Structure-guided development of a viral endonuclease inhibitor that potently restricts human pathogenic mammarenavirus infection in vivo

Mammarenaviruses are zoonotic pathogens that establish persistent infections in rodents and periodically spill over into humans, causing severe disease. Several species, including Lassa virus (LASV) and Junín virus (JUNV) are etiological agents of viral hemorrhagic fever (HF) disease associated with high morbidity and mortality rates. Given the absence of licensed vaccines or effective therapeutics, the development of broad-spectrum antivirals against mammarenaviruses remains an urgent biomedical priority. The mammarenavirus L protein encodes the viral RNA-dependent RNA polymerase (RdRp) and a highly conserved endonuclease (EndoN) domain that plays a critical role in cap-snatching mediated transcription of viral mRNA, making it an attractive antiviral target. Baloxavir acid (BXA), designed to inhibit the EndoN activity of the PA subunit of the influenza polymerase, also inhibited LCMV, JUNV, and LASV multiplication, albeit with limited potency. Guided by this finding, we conducted a medicinal chemistry campaign to modify the chemical scaffold of BXA and identified mCPB916 as a lead compound with broad-spectrum activity against multiple mammarenaviruses. Genetic, structural, and biochemical analyses confirmed mCPB916 targets the EndoN domain of the mammarenavirus L protein. Prophylactic treatment with mCPB916 significantly inhibited viral infection, reduced disease-associated pathology, and improved survival in mouse models of lethal LCMV and LASV infection. Together, these results define the structural basis and in vivo efficacy of mammarenavirus-specific EndoN inhibitors and establishes mCPB916 as a prototype EndoN inhibitor for the development of broad-spectrum antivirals against human pathogenic mammarenaviruses.