Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses

ABSTRACT 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. IMPORTANCE The 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.


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Reviewer #1 (Comments for the Author): Major comments: Line 86: The authors mention the use of the compounds to block cell-cell spread of the virus, but do not elaborate on how this mechanism of infection differs from conventional viral infection.Is this a particle independent mechanism?Line 205: Indicate the figure number here-Also, there is no I3 staining visible in the H42 treated cells infected with the G277C virus.There should be some staining since replication is not completely abrogated at 24h as seen in Fig 5A .Minor comments: Line 36: Mention full name before introducing abbreviations for the virus.Also, there is a typo in the sentence-it should read "... cessation of small pox.." The manuscript can be polished to make reading more fluid.
Reviewer #2 (Comments for the Author): Samolej et al describe the in vitro activity of bisbenzimide compounds and their antiviral activity on vaccinia virus.The paper is well written and informative.My only critique is that no statistical methods were applied to the data.Perhaps this is because the data was normalized to the control in all experiments.Still, it would be interesting to see the statistical comparison of each compound relative to the control and the other compounds.
We thank the reviewers for their helpful comments and suggestions.
Reviewer #1 (Comments for the Author): Major comments: Line 86: The authors mention the use of the compounds to block cell-cell spread of the virus, but do not elaborate on how this mechanism of infection differs from conventional viral infection.Is this a particle independent mechanism?
We have now added additional information to the manuscript regarding VACV infectious virion and replication kinetics.Poxviruses make two types of infectious particle during infection: mature virions (MVs) and extracellular virions (EVs).EVs are first released at 8 hours post infection (hpi) and mediate virus spread.VACV intracellular replication is complete by 24 hpi.

Fig 2A:
The data shown here only has a single time point of 24h.It is important to understand if virus production remains diminished at later time points in the presence of the compounds.Growth curves for up to 72h would be informative.
As above, the VACV replication cycle is ~8 hours and is complete/plateaus by 24 hpi.As such the "24-hour yield" has been the field standard for measuring infectious virion production for over 70 years.(Furness & Youngner 1959) Given that the bisbenzimides block the early stages of infection and prevent virus spread, assaying production beyond 24 h will not provide any additional information.
However, Figure 2B shows representative 48 h plaque formation in the presence of TPOXX, H42, M23, M24, and M25.We observed no sign of plaque formation in the presence of these compounds at 48 hpi.In addition, we have also assessed the impact of H42 on VACV plaque formation at 72 hpi and observed no break-through plaque formation at this extended time point (Yakimovich et al 2017;Figure 4A,B).
Collectively this data indicates that virus production remains diminished at later time points.We have alluded to this extended timeframe of anti-poxvirus activity and added reference to our previous findings with H42 .As this was a gene expression experiment, we use higher MOIs to both synchronize infection and ensure that all cells are infected.As a standard in the field, 24 h yield experiments are performed at an MOI of 1.

Fig 3B:
There are green punctae present in the CHX treated cells-are these incoming viruses?If so, the description in the text should be modified to indicate as such.
We have modified the description in the text to read, "In the presence of CHX, which prevents VACV genome uncoating (Mercer et al 2012), stabilized A5-positive virus cores, but no I3-or A5-positive replication sites were observed" Line 153: I think these should say A5 positive incoming genomes, but no I3 positive replication sites.
I3-positive incoming genomes is correct.As indicated in the manuscript, the early VACV protein I3 is found on uncoated genomes and within VACV factories.The magenta dots in the AraC panel are uncoated I3-positive VACV genomes.
For clarity, we have expanded the description of the findings to read, "In the presence of AraC, I3-positive uncoated genomes and A5-positive incoming cores were observed, but no VACV replication sites were seen".The RevG277C virus is no different from WT.We repaired the mutation in the virus to assure that the G277C virus does not contain any other mutation(s) that may confer the observed phenotypes.
This information has been added to the manuscript.At such low numbers of plaques in the 1x10 -3 -1x10 -2 range we tend to see larger variation in the titers between biological replicates.Despite this, we find that the bisbenzimide are still very effective (3 log) inhibitors of TPOXX-resistant VACV.
Line 205: Indicate the figure number here-Thank you for pointing this out.We have added the figure number to the text.Also, there is no I3 staining visible in the H42 treated cells infected with the G277C virus.There should be some staining since replication is not completely abrogated at 24h as seen in Fig 5A .For consistency, we have swapped the image with another field from the same experiment which displays I3 staining more similar to that seen in the RevG277C sample.

Minor comments:
Line 36: Mention full name before introducing abbreviations for the virus.Also, there is a typo in the sentence-it should read "... cessation of small pox.." We have corrected both instances.
The manuscript can be polished to make reading more fluid.
We have edited the manuscript for readability.
Reviewer #2 (Comments for the Author): Samolej et al describe the in vitro activity of bisbenzimide compounds and their antiviral activity on vaccinia virus.The paper is well written and informative.My only critique is that no statistical methods were applied to the data.Perhaps this is because the data was normalized to the control in all experiments.Still, it would be interesting to see the statistical comparison of each compound relative to the control and the other compounds.
As pointed out by the reviewer, we did not include statistics as all experiments were normalized to control samples.In addition, the data is rather binary in the sense that we either see no effect or obvious and substantial effects with the various compounds.

Fig 2A :
Fig 2A:The data shown here only has a single time point of 24h.It is important to understand if virus production remains diminished at later time points in the presence of the compounds.Growth curves for upto 72h would be informative.

Fig 3 :
Fig 3: Please justify the use of a higher MOI of 20 in this assay as compared to the MOI of 1 in the previous assay.

Fig 3B :
Fig 3B: There are green punctae present in the CHX treated cells-are these incoming viruses?If so, the description in the text should be modified to indicate as such.Line 153: I think these should say A5 positive incoming genomes, but no I3 positive replication sites.

Fig 5 :
Fig 5: What is the difference between the wt virus and the RevG277C virus?It should be explained how they differ to understand their use in this assay.

Fig 5A :
Fig 5A: Please show plaque titers in a log scale on the Y axis.The error bars in the H42 and M23 treated samples are very widely distributed-please comment.

Fig 3 :
Fig 3: Please justify the use of a higher MOI of 20 in this assay as compared to the MOI of 1 in the previous assay.

Fig 5 :
Fig 5: What is the difference between the wt virus and the RevG277C virus?It should be explained how they differ to understand their use in this assay.

Fig 5A :
Fig 5A: Please show plaque titers in a log scale on the Y axis.