| Assay Method Information | |
| | Human D1 Receptor PAM Assay |
| Description: | The PAM activity of the compounds of the present invention may be measured essentially as described in Svensson et al., An Allosteric Potentiator of the Dopamine D1 Receptor Increases Locomotor Activity in Human D1 Knock-in Mices without Casusing Stereotypy or Tachyphylaxis. J. Pharmacol. Exp. Ther. (2017) 360:117-128.More specifically, HEK293 cells that stably express the human D1 receptor (Accession number NM_000794) are generated by gene transduction using the pBABE-bleo retroviral vector and selected with Zeocin (InvivoGen). At approximately 80% confluency, the cells are harvested using TrypLE Express (Gibco), suspended in FBS plus 8% DMSO, and stored in liquid nitrogen. On the day of the assay, cells are thawed and resuspended in STIM buffer (Hanks Balanced Salt Solution supplemented with 0.1% BSA, 20 mM HEPES, 500 μM IBMX, and 100 μM ascorbic acid).Test compound is serially diluted (1:2) with DMSO into assay plates (ProxiPlate-384 Plus, PerkinElmer) using acoustic dispensing (Labcyte) to provide 20 concentrations for full response curves. Test compound (80 nL) is added to 5 μL STIM buffer containing 2000 cells, and 5 μL of a 2× concentration dopamine solution in STIM buffer that will generate an EC20 level response (24 nM in stock solution, or 12 nM final) and a final DMSO concentration in the well of 0.8%. Plates are incubated at room temperature for a total reaction time of 60 min.cAMP production is quantified using HTRF detection (Cisbio) according to the manufacturer's instructions. Generally, lysis buffer containing anti-cAMP cryptate (5 μL) and D2-conjugate (from HTRF kit) (5 μL) is added to the wells, plates are incubated for an additional 60-90 min, and the time-resolved fluorescence is detected using an EnVision plate reader (PerkinElmer). Fluorescence data is converted to cAMP concentrations using a cAMP standard curve and analyzing using a 4-parameter non-linear logistic equation (Genedata Screener, version 13.0.5-standard). For potentiator mode concentration-response curves, results are expressed as percent of the window between a response at EC20 concentration of dopamine alone (normalized to 0%) and the maximum response to dopamine (defined by response to 5 μM dopamine, final concentration, normalized as 100%). |
| Affinity data for this assay | |
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