Discovery of Small-Molecule Orthopoxvirus Resolvase Inhibitors with Antiviral Activity

Poxvirus genome replication and viral maturation require a Holliday junction resolvase. Genetic evidence suggests that targeting the virally encoded resolvase could offer a novel antiviral approach against orthopoxviruses. However, orthopoxvirus resolvases have not been characterized biochemically and pharmacologically, and inhibitors remain unknown. Herein, we have developed and optimized the first <i>in vitro</i> assay directly measuring the activity of mpox virus (MPXV) resolvase (Mpr) and vaccinia virus (VACV) resolvase (A22). The subsequent pilot screen of an in-house compound library using this assay identified multiple inhibitors, each inhibiting both Mpr and A22, and conferring antiviral activity against VACV. Computationally, these inhibitors docked well into the active site of an AlphaFold-generated Mpr structural model. The assay developed herein and the inhibitors identified and characterized provide a valuable platform for developing compounds as broad-spectrum antiviral drug leads against MPXV and other potentially emerging orthopoxviruses, and as probes to investigate the functions of Mpr.