Allosteric Agonists of the Human D1 Dopamine Receptor: qHTS
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 D1 DAR subtype whereas all FDA-approved antipsychotic agents are antagonists of this receptor. Most drugs targeting the D1 DAR are problematic, however, either being less efficacious as more ..
BioActive Compounds: 3713
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: NS064831-01
Assay Submitter (PI): David Sibley
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 D1 DAR subtype whereas all FDA-approved antipsychotic agents are antagonists of this receptor. Most drugs targeting the D1 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 D1 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 primary screening assay measures compound agonism by tracking calcium flux in a force-coupled, inducible Hek293 Trex D1 cell line.
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 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 and is considered to be the agonist portion of the assay (eg, compounds which activate a positive calcium response during this portion are flagged as potential agonists). Following the 180 second read, 1 ul of either an EC20 (positive modulation assay) or EC80 (negative modulation assay) is delivered by an onboard pipette head. Measurements are taken kinetically for an additional 180 seconds.
For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds with more than thirty percent activity, activity score was given as 50.
** Test Concentration.
Data Table (Concise)