Specific Aim: Validate the MOR/14-3-3 LinkLight assay with a pilot HTS to identify molecules modulating MOR trafficking. Significance: The current crisis of opioid use disorders (OUDs) and opioid overdose deaths in the United States urgently demands safer and better therapeutics. To develop better and safer therapeutic agents for the treatment of OUDs, first and foremost is to have new bioassays which are different from the current approaches and are physiologically relevant to the underlying neurobiology of opioid addiction and chronic pain, and to use the bioassays to screen and identify new therapeutic leads for OUD drug development. We have developed MOR/14-3-3 LinkLight assay. We found that 14-3-3 proteins participate in GPCR trafficking and recycling. MOR/14-3-3 association signals can be promoted by MOR antagonists naloxone (live-saving overdose drug) and naltrexone (anti-addiction drug). MOR endomorphins can disrupt MOR/14-3-3 interaction signals, while at the same time promote MOR/?-arrestin interaction. MOR1 endocytosis and trafficking play a critical role in the development of opioid tolerance and dependence. Thus, the MOR/14-3-3 LinkLight bioassay represents an unprecedented opportunity for screen and identification of new therapeutic agents for the treatment of OUDs. Experimental Design: Task 1. Conduct a pilot screen of small molecules modulating MOR1/14-3-3 interaction signals. We plan to screen approximate total 10,000 small molecule libraries including approved drug library, clinic compound library, GPCR compound library, and bioactive compound library. Task 2. Characterize positive hits identified from the screen for G-protein and ?-arrestin pathways. MOR/14-3-3 is a new pathway assay. The hits identified by MOR/14-3-3 LinkLight assay may have different properties for G-protein and ?-arrestin signaling pathways. We will classify agonists and antagonists based on G-protein signaling assay. We have developed MOR/?-arrestin LinkLight assay and the assay will be used to evaluate the hits for ?-arrestin pathway activity. Based on the three pathway assays, we expect to find multi-pathway biased ligands. These multi-pathway biased ligands will enhance our understanding neurobiology of opioid addiction and chronic pain, as well as lead us to develop better and safer drugs. Next Phase Plan: The phase I project will test the feasibility of this novel mechanism-based approach for screening and identifying new molecules for OUDs. Once we demonstrate feasibility, a phase II plan that includes screening larger compound collections, evaluating positive hits for DMPK studies in animals, and evaluating positive hits for animal models of pain, tolerance, withdrawal, and addiction, and in potential collaboration with NCATS will be proposed. We will partner the project with appropriate pharmaceutical companies to accelerate development when possible. Summary: Accumulating evidence suggests that opioid receptor desensitization and trafficking mechanisms are intimately connected to tolerance and addiction. We have developed a novel MOR/14-3-3 LinkLight assay. We found 14-3-3 proteins involved in MOR trafficking. Thus, the MOR/14-3-3 assay can be utilized for screening molecules modulating MOR trafficking. The potential hits identified from the proposed screen could have potentials for therapeutic agent development.