Abstract Monkeypox virus (MPXV) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.032-1.40 μM) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the post-entry step to reduce the intracellular production of virions. Knockdown of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. These evidences propose that IMPDH is a potential target for the development of anti-MPXV agents. Importance Monkeypox is a zoonotic disease caused by infection with the monkeypox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against monkeypox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against monkeypox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-monkeypox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited monkeypox virus propagation, exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested inosine monophosphate dehydrogenase as a potential target for the development of anti-monkeypox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-monkeypox virus activity than mycophenolic acid.

Identification of inosine monophosphate dehydrogenase as a potential target for anti-monkeypox virus agents