Dear Ahmed Elkamhawy,

We are very grateful to you and both reviewers for reviewing our submission and for the considered feedback we have received as a result. A point-by-point reply to the comments can be found below.

Journal requirements

1. The resubmitted version of this report conforms to the PLOS file style and naming guidelines. Thank you for bringing this to our attention.

2. The modelling, docking and docking analysis tools used for this study can be found at https://proteins.swan.ac.uk/modelling-portal/. This website hosts automated structural modelling and docking services based on validated published methods freely available for academic use and credited within the manuscript. An institutional email address is required to sign up and a report detailing the tools found on this website is in press. The code used for MD simulations is freely available through github at https://github.com/bioinfkaustin/gromacs-on-colab.

3. We are sorry that the supplementary data was not available. This has now been corrected, and the files have been appropriately released for public viewing on figshare. The data files can be found at https://doi.org/10.6084/m9.figshare.28363397.v1.

Reviewer 1

We would like to thank Dr. Shovonlal Gayen for their time and for the constructive suggestions. Our responses to their comments are below.

1. We have furnished the introduction with more background information of the protein, polypharmacy as a concept and the widespread use of SGLT2 inhibitors.

2. Significant additions have been made to the results section aiming to address this concern.

3. A discussion of the protein-ligand interactions in greater detail has been included in the revised results section. We hope this addresses both this comment and comment 2 above. New figures have also been included to describe the protein structure and the ligand interactions of the 6 compounds with the lowest median RMSD in the MD studies

4. We acknowledge that through reporting only 10 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 respectively1. The current study is the second performed using a new 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. The first, a repurposing screen of AQP1 was published in PLOS One earlier this year2. This second study of SGLT2 has a significant methodological upgrade to the AQP1 study as each ligand was simulated three times with the median RMSD value used to determine whether a ligand remained bound. There are over 50 publications within the NCBI PubMed database3–54, in addition to our recent report of the aquaporin 1 virtual screen2, which have used MD simulations of 10 nanoseconds to draw conclusions.

Despite a thorough literature review, we have been unable to identify any studies validating the use of RMSF, MMPBSA, and RG in short post-docking MD studies. Without this validation it is unclear whether these additional measurements would meaningfully enhance the study conclusions. Increasing the complexity of data analysis is contrary to the premise of the protocol under development which aims to broaden access to the field of structural bioinformatics and make studies feel more achievable to new researchers to the field.

In conclusion, we assert that three simulations of 10 nanosecond duration with assessment of ligand binding based on median RMSD are provide a validated and reproducible method to judge docked ligand conformation stability. A concise explanation of this in contained within the methods of the revised manuscript.

5. Dapagliflozin, canagliflozin, empagliflozin and ertugliflozin are licensed medicinal compounds known to act against SGLT2. These compounds have been included within all steps of the analysis through design as positive control ligands. The results for these compounds suggest stable binding with the protein. We are unclear which other “standard inhibitors” of SGLT2 reviewer 1 would like included. We cannot think of any more appropriate known inhibitors that these 4 drugs in current clinical use. We would contest that there is a large body of evidence that these compounds act against SGLT2 including experimental crystal structures confirming the interaction55–57.

We note the concern raised in the set questions regarding statistical analysis and would like clarification on whether this relates to points 3, 4, and 5, to which we have provided a robust response, or another aspect of the study.

We were disappointed to learn that this reviewer did not feel the standard of English language within the submitted manuscript met expectations. Unfortunately, no comment regarding any specific language concern or typographical errors has been made to direct improvements to the manuscript or address any perceived deficits. We have therefore concentrated on making strenuous efforts to proofread the manuscript to remove typographical errors from the revised manuscript.

Reviewer 2

We would like to thank Reviewer 2 for their time and for the constructive suggestions. Our responses to their comments are below.

1. We wholeheartedly agree that in vitro validation of these in-silico findings would be extremely valuable. We are unfortunately not able to complete these studies. Our hope in presenting this manuscript is that a laboratory with the expertise and resources to perform in vitro studies of SGLT2 would be inspired to validate these results. We have reinforced our firm belief that these findings need in vitro or clinical validation in the revised manuscript.

2. We have not studied SGLT1, mostly because despite the sequence similarity this protein is not acknowledged as a target for existing SGLT2 selective inhibitors55. However, we agree that a similar study of SGLT1 would be an interesting avenue for further research and now mention this within the discussion section of the revised manuscript.

3. The problem with the supplementary data has been addressed. Thank you for bringing this to our attention. Please see comments to the editor. The data can be found at https://doi.org/10.6084/m9.figshare.28363397.v1.

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