Assay Method Information

Assay Name:  Inhibition Assay
Description:  A TR-FRET-based phosphodiesterase assay kit from Molecular Devices was used to test whether these compounds were indeed direct inhibitors of the PDE4 enzyme. Using Roflumilast as a positive control, compounds of the present invention were tested against fluorescein-labeled cAMP substrate. The principle of the assay is based on the binding of a nucleotide monophosphate generated upon cAMP conversion to 5′ AMP by PDE to an IMAP binding reagent, which in turn is also ligated to a separate complex carrying terbium (Tb)-donor molecule. Proximity of the fluoreceinated 5′AMP to the Tb donor generates Fluorescence Resonance Energy Transfer. A PDE inhibitor will reduce the conversion of cAMP to 5′AMP, thus reducing monophosphate that can bind to the IMAP binding reagent and reduce the resulting FRET signal.
Affinity data for this assay
 

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