BACKGROUND: Natural alkaloids are a structurally diverse class of bioactive compounds with significant therapeutic potential. This study aimed to evaluate the in vitro antiviral activity of various natural alkaloids against coronaviruses, clarify molecular effects via bioassays and docking, and explore structure-activity relationships. Tested compounds included a wide variety of isoquinoline and Amaryllidaceae-type alkaloids. METHODOLOGY: Antiviral activity was assessed using HCoV-229E and pseudotyped lentivirus assays for different strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Cytotoxicity was evaluated with the WST-1 assay. AutoDock was used for molecular docking, online tools assessed drug-likeness, and ChemGPS-NP analyzed physicochemical properties correlated to antiviral clinical drugs. RESULTS: Several bis-benzylisoquinoline alkaloids, especially from Berberis vulgaris L., and specific Amaryllidaceae alkaloids showed protective activity against HCoV-229E (EC50 = 4.1-8.1 μM). Active compounds were further tested against SARS-CoV-2 variants. Aromoline (Compound 16) exhibited strong antiviral activity, inhibiting D614G, Delta, and Omicron variants in pseudovirus assays with IC50 values of 0.47-0.66 μM. Other bis-benzylisoquinoline analogues showed moderate activity (IC50 = 1.24-2.86 μM). Docking studies revealed aromoline's favorable interaction at the SARS-CoV-2 spike/ACE2 interface, forming hydrogen bonds with Gln493 and Ser494 (binding energy -5.34 kcal/mol). ChemGPS-NP analysis highlighted a distinct cluster of active bis-benzylisoquinolines (Compounds 16-19) in chemical space. CONCLUSION: This study highlights the antiviral potential of bis-benzylisoquinoline and Amaryllidaceae alkaloids, particularly aromoline. The findings support their relevance as scaffolds for developing novel anticoronavirus agents and advance the understanding of their structure-activity relationships.

Center for Drug Research and Development College of Human Ecology Chang Gung University of Science and Technology Taoyuan Taiwan

Department of Anesthesiology Chang Gung Memorial Hospital Taoyuan Taiwan

Department of Biochemistry and Molecular Biology College of Medicine Chang Gung University Taoyuan Taiwan

Department of Biotechnology College of Life Science Kaohsiung Medical University Kaohsiung Taiwan

Department of Medical Research Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung Taiwan

Department of Pharmaceutical Biosciences Faculty of Pharmacy Uppsala University Uppsala Sweden

Department of Pharmacognosy and Pharmaceutical Botany Faculty of Pharmacy Charles University Hradec Kralove Czech Republic

Drug Development and Value Creation Research Center Kaohsiung Medical University Kaohsiung Taiwan

Graduate Institute of Health Industry Technology College of Human Ecology Chang Gung University of Science and Technology Taoyuan Taiwan

Graduate Institute of Natural Products College of Medicine Chang Gung University Taoyuan Taiwan

Graduate Institute of Natural Products College of Pharmacy Kaohsiung Medical University Kaohsiung Taiwan

Molecular Infectious Disease Research Center Chang Gung Memorial Hospital Chang Gung University College of Medicine Taoyuan Taiwan

Research Center for Emerging Viral Infections College of Medicine Chang Gung University Taoyuan Taiwan

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