Discussion

Biased GPCR ligands are able to engage with their target receptor in a manner that preferentially activates distinct downstream signalling and offers potential for next generation therapeutics. However, accurate quantification of ligand bias in vitro is complex and current best practice is not amenable for testing large compound numbers. We have therefore sought to adapt ligand bias theory to an industrial scale screening campaign for the identification of new biased µ opioid receptor agonists. Assays with appropriate dynamic range were developed for both Gαi-dependent signalling and β-arrestin recruitment. Intrinsic relative activity was validated as an alternative for the operational model of agonism in calculating pathway bias. We identified 440 hits showing greater than 10-fold bias relative to DAMGO from an HTS. To validate these results, we quantified pathway bias of a subset of these using the operational model of agonism. The high degree of correlation across this subset confirmed that relative activity was a suitable method for identifying genuine biased ligands within a compound collection. This work demonstrates that using relative activity, drug discovery can apply the concept of biased ligand quantification at scale and accelerate the deliberate discovery of novel therapeutics acting via this complex pharmacology.