Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control <i>in situ</i> the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H₃ receptor (H₃R). From 16 newly synthesized photoswitchable compounds, VUF14738 (<b>28</b>) and VUF14862 (<b>33</b>) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H₃R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical <i>trans</i>-/<i>cis</i> conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H₃R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H₃R signaling