Identification of Neferine as a DOR Agonist Activating G_i and G_z Signaling: In Silico and In Vitro Studies

Benzylisoquinoline alkaloids (BIAs) exhibit diverse biological activities, such as neuroprotective effects. The delta-opioid receptor (DOR) has emerged as a promising therapeutic target due to its potential role in enhancing neuroprotection and regeneration. However, reports on the binding of BIAs to the DOR remain scarce. Here, neferine, a BIA from Nelumbo nucifera, as a potential DOR agonist. Molecular docking ranked neferine among the top of 15 BIAs. Initial binding was detected by cellular membrane chromatography and quantitatively confirmed by bio-layer interferometry, with a K_D value of 37.4 μM. ONE vector G protein Optical biosensor revealed that G_i2, G_i3 and G_Z signaling could be activated by neferine through DOR modulation. Consistent with the G_i/z activation, neferine dose-dependently inhibited cAMP accumulation with an EC₅₀ of 0.25 µM. Transcriptomic analysis in DOR-overexpressing HEK293T cells indicated that neferine stimulation predominantly regulates gene networks governing cell cycle and stress adaptation. However, direct transcriptional signature for neuroprotection was not predominant in our system, suggesting that DOR signaling may exhibit context-dependent effects. In conclusion, we identified the neferine as a natural DOR agonist through in silico and in vitro approach, providing a reference for further investigation into its pharmacological potential.