Nicholas D. Holliday

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 β 2 ‐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 β 2 ‐adrenoceptor and EP 2 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 β 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.