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| | Assay of Phosphodiesterase 10 Inhibition In Vitro |
| Description: | Recombinant human phosphodiesterase 10A (PDE10A) purified to homogeneity from Sf9 cells overexpressing PDE10A gene (GenBank/EMBL accession number: NM_001130690) was used for inhibition tests.Inhibitory activity of the compounds towards PDE10A was tested using PDE-Glo (Promega Corporation, Madison, USA) luminescent method on 96-well plates. Test was performed for 8 concentrations of the compounds. Test compounds were dissolved in 100% DMSO and resulted solutions were diluted 5× in concentrated PDE-Glo Reaction Buffer. Eight concentrations of each tested compound were obtained by subsequent dilution. 5 μl of obtained solutions were added into the wells of 96-well plate. Next, 7.5 μl of a solution containing PDE9A enzyme diluted in 1× concentrated PDE-Glo Reaction Buffer were added into the well to obtain the final amount of 2-10 ng (depending on the activity of the enzyme batch used in the study). In order to facilitate interaction between compounds and the enzyme, plates were incubated for 1 minute at room temperature and then 9 minutes at 4° C. Reaction was initiated by addition of 12.5 μl of 2 μM cAMP solution into the well and subsequently plate was incubated at 30° C.After 40 minutes reaction was stopped by addition of 12.5 μl of PDE-Glo Termination Buffer with high concentration of a known PDE10 inhibitor. Plate content was stirred with orbital shaker at 500 RPMs for 10 minutes and then in the next step 12.5 μl of freshly prepared PDE-Glo Detection Solution were added into the well.Plate was incubated for 20 minutes at room temperature before 50 μl of Kinase Glo reagent (Promega Corporation, Madison, USA) was applied into the wells and incubation at room temperature was continued for the next 10 minutes. After incubation, luminescence intensity in wells was measured with the Victor Light (Perkin Elmer Inc.) luminometer.Percent of PDE10A inhibition by tested compounds was determined based on luminescence intensity measurements in wells containing test compounds and in control wells. Results were then fitted using a four-parameter logistic fit in GraphPad Prism 5.03 software (GraphPad Software Inc.). Negative control wells contained all above mentioned reagents except test compounds and positive control wells contained all above mentioned reagents except test compounds and the PDE10A enzyme. Each chemical compound was assayed in at least two independent runs (2×96-well plate in duplicate) with at least 3 wells of each of the controls. |
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