A commercial fluorescence polarization assay was performed using
recombinant PDE3A and PDE3B by BPS Bioscience. Compounds were tested in
duplicate at 9 concentrations in a half-logarithmic dilution series (top
concentration of 10 μM) with final DMSO concentration of 1%. The
enzymatic reactions were conducted at room temperature for 60 minutes in a 50
μl mixture containing PDE assay buffer, 100 nM FAM-cAMP, a PDE enzyme and
the test compound. After the enzymatic reaction, 100 μl of a binding
solution (1:100 dilution of the binding agent with the binding agent diluent)
was added to each mix, and the reaction was performed at room temperature for 15
minutes. Fluorescence intensity was measured at an excitation of 485 nm and an
emission of 528 nm using a Tecan Infinite M1000 microplate reader. Fluorescence
intensity was converted to fluorescence polarization using the Tecan Magellan 6
software.
recombinant PDE3A and PDE3B by BPS Bioscience. Compounds were tested in
duplicate at 9 concentrations in a half-logarithmic dilution series (top
concentration of 10 μM) with final DMSO concentration of 1%. The
enzymatic reactions were conducted at room temperature for 60 minutes in a 50
μl mixture containing PDE assay buffer, 100 nM FAM-cAMP, a PDE enzyme and
the test compound. After the enzymatic reaction, 100 μl of a binding
solution (1:100 dilution of the binding agent with the binding agent diluent)
was added to each mix, and the reaction was performed at room temperature for 15
minutes. Fluorescence intensity was measured at an excitation of 485 nm and an
emission of 528 nm using a Tecan Infinite M1000 microplate reader. Fluorescence
intensity was converted to fluorescence polarization using the Tecan Magellan 6
software.