Molecular Mechanism of the β3AR Agonist Activity of a β‐Blocker

Development of subtype‐selective drugs for G protein‐coupled receptors poses a significant challenge due to high similarity between subtypes, as exemplified by the three β‐adrenergic receptors (βARs). The β3AR agonists show promise for treating the overactive bladder or preterm birth, but their potential is hindered by off‐target activation of β1AR and β2AR. Interestingly, several β‐blockers, which are antagonists of the β1ARs and β2ARs, have been reported to exhibit agonist activity at the β3AR. However, the molecular mechanism remains elusive. Understanding the underlying mechanism should facilitate the development of β3AR agonist drugs with improved selectivity and reduced off‐target effects. In this work, we determined the structures of human β3AR in complex with the endogenous agonist epinephrine or with a synthetic β3AR agonist carazolol, which is also a high‐affinity β‐blocker. Structure comparison, mutagenesis studies and molecular dynamics simulations revealed that the differences on the flexibility of D3.32 directly contribute to carazolol’s distinct activities as an antagonist for the β2AR and an agonist for the β3AR. The process is also indirectly influenced by the extracellular loops (ECL), especially ECL1. Taken together, these results provide key guidance for development of selective β3AR agonists, paving the way for new therapeutic opportunities.