Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses

We previously identified the bisbenzimide Hoechst 33342 (H42) as a potent multi-stage inhibitor of the prototypic poxvirus, the vaccinia virus (VACV), and several parapoxviruses. A recent report showed that novel bisbenzimide compounds similar in structure to H42 could prevent human cytomegalovirus replication. Here, we assessed whether these compounds could also serve as poxvirus inhibitors. Using virological assays, we show that these bisbenzimide compounds inhibit VACV spread, plaque formation, and the production of infectious progeny VACV with relatively low cell toxicity. Further analysis of the VACV lifecycle indicated that the effective bisbenzimide compounds had little impact on VACV early gene expression but inhibited VACV late gene expression and truncated the formation of VACV replication sites. Additionally, we found that bisbenzimide compounds, including H42, can inhibit both monkeypox and a VACV mutant resistant to the widely used anti-poxvirus drug TPOXX (Tecovirimat). Therefore, the tested bisbenzimide compounds were inhibitors of both prototypic and pandemic potential poxviruses and could be developed for use in situations where anti-poxvirus drug resistance may occur. Additionally, these data suggest that bisbenzimide compounds may serve as broad-activity antiviral compounds, targeting diverse DNA viruses such as poxviruses and betaherpesviruses.IMPORTANCEThe 2022 mpox (monkeypox) outbreak served as a stark reminder that due to the cessation of smallpox vaccination over 40 years ago, most of the human population remains susceptible to poxvirus infection. With only two antivirals approved for the treatment of smallpox infection in humans, the need for additional anti-poxvirus compounds is evident. Having shown that the bisbenzimide H33342 is a potent inhibitor of poxvirus gene expression and DNA replication, here we extend these findings to include a set of novel bisbenzimide compounds that show anti-viral activity against mpox and a drug-resistant prototype poxvirus mutant. These results suggest that further development of bisbenzimides for the treatment of pandemic potential poxviruses is warranted.