Specializing in both standard and custom in vitro functional assays, we are focused on providing the right assays for assessing a GPCR therapeutic’s potential efficacy and liabilities to help identify candidate compounds for preclinical and clinical development. Combined with our radioligand binding assays, these functional assays can provide an integrated assessment of a drug candidate’s in vitro pharmacology profile.
In Vitro Assays And Screening List
Primary & Secondary Radioligand Binding Assays
Compounds are typically subjected to primary functional screening assay at targets (receptors) selected by investigators. In primary screening assays, compounds are tested in quadruplicate at a final concentration of 10 μM or at specific concentrations upon request for agonist and antagonist activities. Results are normalized and transformed to percentage values. For agonists, the reference agonist activity at 10 μM is set as 100% and basal activity with buffer as 0%. For antagonists, the basal activity with buffer only is set as 100% inhibition and the activity of the EC80 of the reference agonist as 0% inhibition. For allosteric potentiators, the activity of the EC20 of a reference agonist is set as 0% potentiation. Compounds with a minimum of 30% agonist activity, or 50% antagonist activity, or 30% potentiation above control are selected for secondary screening using concetnration-response assays.
In secondary functional assays, potential agonist hits are subjected to full concentration-response (8 concentrations in quadruplicate) assay to determine their efficacy and potency. Potential antagonist hits are subjected to full concentration-response (8 concentrations in quadruplicate) assay to determine IC50 values against EC50to EC80 concentrations of a reference agonist. Potential antagonist hits and positive allosteric modulators are further characterized if necessary.
Both primary and secondary functional binding assays are carried out in a final of volume of 125 μl per well in appropriate binding buffer. [35S]-GTPγS is at a concentration specific for the target receptor (unless stated otherwise). Basal and nonspecific binding are determined in the absence and presence of 10 μM of GTPγS. Agonist & Antagonist activity is determined by receptor specific compounds.In brief, plates are incubated at a specific temperature in the dark for a specific amount of time (receptor specific). Reactions are stopped by vacuum filtration onto buffer soaked 96-well Unifilters using a Filtermate harvester, followed by a wash with cold wash buffer. Scintillation cocktail is then added onto air-dried filters. Radioactivity is counted in a Microbeta counter.
Key details of our functional binding assay services:
How We Work
Compound Characterization Study Goal Determine ex vivo receptor occupancy of CMPDx at Orexin-1 (OX1) receptors using radiolabeled Orexin antagonist Created by potrace 1.10, written by Peter Selinger 2View More
Compound Characterization 1
Call Us On