| Assay Method Information | |
| | In Vitro Pharmacology Assay |
| Description: | In one embodiment, the compounds provided herein were assayed for their ability to inhibit human PDE-10A. In one embodiment, the activities of the compounds were determined using the Molecular Devices IMAP PDE Fluorescence Polarization assay using recombinant human PDE-10 enzyme expressed in a baculoviral system. Briefly, 10 μL of a compound (0.2 nM-20 μM) was added to either a 96-well half area black plate or a 384-well black plate along with 10 μL of Fluorescein-labeled cAMP/cGMP substrate as per manufacturer's instructions and 10 μL of PDE enzyme (activity 0.1 U). Following a 40-minute incubation at 37° C., 60 μL of IMAP binding reagent was added. The plate was then read on a Perkin Elmer Victor (480-535 nm). The data was analyzed using Prism Software (GraphPad Inc, San Diego, Calif.).In one embodiment, the compounds provided herein were run through a whole cell PDE-10 assay to assess their abilities to elevate intracellular concentrations of cAMP after PDE-10 blockade. Briefly, intracellular cGMP levels in HEK293 cells over-expressing PDE-10A were measured in a cell-based assay. Cells were plated into 96-well plates at a density of 100,000 cells per well and incubated at 37° C. overnight. The following day, cells were treated with a compound provided herein in fresh culture medium for 30 minutes. Sodium nitroprusside was then added from a 5× stock to a final concentration of 200 μM and the cells were incubated for 2 minutes exactly. The reaction was then stopped by addition of 200 μL Lysis Reagent A (GE Healthcare) and the intracellular cGMP concentration was determined using a cGMP EIA kit (GE Healthcare) according to the manufacturer's instructions. |
| Affinity data for this assay | |
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