(1167-D) Competition assay using Fluorescence Polarization to determine the Residence Times for Calcitonin and AMYR agonist, AM833
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: There is increasing evidence that targeting the amylin receptors, may improve weight control. AM833, a lipidated peptide analogue of Amylin and a non-selective agonist of Amylin and Calcitonin G-Protein Coupled Receptors (GPCR’s), is a potential drug that could be used for this purpose. The peptide is currently undergoing phase II clinical trials for the treatment of obesity.
In this study, the residence times (1/Koff) of AM833, at the Calcitonin receptor (CTR) and Amylin receptors, AMY1R and AMY3R, were evaluated. Residence time is the time taken for 50% of a ligand to dissociate from a receptor and relies on the determination of the association and dissociation rate constants Kon and Koff for the receptor/drug interaction. One method for determination of Kon and Koff rates is a competition assay between a fluorescently labelled ligand with known Kon and Koff rates and the unlabelled ligand of interest (e.g. AM833) for a particular GPCR. To generate the signal for agonist/receptor interaction, FRET (fluorescence resonance energy transfer) and BRET (bioluminescence resonance energy transfer) techniques have been used for competition assays in microplate readers. However, these competition assays require both a labelled agonist and labelled GPCR.
Here we describe the use of fluorescence polarization (FP) to determine agonist/receptor Kon and Koff rates in a kinetic competition assay, with the advantage that labelling of the receptor is unnecessary. The results show the ability to characterize the binding kinetics of several different agonists with implications for understanding drug safety and efiicacy.