22-thiocyanatosalvinorin-a and 3-(2-((cyclobutylmethyl)(phenethyl)amino)ethyl)phenol

A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Anticonvulsants; Arrestins; Behavior, Animal; Brain; Cell Line, Tumor; Diterpenes, Clerodane; High-Throughput Screening Assays; Humans; Ligands; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenethylamines; Phosphoproteins; Phosphoric Monoester Hydrolases; Proteomics; Receptors, Opioid, kappa; Signal Transduction; TOR Serine-Threonine Kinases