HTS Assay for Allosteric Antagonists of the Human D2 Dopamine Receptor: SAR in D3 Beta Arrestin Assay
There is great interest in identifying small molecule ligands for G-protein coupled receptors (GPCRs) as nearly 50% of all FDA-approved drugs target these important receptor proteins. Unfortunately, many of the ligands that are used as drugs, or as pharmacological tools, are not selective and exhibit problematic or limiting side effects due to inappropriate signaling activities. Amongst the more ..
BioActive Compounds: 12
Depositor Specified Assays
There is great interest in identifying small molecule ligands for G-protein coupled receptors (GPCRs) as nearly 50% of all FDA-approved drugs target these important receptor proteins. Unfortunately, many of the ligands that are used as drugs, or as pharmacological tools, are not selective and exhibit problematic or limiting side effects due to inappropriate signaling activities. Amongst the dopamine receptors (DARs), the D2 is arguably one of the most validated drug targets in neurology and psychiatry. For instance, all receptor-based antiparkinsonian drugs primarily work via stimulating the D2 DAR, whereas all FDA-approved antipsychotic agents are antagonists of this receptor. The D2 DAR is also therapeutically targeted in other disorders such as restless legs syndrome, tardive dyskinesia, Tourette's syndrome, and hyperprolactinemia. However, most drugs targeting the D2 DAR are problematic, either being less efficacious than desired or possessing adverse side effects due to the activation or blockade of inappropriate signaling pathways. A novel approach for attaining greater selectivity of therapeutic agents targeting the D2 DAR is needed to identify and develop ligands that exhibit functionally-selective properties.
This is an selectivity assay to test the lead compounds against the D3 receptor, using a beta arrestin assay.
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
PI Name: David Sibley, National Institutes of Neurological Disorders and Stroke
DiscoveRx D3 cells were thawed from frozen stock and seeded at 2,100 cells/ 3 uL/well media (DiscoveRx Plating Reagent 2) with MultiDrop Combi dispenser (Thermo Scientific, Logan, UT) onto white tissue culture treated 1536-well Aurora plates (Brooks Automation, Chelmsford, MA) and allowed to attach overnight at 37 deg C, 5% CO2. Next, 23 nL/well of compound solutions in DMSO were added with a pintool transfer (Kalypsis, San Diego, CA). As the positive control, 1 uL/well dopamine (EC100, 57 uM final concentration) diluted in buffer (HBSS + 10 mM HEPES + SMB + 0.1% Tween-20) was dispensed to 1 column of each plate. The rest of the plate was dispensed with buffer only, 1 uL/well as well. The cells were stimulated by compounds or dopamine control for 90 minutes at 37 deg C, 5% CO2, after which 1.5 ul/well of DiscoveRx detection reagent was added with BioRAPTR FRD dispenser. The detection reagent was prepared by mixing of Galacton Star Substrate, Emerald II solution and PathHunter buffer (supplied by the assay kit) together at 1:5:19 proportion accordingly just prior to dispensing. The plates were incubated at ambient temperature for 1 hour and then the luminescent signal was read on ViewLux plate reader (PerkinElmer, Waltham, MA).
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)