Aims: This ongoing PhD project aims to identify and characterise potential preclinical G protein-coupled receptor heteromer candidates as therapeutic targets for chronic inflammation and fibrosis. Chronic inflammation and organ fibrosis underpins much of the morbidity of many common diseases, such as chronic-obstructive pulmonary disease, diabetes, stroke, cirrhosis, heart disease and chronic kidney disease. These diseases cause 18% of all mortality worldwide (1). There is clearly an unmet need in controlling the pathophysiological inflammatory axes of these diseases effectively. G protein-coupled receptors (GPCRs) are prominent targets of existing therapeutics, owing to their cell membrane expression, and profound potential for modulating physiology. Some GPCRs are shown to influence the function of other GPCRs through heteromerisation, presenting new opportunities for pharmacological intervention.
Methods: Our objective is to identify novel GPCR heteromers with Receptor-Heteromer Identification Technology (HIT) and profile their unique pharmacology using a suite of bioluminescence resonance energy transfer (BRET)-based biosensors. Receptor-HIT is a proprietary assay format to identify receptor-receptor proximity. The β-arrestin2 recruitment BRET HIT assay was conducted to screen combinations of GPCRs with relevance to chronic inflammation and fibrosis. From this screen, heteromer candidates proceeded toward pharmacological characterisation using BRET-based biosensors. These include sensors for GPCR intracellular trafficking, G protein activation and second-messenger generation.
Results: The heteromer candidate identified demonstrate unique pharmacology dependent on protomer co-stimulation with endogenous agonists. An asymmetrical perturbance to internalisation was observed upon co-stimulation of protomers with the BRET trafficking sensor. The G protein activation BRET sensor reveals a similar effect on upon co-stimulation, with this effect extending to a complimentary second-messenger BRET sensor.
Conclusions: This project has identified a novel candidate with pharmacology consistent with the criteria to classify it as a putative GPCR heteromer. This candidate will undergo further scientific and commercial validation.