Lassa fever and Argentine hemorrhagic fever treatment in guinea pigs using broad-spectrum cap-dependent endonuclease inhibitors

encompasses several highly pathogenic viruses that cause lethal hemorrhagic fevers. Due to their limited prevention and treatment options and high pathogenicity, these viruses require handling in biosafety level-4 facilities. Within the bunyaviruses, arenaviruses are particularly notable for their pathogenicity and ability to cause severe hemorrhagic disease in humans. The cap-dependent endonuclease (CEN) is a unique and crucial enzyme involved in the replication cycle of these viruses. As humans do not possess a similar enzyme, CEN represents an ideal target for antiviral drug development with reduced risk of side effects. Recently, we identified a promising CEN inhibitor (CENi) demonstrating potent inhibition of virus replication. In this manuscript, we demonstrate the successful therapeutic efficacy of CENis against Lassa fever and Argentine hemorrhagic fever virus infections in guinea pig models of lethal hemorrhagic fever. In addition, we identified several CENis with antiviral activity against other highly pathogenic arenaviruses. These findings further support the potential of CENis as therapeutic agents for arenavirus infections that cause severe and often lethal hemorrhagic fever. Collectively, our results suggest that CENis are promising candidates for pan-arenavirus therapy and may also have broader utility against other CEN-containing viruses for which no approved antiviral treatments currently exist.IMPORTANCEArenaviruses, such as Lassa virus and Junin virus, cause severe hemorrhagic fevers in humans and are associated with high mortality rates and limited treatment options. Because of their pathogenicity and potential for outbreaks, these viruses represent an important global health threat. The viral cap-dependent endonuclease (CEN) plays a critical role in arenavirus replication by enabling the cap-snatching process required for viral mRNA synthesis. Notably, mammalian cells lack a comparable enzyme, making CEN an attractive target for antiviral drug development. In this study, we demonstrate that inhibitors targeting the arenavirus CEN effectively suppress viral replication and provide therapeutic protection in animal models of lethal Lassa virus and Junin virus infections. We also identify compounds with antiviral activity against multiple highly pathogenic arenaviruses. These findings establish CEN inhibition as a promising strategy for the development of broad-spectrum antivirals against arenaviruses that cause life-threatening hemorrhagic fevers.