Label-free cell phenotypic study of opioid receptors and discovery of novel mu opioid ligands from natural products

Mu opioid receptor (MOR) is mainly a drug target for analgesia. Opioid-like agonists such as morphine have been clinically used for analgesia but have potential adverse effects. MOR antagonists have been demonstrated to alleviate these side effects. Plants (Carthamus tinctorius L, Cynanchum otophyllum C. K. Schneid., Coffea arabica L., Prinsepia utilis Royle and Lepidium meyenii Walp.) and Ganoderma fungi (Ganoderma hainanense J. D. Zhao, Ganoderma capense (Lloyd) Teng, Ganoderma cochlear (Blume et Nees) Bres., Ganoderma resinaceum Boud and Ganoderma applanatum (Pers.) Pat.) are traditional medicines with beneficial effects on immunoregulation, analgesia and the nervous system, but whether MORs are engaged in their effects remains unknown. This work aimed to identify MOR ligands among compounds isolated from the above-mentioned 10 species, and to investigate selectivity against four opioid receptor subtypes. By analyzing the structure-activity relationship and off-target effects, we could provide a new direction for the future development of MOR drugs. Four opioid receptor subtype models, including MOR, delta (DOR), kappa (KOR) and nop (NOR), were established with a label-free phenotypic dynamic mass redistribution assay to systematically profile the pharmacological properties of known ligands. Then, 82 natural compounds derived from the 10 species were screened against MOR to identify new ligands. The selectivity of the new ligands was characterized against the four subtypes, and off-target effects were also investigated on eight G protein-coupled receptors (GPCRs). The pharmacological properties of known ligands on transfected HEK293T-MOR, HEK293-DOR, HEK293-KOR and HEK293-NOR cell lines were characterized. Seven compounds purified from Ganoderma cochlear (Blume et Nees) Bres. and Carthamus tinctorius L were MOR antagonists with micromolar potency. Among them, compound 35 showed the strongest antagonistic activity on MOR with an IC50 value of 10.0 ± 3.0 μM. To a certain extent, these seven new antagonists, exhibited antagonistic activity on the other opioid receptor subtypes, and they had almost no effect on other GPCRs, including CB1, CB2, M2 and beta2AR. Additionally, a compound from Lepidium meyenii Walp. displayed MOR agonistic activity. The established screening models opened new avenues for the discovery and evaluation of opioid receptor ligand selectivity. Together, the novel MOR antagonists and agonists will enrich the inventory of MOR ligands an