Supplemental Digital Content is Available in the Text.Fibromyalgia is associated with a dysregulated NK cell activation signature and their accumulation at peripheral nerves.

Background and Purpose The μ‐opioid receptor (μ receptor) is the primary target for opioid analgesics. The 7‐transmembrane (TM) and 6TM μ receptor isoforms mediate inhibitory and excitatory cellular effects. Here, we developed compounds selective for 6TM‐ or 7TM‐μ receptors to further our understanding of the pharmacodynamic properties of μ receptors. Experimental Approach We performed virtual screening of the ZINC Drug Now library of compounds using in silico 7TM‐ and 6TM‐μ receptor structural models and identified potential compounds that are selective for 6TM‐ and/or 7TM‐μ receptors. Subsequently, we characterized the most promising candidate compounds in functional in vitro studies using Be2C neuroblastoma transfected cells, behavioural in vivo pain assays using various knockout mice and in ex vivo electrophysiology studies. Key Results Our virtual screen identified 30 potential candidate compounds. Subsequent functional in vitro cellular assays shortlisted four compounds (#5, 10, 11 and 25) that demonstrated 6TM‐ or 7TM‐μ receptor‐dependent NO release. In in vivo pain assays these compounds also produced dose‐dependent hyperalgesic responses. Studies using mice that lack specific opioid receptors further established the μ receptor‐dependent nature of identified novel ligands. Ex vivo electrophysiological studies on spontaneous excitatory postsynaptic currents in isolated spinal cord slices also validated the hyperalgesic properties of the most potent 6TM‐ (#10) and 7TM‐μ receptor (#5) ligands. Conclusion and Implications Our novel compounds represent a new class of ligands for μ receptors and will serve as valuable research tools to facilitate the development of opioids with significant analgesic efficacy and fewer side‐effects.

The G protein‐coupled μ‐opioid receptor (μ‐OR) mediates the majority of analgesia effects for morphine and other pain relievers. Despite extensive studies of its structure and activation mechanisms, the inherently low maturation efficiency of μ‐OR represents a major hurdle to understanding its function. Here we computationally designed μ‐OR mutants with altered stability to probe the relationship between cell‐surface targeting, signal transduction, and agonist efficacy. The stabilizing mutation T315Y enhanced μ‐OR trafficking to the plasma membrane and significantly promoted the morphine‐mediated inhibition of downstream signaling. In contrast, the destabilizing mutation R165Y led to intracellular retention of μ‐OR and reduced the response to morphine stimulation. These findings suggest that μ‐OR stability is an important factor in regulating receptor signaling and provide a viable avenue to improve the efficacy of analgesics.

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