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 in vitro 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.