of 613/55 nm or 618/40 nm and a 5 s. For inhibition curves, compounds were diluted using a 3-fold serial dilution and tested at 11 concentrations. Curves were analysed as describe above using ActivityBase and XLfit , and results were expressed as pIC50 values. |
| Affinity data for this assay | |
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