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 agonists 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 D^3.32 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.