u-50488 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

We previously reported on a series of small molecules targeting the κ-opioid (KOP) receptor featuring a diphenethylamine scaffold and showed the promise of these ligands as effective analgesics with reduced liability for adverse effects. This study expands the structure-activity relationships on our original series by presenting several modifications in the lead compounds 1 (HS665) and 2 (HS666). A library of new diphenethylamines was designed, synthesized, and pharmacologically evaluated. In comparison with 1 and 2, the KOP receptor affinity, selectivity, and agonist activity were modulated by introducing bulkier N-substituents, a 2-fluoro substitution, and additional hydroxyl groups at positions 3' and 4'. Several analogues showed subnanomolar affinity and excellent KOP receptor selectivity acting as full or partial agonists, and one as an antagonist. The new diphenethylamines displayed antinociceptive efficacies with increased potencies than U50,488, 1 and 2 in the writhing assay and without inducing motor dysfunction after sc administration in mice.

Topics: Analgesics; Animals; CHO Cells; Cricetulus; Humans; Male; Mice; Phenethylamines; Receptors, Opioid, kappa; Structure-Activity Relationship

Here we report on the design, synthesis, and biological characterization of novel κ opioid (KOP) receptor ligands of diphenethylamines. In opioid receptor binding and functional assays, the N-cyclobutylmethyl substituted derivative 4 (HS665) showed the highest affinity and selectivity for the KOP receptor and KOP agonist potency. Compound 4 inhibited acetic acid induced writhing after subcutaneous administration in mice via KOP receptor-mediated mechanisms, being equipotent as an analgesic to the KOP agonist U50,488.

Topics: Acetic Acid; Analgesics; Animals; Drug Discovery; Mice; Nociception; Pain; Phenethylamines; Receptors, Opioid, kappa