| Assay Method Information | |
| | Determination of the inhibition of the compounds on PDE4B1 activity by using cAMP HTRF assay |
| Description: | The inhibitory effect of the compounds on human PDE4B1 enzyme activity was determined by quantifying the 5′-adenosine monophosphate (5′-AMP) formed from 3′,5′-cyclic adenosine monophosphate (cAMP).The test compound or water (control) and human recombinant PDE4B1 enzyme (4.8 U) were mixed in a buffer solution (pH 7.4) consisting of 1× Hanks' balanced salt solution (HBSS), 5 mM HEPES, 3 mM MgCl2, and 0.1% BSA and incubated for 10 min. A cAMP enzyme substrate (final concentration 40 nM) was added, and the mixture was incubated at room temperature for 60 min. Then a fluorescent acceptor (Dye2 labeled with cAMP), a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) and a non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methyl xanthine; final concentration 1 mM) were added. After 60 min, the fluorescence transfer related to the amount of remaining cAMP was measured on a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity is calculated from the ratio of the signals measured at 665 nm and 620 nm. The results are expressed as percent inhibition of the enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basic control. The IC50 value (IC50=the concentration that caused the half-maximum inhibition of the specific activity of the control) is derived from dose-response measurements with eight different concentrations (n=2; repeated 2 times). |
| Affinity data for this assay | |
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