Identification and characterization of a dual antagonist for mu and kappa opioid receptors from Menispermum dauricum DC.

Abstract

Opioid receptors, particularly mu-opioid receptors (MOR), represent primary targets for analgesia. Opioid analgesics cause peripheral adverse effects that current symptomatic therapies fail to address, highlighting the need for novel acting MOR antagonists. The aim of this study was to identify and characterize MOR antagonists from alkaloids of Menispermum dauricum DC. We established a high-throughput screening platform targeting all four opioid receptor subtypes using FLIPR assays. A library of 20 alkaloids derived from Menispermum dauricum DC was subsequently screened against MOR, leading to the identification of an alkaloid not previously characterized as a functional MOR antagonist. Its antagonistic activity was validated using complementary label-free phenotypic dynamic mass redistribution (DMR) assays and cAMP-based functional assays. MOR antagonism was consistently reproduced across FLIPR, DMR, and cAMP assays, with pIC₅₀ values of 5.23 ± 0.04, 5.62 ± 0.05, and 5.52 ± 0.17, respectively. Selectivity profiling showed that the compound also antagonized KOR (pIC50 = 5.47 ± 0.04 and 4.91 ± 0.05 in FLIPR and cAMP assays, respectively), with no detectable activity at delta-opioid receptor (DOR) or nociceptin/orphanin FQ receptor (NOPR). Moreover, it displayed negligible cross-reactivity against five unrelated GPCRs. Molecular docking and molecular dynamics simulations were further performed to delineate its binding mode with MOR and to suggest key residues governing ligand-receptor interactions. Collectively, these findings establish compound 1 as a promising natural product-derived scaffold for further optimization.

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