Exoticin as a selective agonist of 6TM μ opioid receptors identifies endogenous chaperones essential for its activity

Background: Classical opioids are effective analgesics but carry various side effects, necessitating safer alternatives. Truncated six-transmembrane mu opioid receptors (6TM-mu ORs) mediate potent analgesia with fewer side effects and are a promising therapeutic target. However, few ligands known selectively target 6TM-mu ORs. Moreover, endogenous chaperones are believed essential for 6TM-mu OR ligand binding and function. Purpose: To identify a 6TM-mu OR selective agonist and elucidate requisite endogenous chaperones. Methods: Virtual screening was used to identify promising selective 6TM-mu OR agonists from traditional Chinese medicines. The role of 6TM-mu OR in Exoticin analgesia was validated in loss- and gain-of-function models. APEX2 proteomics profiled proximal proteins under Exoticin or IBNtxA. Interactions were further characterized in vivo and in vitro. Results: Exoticin was shortlisted for its selective binding to 6TM-mu OR and ability to induce 6TM-mu OR-dependent signal transduction. Exoticin analgesia was sensitive to (3-FNA and absent in E11 KO mice, but restored in mice infected with AAV-mu OR1G. Slc3a2, Lrrc59, and Ppp1cb co-interacted with 6TM-mu OR1G and were equally essential for Exoticin binding and 6TM-mu OR1G activity. Conclusion: Exoticin is a promising selective agonist of 6TM mu opioid receptors with broad-spectrum analgesic efficacy but few side effects. Slc3a2, Lrrc59, Ppp1cb are endogenous chaperones essential for 6TM-mu OR ligand binding and function.