Development of fluorescent peptide G Protein Coupled Receptor activation biosensors for NanoBRET characterisation of intracellular allosteric modulators

Abstract

G protein coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at this class of receptors offer further potential for exploitation. In particular GPCR intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor-effector interface (e.g. G protein) and so inhibit agonist responses non-competitively. This potentially offers a tailored mode of action and greater selectivity between conserved receptor subtypes compared to classical orthosteric ligands. However, while specific examples of the IAM class of ligands are well described (particularly for chemokine receptors), a more general methodology for assessing compound interactions at the GPCR IAM site is lacking. Here fluorescent labelled peptides based on the Gα peptide C terminus are developed as novel binding and activation biosensors for the GPCR IAM binding site. In TR-FRET binding studies, unlabelled peptides derived from the GαS subunit C-terminus were first characterised for their ability to positively modulate agonist affinity at the β2-adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labelled tracer was synthesized based on the 19 amino acid C terminal 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 (correlated with ligand efficacy), but not to the Gi coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-GαS19cha18 enabled direct assessment of the affinity of unlabelled ligands for β2-adrenoceptor IAM site. Thus the NanoBRET platform using fluorescent-labelled G protein peptide mimetics offers novel potential for medium-throughput affinity screens to identify new 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.

Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2

“…This high affinity may be the result of a slow dissociation rate from the receptor. , However, to directly determine NAM affinity and binding kinetics, a molecular tool that binds competitively with unlabeled ligands to the intracellular allosteric binding site is needed, which can then be used in real-time analysis of ligand binding. High-affinity fluorescent probes are suitable for this purpose and can be employed in several GPCR binding techniques, including bioluminescence resonance energy transfer (BRET) assays. − These assays present considerable advantages over radioligand binding assays, traditionally used to characterize ligand binding. A better safety profile allows an easier performance of the assay and disposal of waste materials. , Furthermore, fluorescent ligand-based resonance energy transfer assays have the key capability to monitor specific binding in a homogeneous format, without the need to separate bound from the free tracer, and the binding from a single sample can be monitored continuously in real time, enabling kinetic analysis to be performed in a straightforward manner.…”

“…High-affinity fluorescent probes are suitable for this purpose and can be employed in several GPCR binding techniques, including bioluminescence resonance energy transfer (BRET) assays. 48 − 53 These assays present considerable advantages over radioligand binding assays, traditionally used to characterize ligand binding. A better safety profile allows an easier performance of the assay and disposal of waste materials.…”