Aims: G protein-coupled receptors (GPCRs) are traditionally investigated as monomers; however, their signalling is not confined to a monomeric profile. A growing body of evidence suggests that GPCR heteromers, complexes formed by two different receptors, may exhibit unique signalling with potential clinical relevance. Receptor heteromerisation has been proposed to explain some cases of toxicity and loss of efficacy observed during clinical development. This additional layer of signalling may also contribute to the pathophysiological complexity of chronic inflammatory and fibrotic diseases, where intervention considering only monomers may be insufficient to achieve efficacy. This project aimed to identify and characterise novel GPCR heteromers with a strong mechanistic justification and translational potential. Due to the potential commerciality of the project, receptor and molecule identities are withheld.
Methods: Receptor-receptor proximity was assessed using Receptor-Heteromer Investigation Technology (HIT). A β-arrestin2 recruitment bioluminescence resonance energy transfer (BRET)-based Receptor-HIT assay was used to screen receptor combinations for heteromer formation. Select candidate heteromers identified in the screen underwent further pharmacological characterisation using BRET-based biosensors to investigate intracellular trafficking, G protein activation, second messenger and effector signalling.
Results: Het-3X was identified from a β-arrestin2 recruitment BRET Receptor-HIT screen and was selected for detailed investigation. Intracellular trafficking analysis demonstrated asymmetric modulation of receptor internalisation following coactivation of the component receptors. Assessment of the G protein and effector signalling pathways revealed complementary and pathway-selective effects upon Het-3X activation, including an asymmetric reduction in agonist potency between protomers. The heteromer-specific signalling of Het-3X was modified with a specific antagonist to one of the component receptors.
Conclusions: Het-3X is a novel GPCR heteromer candidate that exhibits a modified signalling profile consistent with criteria for classifying putative receptor heteromers. This finding represents a promising development in the class of receptor heteromer therapeutic targets and informs a protectable intellectual property position currently being considered.