James P. Farmer
Development of fluorescent peptide G protein-coupled receptor activation biosensors for NanoBRET characterization of intracellular allosteric modulators
Farmer, James P.; Mistry, Shailesh N.; Laughton, Charles A.; Holliday, Nicholas D.
Authors
Dr SHAILESH MISTRY Shailesh.Mistry@nottingham.ac.uk
Associate Professor
CHARLES LAUGHTON CHARLES.LAUGHTON@NOTTINGHAM.AC.UK
Professor of Computational Pharmaceutical Science
DR NICHOLAS HOLLIDAY nicholas.holliday@nottingham.ac.uk
Associate Professor
Abstract
G protein-coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at these receptors offer further potential for exploitation. Intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor-effector interface (e.g., G protein) and inhibit agonist responses noncompetitively. This potentially offers greater selectivity between receptor subtypes compared to classical orthosteric ligands. However, while examples of IAM ligands are well described, a more general methodology for assessing compound interactions at the IAM site is lacking. Here, fluorescent labeled peptides based on the Gα peptide C terminus are developed as novel binding and activation biosensors for the GPCR-IAM site. In TR-FRET binding studies, unlabeled peptides derived from the Gαs subunit were first characterized for their ability to positively modulate agonist affinity at the β2 -adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labeled tracer was synthesized based on the 19 amino acid Gαs peptide (TMR-Gαs19cha18, where cha=cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-Gαs19cha18 was recruited to Gs coupled β2 -adrenoceptor and EP2 receptors in an agonist-dependent manner, but not the Gi-coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-Gαs19cha18 enabled direct assessment of the affinity of unlabeled ligands for β2 -adrenoceptor IAM site. Thus, the NanoBRET platform using fluorescent-labeled G protein peptide mimetics offers novel potential for medium-throughput screens to identify IAMs, applicable across GPCRs coupled to a G protein class. Using the same platform, Gs peptide biosensors also represent useful tools to probe orthosteric agonist efficacy and the dynamics of receptor activation.
Journal Article Type | Article |
---|---|
Acceptance Date | Sep 19, 2022 |
Online Publication Date | Oct 2, 2022 |
Publication Date | Nov 1, 2022 |
Deposit Date | Oct 3, 2022 |
Publicly Available Date | Oct 5, 2022 |
Journal | FASEB journal : official publication of the Federation of American Societies for Experimental Biology |
Print ISSN | 0892-6638 |
Electronic ISSN | 1530-6860 |
Publisher | Wiley |
Peer Reviewed | Peer Reviewed |
Volume | 36 |
Issue | 11 |
Article Number | e22576 |
DOI | https://doi.org/10.1096/fj.202201024R |
Keywords | RESEARCH ARTICLE, RESEARCH ARTICLES, β2‐Adrenoceptor, Allosteric modulators, Biosensors, CXCR2, GPCRs, Prostanoid receptor EP2 |
Public URL | https://nottingham-repository.worktribe.com/output/12024311 |
Publisher URL | https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202201024R |
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https://creativecommons.org/licenses/by-nc-nd/4.0/
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