ABSTRACT Opioids until recently were the drugs of choice to treat moderate to severe chronic pain. Decades of intensive research has led to the pharmacological characterization of countless synthetic and semi-synthetic opioid derivatives. However, an ideal compound, devoid of serious side-effects such as respiratory depression, dependence, and addiction, has not yet been identified. Opioid activation of mu opioid receptor (MOR) G protein signaling pathways is believed to cause both desirable analgesia as well as undesirable adverse effects like respiratory depression. Since MOR can couple with six Gi/o/z subtypes, namely Gi1, Gi2, Gi3, GOA, GOB and Gz, we hypothesize that it may be possible to achieve functional selectivity by activating an individual Gα-isoform and/or a combination of these isoforms. Our preliminary data shows that bitopics of fentanyl show unique Gi/o/z signaling as well as a behavioral profile distinct from typical opioids. To this end, we have also obtained cryoEM structures of MOR bound to one lead bitopics, C6 guano (3.3Å), and an analog of C6 guano named LG94 (3.4Å) getting insights into distinct modes of ligand binding that also involves engagement of an allosteric binding site. We have identified two distinct modes of engaging the sodium binding allosteric site in MOR and identified an antagonist as well. In this study, we will interrogate Gα-subtype bias through screening of additional analogs, across both fentanyl and morphinan based templates as well as novel indole-based templates and targeting the allosteric site with additional bitopics with higher potency and optimal drug like properties. We will also develop a first in class antagonist on the fentanyl template. High resolution cryoEM will be used on promising analogs to enable structure-function studies and ligand binding fine-tuning. This, in turn, will enable us to design analogs that are more effective and possibly display a more advantageous side-effect profile (no respiratory depression while showing analgesia) than their parent compounds. These probes will also allow us to build a better framework for understanding MOR Gα-subtype signaling and facilitate identification of useful preclinical candidates for the treatment of pain/OUD in the near future. Furthermore, any novel antagonists could become tools in fighting opioid-related substance abuse.