Effects of Statin Combinations on Zika Virus Infection in Vero Cells
Round 1
Reviewer 1 Report
The authors stated that if and when the statins are used as a treatment for Zika infections, a combination of statins that show synergy will require lower doses. Although this is a reasonable presumption, the in-vitro findings are generally hard to extrapolate to in-vivo conditions. For example, authors should discuss if any statin combination therapies are already in clinical use and shall examine the dosage and toxicity associated with such treatments.
Authors should take clinically available pharmacokinetic data from patients treated with statins and compare them to see if the serum study state concentrations (Css) or Vd of these statins are within the ranges used in this study. If the drug concentrations used in this study are not clinically relevant, authors should note the differences.
The authors have conducted the experimental methodology, and data analysis is appropriate for testing synergy. However, authors must provide confidence intervals for EC50, and FIC50, which will give lower and higher value ranges for CI for a given drug combination.
The author shall explain why the EC50 number for SIM shows more than a two-fold difference between the two experiments. EC50 of SIM alone in ATO + SIM is at 1.183uM, and EC50 of SIM alone is at 2.544 in the FLU+SIM experiment.
Authors have given no mechanistic explanation on why certain drug combinations tend towards synergy while some do not. It's known that 21 different HMGR residues present in the catalytic binding pocket can interact with statin drug active groups. The interaction strength and distance between functional drug groups and HMGR resides will define the binding energies of each drug. These binding energies of the drugs are correlated with their IC50 values. Given this, It will be helpful to see a table that shows the IC50 of statins on HMGR and their EC50 values for anti-Zika infections. Explain if the IC50 values correlate with their EC50 for anti-Zika infections.
It is quite puzzling to see the synergy between some drug combinations since all statins bind to the same catalytic binding pocket present in HMGR. One would expect competition between two drugs for the same binding pocket, and the drug molecule with higher binding energy should exclude the one with lower. The presence of synergy indicates that drugs are interacting at different sites and the interaction of one drug increases the binding energies and lower IC for another. Or the effect of drugs is not exclusively via HMGR. Alternative cellular pathways are affected. The authors shall explain how drugs interact with the exact catalytic pocket to exhibit synergy.
Suppose the synergies seen in this paper are solely due to the inhibition of HMGR. In that case, the cholesterol levels must have to be lower in those drug combinations, considerably lower than those combinations where synergy is not seen. Authors should minimally measure total cellular cholesterol levels in the presence of statins and their combinations. Explain if the cholesterol levels are in congruence with synergy or lack thereof.
Authors should give different numbers for each table presented per experiment. All tables are denoted as Table #1.
Overall, the authors have shown unexpected results in this paper but failed to provide any mechanistic explanation or the relevance of finding for in-vivo application. A more substantial discussion must be provided while addressing my concerns provided above.
Author Response
The responses to the review are in the attached file. Line numbers are based on the file with tracked changes in balloons.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In the manuscript, “Effects of statin combinations on Zika Virus infection in Vero cells,” authors Erica Espano and Jeong-Ki Kim test whether combinations of active and prodrug statins have synergistic antiviral effects against ZIKV. The rationale being that statins themselves have been shown previously by these authors and others to have an anti-viral effect on ZIKV yet are also known to have safety concerns in pregnancy. The combinations tested were ATO + MEV, ATO + SIM, FLU + MEV, and FLU + SIM, with ATO and FLU exhibiting synergistic effects against ZIKV with MEV and SIM. The authors suggest following up with combinations with FLU since its antiviral activity occurs at lower doses. The manuscript is clear and well-written, and the experiments are well designed and executed. Only minor comments and concerns are noted.
1. Other reports have shown anti-viral effects on flaviviruses including one on ZIKV not published by the authors. These publications and a sentence or two about their findings should be referenced in the introduction even if discusses later in the discussion.
2. Since the statins described appear to act at the same rate-limiting step, it’s difficult to understand how they could have different targets in the ZIKV life cycle (lines 46-47). It’s possible for them to have different effects due to potency, but different targets seem unlikely. The authors may want to clarify for better understanding.
3. Are prodrug statins considered safer in pregnancy than active statins?
4. In section 2.1. Cells, virus, and reagents, please add a sentence starting on line 77 about how the virus was tittered.
5. In section 2.2. Fixed ration combinations, the combination ‘C4-2:1;’ line 89 should read ‘C4-2:3;’.
6. For the ATO + MEV combination, the ranges of the x-axes could be modified to better see the combination curves. For example, the Figure1a x-axis could be changed to 1.0 to possibly 4.0 without interfering with the data points. The Figure 1b x-axis could range from 1.5 to 4.5. Subscript the 10 in Log10 in Figure 1a.
7. For the ATO + SIM combination, please check whether the following statement is correct: These combinations had similar CI values (0.84 to 0.88), with higher fractions of SIM over ATO appearing to be more beneficial to the combination. It appears the opposite is true-that higher fractions of ATO are more beneficial. Is a value of 0.84 a meaningful improvement over 0.88? Modifying the range of x-axes of Figure 2a to 1.0 to 4.0 and Figure 2b to 0.5 to 4.0 would make the combination curves easier to see, though again a minor concern.
8. Figures 3a-b and 4a-b, modify the x axes as appropriate to better visualize the curves.
Author Response
The responses to the review are in the attached file. Line numbers are based on the file with tracked changes in balloons.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Thank you for addressing my concerns and proactively editing the paper with the necessary changes. Although measuring cholesterol levels and understanding the mechanism of action is beyond this paper, I think it will help the scientific community to understand the mechanism of action. I encourage the researchers to explore the mechanism of Zika inhibition in the presence of synergistic drug combinations.
