Development of fluorescent peptide G protein-coupled receptor activation biosensors for NanoBRET characterization of intracellular allosteric modulators

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 G alpha 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 G alpha s subunit were first characterized for their ability to positively modulate agonist affinity at the beta(2)-adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labeled tracer was synthesized based on the 19 amino acid G alpha s peptide (TMR-G alpha s19cha18, where cha = cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-G alpha s19cha18 was recruited to Gs coupled beta(2)-adrenoceptor and EP2 receptors in an agonist-dependent manner, but not the Gi-coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-G alpha s19cha18 enabled direct assessment of the affinity of unlabeled ligands for beta(2)-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.

Automated summary

This study developed fluorescent-labeled peptides as novel binding and activation biosensors for the GPCR-IAM site, allowing the assessment of the affinity of unlabeled ligands for the receptor and providing a new method for screening IAMs. Moreover, this method can be used to investigate the efficacy of orthosteric agonists and the dynamics of receptor activation.