The traditional Chinese medicine Stephania epigaea (S. epigaea) has been used for medicinal purposes, including the treatment of abdominal pain and malaria. Increasing evidence suggests a relationship between pain relief, gut regulation, and the dopaminergic system, particularly the dopamine D1 receptor (D1R). However, it remains unclear whether and how active components of S. epigaea exert their effects through dopamine receptors. This study aims to discover active alkaloids targeting D1R from S. epigaea, evaluate their potencies, and analyze their structure-activity relationships (SARs). Twenty-four alkaloids were screened on an HEK293-D1R cell model using label-free cell phenotypic assays, and six were identified for the first time as exhibiting D1R antagonistic activity, including capaurimine, (-)-discretine, (+)-corydaline, N-methyllaurotetanine, (+)-isocorydine and O-methylbulbocapnine. Among these, protoberberine-type alkaloids capaurimine and (-)-discretine were found to be potent D1R antagonists, with IC50 values of 0.16 ± 0.02 μM and 0.25 ± 0.01 μM, respectively. The aporphine-type alkaloid N-methyllaurotetanine also exhibited micromolar-level D1R antagonistic activity (IC50 = 1.65 ± 0.20 μM). Their kinetic binding profiles were characterized using co-stimulation assay, confirming them as competitive D1R antagonists. Additionally, these compounds were docked with the crystal structure of human D1R, allowing for an analysis their SARs. These findings provide potential mechanisms underlying the analgesic and gastrointestinal effects of S. epigaea and offer valuable information for D1R antagonist drug design.
Keywords: Alkaloids; Antagonists; Dopamine D1 receptor; Label-free cell phenotypic assay; Stephania epigaea.
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