Identification of Biased Kappa Opioid Receptor Ligands For In Vivo Probing of Specific Signal Transduction Pathways

The κ opioid receptor (KOR)-dynorphin system has been implicated in the control of affect, cognition, motivation, and is thought to be dysregulated in mood and psychotic disorders, as well as in various phases of opioid dependence. KOR agonists exhibit analgesic effects but the adverse effects produced by KOR agonists, including sedation, dysphoria, and hallucinations have limited their clinical use. Interestingly, KOR-mediated dysphoria, assessed in rodents as aversion, has recently been attributed to the activation of the p38 MAPK pathway following arrestin recruitment to the activated KOR. Therefore, KOR-selective G protein biased agonists, which do not recruit arrestin, have been proposed to be more effective analgesics, without the adverse effects triggered by the arrestin pathway. As an initial step toward identifying novel biased KOR agonists, we applied a multi-faceted screening strategy utilizing both in silico and parallel screening approaches. We identified several KOR-selective ligand scaffolds with a range of signaling bias in vitro. The arylacetamide-based scaffold includes both G protein and β-arrestin-biased ligands, while the endogenous peptides and the diterpene scaffolds are G protein-biased. Interestingly, we found scaffold screening to be more successful than library screening in identifying biased ligands. Many of the identified functionally selective ligands are potent selective KOR agonists that are active in the central nervous system. Therefore, we sought to determine if G protein-biased ligands have therapeutic potential by assessing β-arrestin 2 KO mice and KOR-selective biased ligands in multiple behavioral paradigms. We found that KOR-mediated G protein signaling induces analgesia and aversion, while β-arrestin 2 signaling causes motor incoordination. Additionally, unlike unbiased KOR agonists, the G protein-biased ligand RB 64 does not induce sedation and anhedonia-like effects, suggesting that a signaling mechanism other than G protein signaling mediates these effects. These studies suggests that many of the negative side effects of KOR agonism can be alleviated with the use of KOR-selective G protein-biased ligands.Doctor of Philosoph