Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.

Biology Centre of the Czech Academy of Sciences Institute of Parasitology Branišovská 1160 31 CZ 370 05 Ceske Budejovice Czech Republic

Department of Chemistry and Biochemistry Mendel University in Brno Zemědělská 1665 1 CZ 613 00 Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 735 5 CZ 625 00 Brno Czech Republic

Faculty of Chemical Technology Institute of Organic Chemistry and Technology University of Pardubice Studentská 573 CZ 532 10 Pardubice Czech Republic

KP Therapeutics s r o Purkyňova 649 127 CZ 612 00 Brno Czech Republic

Laboratory of Emerging Viral Diseases Veterinary Research Institute Hudcova 296 70 CZ 621 00 Brno Czech Republic

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