Category: Assay Development and Screening
A multiplex high throughput cAMP assay for identification of agonists, antagonists, and allosteric modulators of G protein-coupled receptors
Tina Desai1, Gregory Kabachinski2, Kevin Cusack2, Dawn George2, Michael Hoemann2, Christopher Harris2, Nathaniel Elsen1,Sujatha Gopalakrishnan1
G protein-coupled receptors (GPCRs) are highly druggable membrane-bound signaling proteins. Traditionally, these targets are pursued by direct agonism or antagonism at the orthosteric site. While this has worked for many targets, many efforts to develop selective ligands have failed due to the conservation of the orthosteric binding site across GPCR classes. Therefore, allosteric modulation of GPCRs is an alternative approach for enhancing traditional GPCR drug discovery. An allosteric modulator stabilizes a particular GPCR confirmation and promotes an active or inactive state. Since allosteric modulators do not bind to the orthosteric site, they provide additional opportunities for selectivity against closely related receptors. Additionally, there have been examples reported of interconverting GPCR agonists and antagonists, where a small change in chemical structure significantly alter the functional activity at the receptor. This approach is also a method to identify novel ligands for GPCR discovery. Here, we describe a high throughput cAMP screen to identify agonists, antagonists and allosteric modulators for a GPCR target in a single assay, with target agonism being the desired functional outcome. Using a cAMP assay at EC20 of the natural ligand, we were able to separate the hits into different ligand classes showing distinct profile. The hits were further characterized into biased agonists based on whether they selectively confer activity in G protein activation vs beta arrestin recruitment assays. This multiplexed screening approach not only saved cost, but also saved time, as a single screen of the compound library was able to identify novel agonists, partial agonists, antagonists, positive allosteric modulators (PAMs) and ago-PAMS.
1AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064
2AbbVie Bioresearch Center, 100 Research Drive, Worcester, MA 01605
Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Tina Desai– Scientist , AbbVie, North Chicago, IL