In Silico to In Cerebro—Development of the Orexin Receptor Antagonist “Photorexant” for Photomodulation In Vitro and in Mouse Brains

ABSTRACT In this study, we report the first photoswitchable small molecule dual ligands for the orexin receptor subtypes 1 (OX1R) and 2 (OX2R) as molecular tools for optical control of orexin signaling. The orexin system is critical for several physiological processes and has been increasingly implicated in the pathogenesis of various psychiatric disorders. We implemented a prospective structure‐based design approach, generating azobenzene‐containing derivatives of the FDA‐approved dual orexin receptor antagonist suvorexant with a computational workflow that featured a novel fragmentation and azobenzene‐fusion strategy. The derivatives were subsequently evaluated by molecular docking to ensure preservation of the characteristic binding mode of suvorexant. Top candidates were synthesized and characterized by photophysical and pharmacological methods, namely our recently developed β‐arrestin 2 and miniGα q recruitment assays for both receptor subtypes. Optimization of the photoswitchable moiety yielded the highly potent antagonist “photorexant,” exhibiting an up to 11‐fold activity difference between photoisomers. A newly optimized assay protocol for the characterization of photoswitchable GPCR antagonists demonstrated dynamic, reversible photomodulation of orexin receptors by photorexant in vitro. Electrophysiological studies in hypothalamic and locus coeruleus neurons in mice demonstrated photomodulation of its antagonistic properties in vivo, positioning photorexant as an excellent tool for further investigation of orexin signaling.