HTS Assay for Allosteric Antagonists of the Human D2 Dopamine Receptor: SAR in Primary Assay.
Dopamine receptors (DARs) are involved in the etiology and/or therapy of a number of neuropsychiatric and endocrine disorders. For instance, all receptor-based antiparkinsonian drugs work via stimulating the D2 DAR subtype whereas all FDA-approved antipsychotic agents are antagonists of this receptor. Most drugs targeting the D2 DAR are problematic, however, either being less efficacious as more ..
BioActive Compounds: 46
Depositor Specified Assays
Dopamine receptors (DARs) are involved in the etiology and/or therapy of a number of neuropsychiatric and endocrine disorders. For instance, all receptor-based antiparkinsonian drugs work via stimulating the D2 DAR subtype whereas all FDA-approved antipsychotic agents are antagonists of this receptor. Most drugs targeting the D2 DAR are problematic, however, either being less efficacious as desired or possessing limiting side effects, most of which are due to reactivity at other receptors. One approach towards improved receptor specificity is to identify allosteric ligands that bind to less conserved regions of the receptor and therefore have the potential to be much more selective. The goal of this project is to use high throughput screening approaches to identify and develop novel, highly selective small molecule allosteric modulators of the D2 DAR for use as in vitro and in vivo pharmacological tools and in proof-of-concept experiments in animal models of neuropsychiatric disease. There are three different types of allosteric modulators that we are seeking, two of which will stimulate or augment receptor signaling, allosteric agonists and potentiators, while the third, allosteric antagonists, will attenuate receptor signaling. The present project is looking for compound antagonism after an EC80 addition of dopamine by tracking calcium flux in a force-coupled, inducible Hek293 Trex D2 cell line, which act through an allosteric site.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: NS064831
Assay Submitter (PI): David Sibley
Freshly passaged cells are plated in 1536 well black, clear bottom plates at a density of 4000 cells/well in 3 ul of complete media containing 1x tetracycline inducer. After an overnight incubation at 30 deg C in 5% CO2, cells are loaded with 2 ul of the no wash Calcium Assay Kit (ABD Bioquest) and incubated at room temperature for 30 to 90 minutes. Agonist read: A 10 cycle (1 second/cycle) baseline measurement is taken and then the FDSS online 1536 pintool delivers 23 nl of compound library (as well as dopamine controls) to the plate. The plate is measured for kinetically at 1 cycle/sec for 180 seconds. Following the 180 second read, 1 ul of an EC80 (negative modulation assay) is delivered by an onboard pipette head. Measurements are taken kinetically for an additional 180 seconds.
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)