A high-affinity, cis-on photoswitchable beta blocker to optically control β-adrenergic receptors in vitro and in vivo.

This study introduces (S)-Opto-prop-2, a second-generation photoswitchable ligand designed for precise modulation of β-adrenoceptor (βAR). Synthesised by incorporating an azobenzene moiety with propranolol, (S)-Opto-prop-2 exhibited a high PSS (photostationary state for cis isomer) percentage (∼90 %) and a favourable half-life (>10 days), facilitating diverse bioassay measurements. In vitro, the cis-isomer displayed substantially higher βAR binding affinity than the trans-isomer (1000-fold), making (S)-Opto-prop-2 one of the best photoswitchable GPCR (G protein-coupled receptor) ligands reported so far. Molecular docking of (S)-Opto-prop-2 in the X-ray structure of propranolol-bound βAR followed by site-directed mutagenesis studies, identified D113, N312 and F289 as crucial residues that contribute to ligand-receptor interactions at the molecular level. In vivo efficacy was assessed using a rabbit ocular hypertension model, revealing that the cis isomer mimicked propranolol's effects in reducing intraocular pressure, while the trans isomer was inactive. Dynamic optical modulation of βAR by (S)-Opto-prop-2 was demonstrated in two different cAMP bioassays and using live-cell confocal imaging, indicating reversible and dynamic control of βAR activity using the new photopharmacology tool. In conclusion, (S)-Opto-prop-2 emerges as a promising photoswitchable ligand for precise and reversible βAR modulation with light. The new tool shows superior cis-on binding affinity, one of the largest reported differences in affinity (1000-fold) between its two configurations, in vivo efficacy, and dynamic modulation. This study contributes valuable insights into the evolving field of photopharmacology, offering a potential avenue for targeted therapy in βAR-associated pathologies.