| Assay Method Information | |
| | Inhibition of PDE4 |
| Description: | The inhibition of PDE4 (e.g., PDE4A, PDE4C) activity by the compounds (e.g., compound-1, compound-2, compound-3, compound-4, compound-5, and compound-6) are monitored using the PDE fluorescence polarization assay kits from BPS Bioscience (San Diego, USA) following the provided procedure. This assay is based on the selective binding of the fluorescent dye FAM-labelled AMP generated by the PDEs from the FAM-cAMP to its binding beads.Briefly, compounds (2.5 ul of different concentrations diluted in the assay buffer) were mixed with 12.5 uL of the FAM-cAMP substrate (200 nM in the assay buffer) in a 384-well plate, 10 ul of the human recombinant PDE4A (BPS, Catalog #60340) or PDE4C (BPS, Catalog #60384) was added. The plate was incubated for 1 hr at room temperature. 50 ul of the binding agent for FAM-AMP was added. The mixture was incubated at room temperature for 20 min. The amount of binding agent bound FAM-AMP was measured by the fluorescence polarization method on the Envision spectrometer. The potency (IC50 value, Table1) of exemplified compounds was calculated from the dose-response curve using the 4-parameter non-linear regression fitting routine. |
| Affinity data for this assay | |
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