In vivo veritas, the next frontier for functionally selective GPCR ligands

• We present general guidelines for the validation of biased GPCR ligands in animal models.
• Seven criteria for the validation of biased ligands in vivo are proposed.
• Detailed protocol for the evaluation of the effects of biased GPCR ligands on protein phosphorylation in vivo is provided.

The realization that G-protein coupled receptors (GPCR) engage several cell signaling mechanisms simultaneously has led to a multiplication of research aimed at developing biased ligands exerting a selective action on subsets of responses downstream of a given receptor. Several tools have been developed to identify such ligands using recombinant cell systems. However the validation of biased ligand activity in animal models remains a serious challenge. Here we present a general strategy that can be used to validate biased ligand activity in vivo and supports it as a strategy for further drug development. In doing so, we placed special attention on strategies allowing to discriminate between G-protein and beta-arrestin mediated mechanisms. We also underscore differences between in vitro and in vivo systems and suggest avenues for tool development to streamline the translation of biased ligands development to pre-clinical animal models.