Editor Comments

We thank the editor for their comments and clarification of requirements. In response:

1. The article format has been altered to comply with the journal specifications

2. The code used for MD simulations is freely available through github at https://github.com/bioinfkaustin/gromacs-on-colab.

3. References to figure 1 and figure 5 can now be found in the text.

4. The supplementary data has been moved to a separate file as requested

Reviewer 1

We thank Reviewer 1, Dr Rout, for his comments. In response:

1. The abstract has been entirely rewritten.

2. The requested additions have been made to the introduction

3. This information can be found in the methods and results section of the revised report.

4. We acknowledge that through reporting only 15 nanoseconds of data, the molecular dynamics (MD) elements of this study include relatively short simulations. This important aspect of study design was not a choice taken lightly. MD studies are a valuable addition to the assessment of docking results and have been validated for this purpose1. A validation study using the DUD-E dataset identified a root mean square deviation (RMSD) threshold of 5.5Å was robust in discerning true active poses in MD simulations of 10, 50 and 100 nanosecond durations. In this study the difference in sensitivity measured by the area under a ROC curve (AUC) between each simulation length was relatively small. The calculated AUC values for experiments using the DUD-E dataset were 0.806, 0.840 and 0.836 for simulations of 10, 50 and 100 nanoseconds respectively(25).

The current study is the first performed using a study protocol for high-throughput docking-MD screening that aims to make such structural bioinformatics studies achievable and accessible to new researchers to the field and researchers in training. This study is not alone in selecting a short duration of MD simulation to evaluate docking results with over 50 examples of studies using 10 nanosecond simulations identified on the NCBI PubMed database at the time of writing2–53.

5. Despite a thorough literature review, we have been unable to identify studies validating the use of root mean square fluctuation (RMSF) and RF in short post-docking MD studies. Without this validation it is unclear whether these additional measurements would meaningfully enhance the study conclusions. As outlined above increasing the complexity of data analysis is contrary to the premise of the protocol under development. In response to the points made, more detail has been included regarding selected observed hydrogen bond patterns before and after the MD simulations which can be found in a dedicated section within the results.

6. A dedicated section has been included with information on the validation of the model. This includes confidence scores obtained from I-TASSER and RMSD values against reference structures.

7. As explained in our response to point 4 (above), with the validation provided and strong support across a raft of literature, we sincerely believe that creditable conclusions can be made from 10 nanosecond simulations in this validated setting1, see point 4 above. We accept that wherever possible, the results of any computational study, but especially those investigating ligand binding, should be validated by laboratory experiments or clinical data due to the inherent limitations of the methods. Both the original and rewritten conclusions make this point plainly.

8. The discussion has been extended by the inclusion of a section discussing the rationale for the methods used.

Reviewer 2

We thank Reviewer 2 for their comments. In response:

1. A robust defence of the methods employed within this study and their rationale can be found above.

2. The abstract and conclusion sections have been entirely rewritten. Redrafting these sections has taken place without knowledge of the specific concerns of the reviewers and we hope that the new versions are satisfactory. More widely, we have also extended the introduction, including additional references to appropriate studies.

3. We assume that the “recent relevant paper” referred to here is the same investigation of a catfish aquaporin recommended by Reviewer 154. We have referred to this article in the text.

Suggestions within the document

4. The abstract has been completely rewritten given uncertainty about which elements of the abstract required improvement.

5. We have extended the introduction, including additional references to appropriate studies.

6. We again assume that the recent publication referenced here is the mechanistic study of water transport through catfish aquaporin by Behera and colleagues 202254. This study investigating the mechanism of water transport through AQP1 is fundamentally different in its aims and design to the assessment of stability of docked ligand conformations in a screening study. As explained in the response to reviewer 1 the differences between 10 and 100 nanosecond MD simulations for discerning true binding from docking study results are modest1.

7. The conclusion has been rewritten as mentioned in point 3.

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