Integrative Network Pharmacology, Molecular Docking, and Dynamics Simulations Reveal the Mechanisms of Cinnamomum tamala in Diabetic Nephropathy Treatment: An In Silico Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The presented manuscript describes the identification of Cinnamomum tamala bioactive compounds and exploration of their therapeutic mechanisms in diabetic nephropathy in silico. The Introduction is concise but thorough and gives enough information about the subject. The Methods section is written in great detail, which is commendable. The study itself seems well designed, the methods used appropriately and the results clearly presented and discussed in comparison to pertinent data. There are only some minor points to correct given below:

1) The order of authors in the manuscript is not the same as in the submission system. Please check this and correct where needed. Likewise, assign the correspondence to one author or check with the editors if more than one is okay with them.

2) There should be no subtitles in the abstract!

3) “Ligands were prepared using the OPLS4 force field, and their conformations were generated for pH 7.0 ± 2.0.”

Is this a standard option in the software? pH 7.0 ± 2.0 covers quite large pH area, does it generate only one structure for the whole span or gives multiple?

4) The description of Figure 9 lacks some details, what is represented on the A, and what on the B part? Please add.

Author Response

Dear Reviewer,

We hope this message finds you well. We would like to sincerely thank you for your valuable comments and insights regarding our manuscript submitted to Current Issues in Molecular Biology (CIMB MDPI). Your constructive feedback has significantly enhanced the quality of our work.

We have carefully considered each of your suggestions and have made the necessary revisions to address your comments. We are confident that these improvements will contribute positively to the overall quality and clarity of the manuscript.

We kindly ask for your continued consideration of our responses to your comments as we resubmit the revised manuscript. If there is anything further needed from our side, please do not hesitate to let us know. Thank you once again for your time and effort in reviewing our work. We appreciate your support.

• The order of authors in the manuscript is not the same as in the submission system. Please check this and correct where needed. Likewise, assign the correspondence to one author or check with the editors if more than one is okay with them.

Response: During the submission process, there was a glitch that caused the author order to appear differently. We kindly request that the order given in the manuscript be considered. Additionally, there should be no issue with assigning two corresponding authors.

• There should be no subtitles in the abstract!

Response: As per your comment, we have made the necessary corrections to the abstract. Thank you for bringing it to our attention.

• “Ligands were prepared using the OPLS4 force field, and their conformations were generated for pH 7.0 ± 2.0.”

Is this a standard option in the software? pH 7.0 ± 2.0 covers quite large pH area, does it generate only one structure for the whole span or gives multiple?

Response: According to the literature, this is the standard approach to follow, and it has been cited in the procedure. Throughout the entire span, only one structure has been generated, and no significant changes were observed.

• The description of Figure 9 lacks some details, what is represented on the A, and what on the B part? Please add.

Response: Thank you for noticing. We have made the necessary change by specifying parts A and B in the description of Figure 8.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors have investigated the therapeutic potential of Cinnamomum tamala (CT) in the treatment of diabetic nephropathy (DN), a serious complication of diabetes. The study employs a combination of computational methods and bioinformatics analyses to identify bioactive compounds within CT and their potential mechanisms of action. Using the IMPPAT database, the authors identified six active compounds in CT, and through PharmMapper, they predicted 347 potential targets. By intersecting these with DN-related targets from GeneCards, they narrowed down 70 relevant therapeutic targets, including key proteins such as MMP9, EGFR, and AKT1.

One of the key strengths of the study is its identification of the PPAR and PI3K-AKT signaling pathways as the main pathways through which CT could exert its effects on DN. These pathways are crucial for regulating cellular metabolism, development, and inflammation, which are central processes in the progression of DN. The study also integrates protein-protein interaction network analysis using STRING and Cytoscape, providing further insights into the relationships between these therapeutic targets. The study is well-presented and offers a solid foundation for future research, especially with its use of molecular docking and dynamics simulations to validate key compound-target interactions. However, while the study is promising, I would like the authors to address the following points to improve the manuscript:

(1)  The authors can validate the docking procedure by docking the native ligand from the PDB structure and comparing the RMSD between the docked pose and the crystal pose.

(2)  The authors may perform binding free energy calculations through MM-GBSA or MM-PBSA methods on the converged trajectories from MD simulations.

(3)  The authors can add the cytotoxic effects of the CT. If the information is not available through the literature, the authors can perform predictions through the Swiss ADME web server.

(4)  The authors can predict the off-targets of CT through SwissTargetPrediction.

 

(5)  The authors should provide the correct references for the softwares that they have used. 

Author Response

Dear Reviewer,

We hope this message finds you well. We would like to sincerely thank you for your valuable comments and insights regarding our manuscript submitted to Current Issues in Molecular Biology (CIMB MDPI). Your constructive feedback has significantly enhanced the quality of our work.

We have carefully considered each of your suggestions and have made the necessary revisions to address your comments. We are confident that these improvements will contribute positively to the overall quality and clarity of the manuscript.

We kindly ask for your continued consideration of our responses to your comments as we resubmit the revised manuscript. If there is anything further needed from our side, please do not hesitate to let us know. Thank you once again for your time and effort in reviewing our work. We appreciate your support.

• The authors can validate the docking procedure by docking the native ligand from the PDB structure and comparing the RMSD between the docked pose and the crystal pose.

Response: We have compared the docked pose and crystal pose with the native ligand from the PDB file of the best docked structure, along with the ligand pose. We respectfully request the reviewer to consider this, as conducting this analysis again would be highly time-consuming and may require repeating the entire study. We will ensure to incorporate this approach in our future investigations.

• The authors may perform binding free energy calculations through MM-GBSA or MM-PBSA methods on the converged trajectories from MD simulations.

Response: We have incorporated the MM-GBSA method on the trajectories for MD simulation, and this has been highlighted in the manuscript.

• The authors can add the cytotoxic effects of the CT. If the information is not available through the literature, the authors can perform predictions through the Swiss ADME web server.

Response: We have included the cytotoxicity effects of the compound in the introduction section, based on findings from the literature survey.

(4)  The authors can predict the off-targets of CT through SwissTargetPrediction.

 Response: At the outset of the target selection process, we identified targets that are actively related to the CT compounds. Re-evaluating this through SwissTargetPrediction may lead to the repetition of the entire study, as the limitations of software availability would render it a time-consuming process. We kindly request the reviewer to consider this.

(5)  The authors should provide the correct references for the softwares that they have used. 

Response: The correct references for the software have been updated. We are very grateful for your attention to this matter.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Integrative Network Pharmacology, Molecular Docking, and Dynamics Simulation Reveal the Mechanisms of Cinnamomum tamala in Diabetic Nephropathy Treatment: An In-silico Study” has many mistakes, authors need to rectify many portions.

 

1.                Are there missing steps or further explanation needed when describing the use of Venn diagrams to identify overlapping targets?

2.                Does the phrase "a docking score of less than -5 kcal/mol indicated excellent binding activity" need more context or references to support this threshold?

3.                Should the preparation of ligands and proteins for molecular docking clarify why specific force fields like OPLS4 were chosen?

4.                The authors calculated pharmacodynamic properties in the manuscript and could also provide insights into pharmacokinetic aspects.

5.                The methodology section should be written more concisely.

6.                The methods section lacks several key citations relevant to MD simulations, including references for GROMACS, CHARMM, water models, and ion parameters.

7.                The author required to update recent references can be seen PMID: https://doi.org/10.1039/D4NJ02065K, https://doi.org/10.1080/07391102.2023.2290621, PMID: 36936534.

8.                The manuscript is predominantly focused on experimental reports, and the results section is lengthy and difficult to follow. Some of the information could be moved to the supporting information.

9.                The PDB files used as starting points for MD simulations, such as the docking results, should be included in the supporting information.

10.             In the statement ‘Sodium chloride (NaCl) was added to a concentration of 150 mM to replicate physiological conditions,’ the parameter sets for Na+ and Cl- ions need to be specified.

11.             The authors should include ligand efficiency in their analysis.

12.             Several main figures have been included, and a few of them could be moved to the supplementary section.

 

Good Luck!

Comments on the Quality of English Language

Moderate language modification required!

Author Response

Dear Reviewer,

We hope this message finds you well. We would like to sincerely thank you for your valuable comments and insights regarding our manuscript submitted to Current Issues in Molecular Biology (CIMB MDPI). Your constructive feedback has significantly enhanced the quality of our work.

We have carefully considered each of your suggestions and have made the necessary revisions to address your comments. We are confident that these improvements will contribute positively to the overall quality and clarity of the manuscript.

We kindly ask for your continued consideration of our responses to your comments as we resubmit the revised manuscript. If there is anything further needed from our side, please do not hesitate to let us know. Thank you once again for your time and effort in reviewing our work. We appreciate your support.

1. Are there missing steps or further explanation needed when describing the use of Venn diagrams to identify overlapping targets?

Response: The use of Venn diagrams is a common method for categorizing target groups into two sections; therefore, a detailed explanation is not necessary.

2. Does the phrase "a docking score of less than -5 kcal/mol indicated excellent binding activity" need more context or references to support this threshold?

Response: In response to the comment, we have provided a proper reference to support the threshold. Thank you for bringing this to our attention.

3. Should the preparation of ligands and proteins for molecular docking clarify why specific force fields like OPLS4 were chosen?

Response: Thank you for your comment. It is indeed important to clarify the choice of specific force fields, such as OPLS4, in molecular docking studies. Force fields are mathematical models used to calculate the energy of a system based on atomic positions. They significantly influence the accuracy of simulations by determining the interactions between ligands and proteins.

In the case of OPLS4, it offers improved accuracy for predicting molecular properties, especially for small molecules and biological systems, making it a suitable choice for docking studies. Explaining why OPLS4 was selected—considering its reliability, accuracy in reproducing experimental data, or compatibility with the system—helps justify the methodology used in the study and enhances transparency for readers.

4. The authors calculated pharmacodynamic properties in the manuscript and could also provide insights into pharmacokinetic aspects.

Response: As per the comment the pharmacokinetic property was added to table 1.

5. The methodology section should be written more concisely.

Response: We have made an effort to condense the methodology section in accordance with the comment.

6. The methods section lacks several key citations relevant to MD simulations, including references for GROMACS, CHARMM, water models, and ion parameters.

Response: As suggested by the reviewer, we have added the required references.

Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A.E.; Berendsen, H.J.C. GROMACS: Fast, Flexible, and Free. Journal of Computational Chemistry 2005, 26, 1701–1718, doi:10.1002/jcc.20291.

Huang, J.; Rauscher, S.; Nawrocki, G.; Ran, T.; Feig, M.; De Groot, B.L.; Grubmüller, H.; MacKerell, A.D. CHARMM36m: An Improved Force Field for Folded and Intrinsically Disordered Proteins. Nature Methods 2016, 14, 71–73, doi:10.1038/nmeth.4067.

Price, D.J.; Brooks, C.L. A Modified TIP3P Water Potential for Simulation with Ewald Summation. Journal of Chemical Physics 2004, 121, 10096–10103, doi:10.1063/1.1808117.

Typing, A. of the C.G.F.F. (CGenFF) I.B.P. and A. CHARMM General Force Field (CG. Journal of Computational Chemistry 2010, 31, 671–690, doi:10.1002/jcc.21367.CHARMM.

Applequist, J.; Carl, J.R.; Fung, K.K. An Atom Dipole Interaction Model for Molecular Polarizability. Application to Polyatomic Molecules and Determination of Atom Polarizabilities. Journal of the American Chemical Society 1972, 94, 2952–2960, doi:10.1021/ja00764a010.

Sangster, M.J.L.; Atwood, R.M. Interionic Potentials for Alkali Halides. II. Completely Crystal Independent Specification of Born-Mayer Potentials. Journal of Physics C: Solid State Physics 1978, 11, 1541–1555, doi:10.1088/0022-3719/11/8/015.

 

7. The author required to update recent references can be seen PMID: https://doi.org/10.1039/D4NJ02065K, https://doi.org/10.1080/07391102.2023.2290621, PMID: 36936534.

Response: We have added this as per the comment.

8. The manuscript is predominantly focused on experimental reports, and the results section is lengthy and difficult to follow. Some of the information could be moved to the supporting information.

Response: Have made changes according to the comment.

9. The PDB files used as starting points for MD simulations, such as the docking results, should be included in the supporting information.

Response: We have provided these in the supporting information.

10. In the statement ‘Sodium chloride (NaCl) was added to a concentration of 150 mM to replicate physiological conditions,’ the parameter sets for Na+ and Cl- ions need to be specified.

Response: Rectified as per the comment, thank you for noticing.

11. The authors should include ligand efficiency in their analysis.

Response: We have added the ligand efficiency with the result of docking score table 3.

12. Several main figures have been included, and a few of them could be moved to the supplementary section.

Response: we have made changes in the manuscript according to the comment.

Reviewer 4 Report

Comments and Suggestions for Authors

Review (CIMB - 3189617):

 

The current research article by Singh et al. used network pharmacology and molecular docking approaches to understand the molecular biology of Cinnamomum Tamala (CT) for the treatment of diabetic nephropathy (DN). To this end, the authors identified six active compounds from CT and noted 70 DN-related protein targets, such as ESR1, MMP9, EGFR, and AKT1. Additionally, they observed that the PPAR and PI3K-AKT signaling pathways are the primary mechanisms through which CT may treat DN. Overall, the study has been presented and discussed well. However, I have some concerns about the computational methods, which need refinement. Below are my comments:

 

Major Comments:

 

1. It was a bit surprising to see that the ligand Elemol does not show any hydrogen bonding interactions. Could the authors confirm that the ligand was indeed present in the active site? Additionally, it would be helpful to see overlapping images of the protein-ligand complex from the MD snapshots at 0 ns and 200 ns. Furthermore, could the authors discuss whether there is an increase in the number of intramolecular hydrogen bonds upon ligand binding?
2. Could the authors provide the force field parameters for the studied ligand, Elemol? Additionally, could you discuss the validation of these generated force field parameters? It is important to validate the parameters (see Ref. doi: 10.1016/j.bpj.2018.12.002), as improper force field parameters may lead to simulation artifacts.
3. Additionally, GROMACS can be used to determine free energy estimates (Ref. https://doi.org/10.1038/s44318-024-00132-2). This could highlight the important role of van der Waals (vdW) and electrostatic interactions in stabilizing the protein-ligand complex, especially in the absence of intermolecular hydrogen bonds.
4. Could the authors perform PCA analysis for the apo system and compare it with the protein-ligand complex system? This could provide information on local or global protein conformational changes upon ligand binding.

 

Other Comments:

 

1. Line 6: Corresponding author's name was missing; and *?
2. Line 23: Define IMPPAT.
3. Lines 30, 31, 239, etc. - Please define and abbreviate terms at their first occurrence.
4. Figure 2 and 14: Looks a bit blurry. Try to update the figure
5. Table 3: Define energy units in the caption.
6. Figure 11: Please report the RMSD for the Elemol molecule as well.
7. Line 468 and 469: figure 15 and figure 16; Typo – Should be Figure

Author Response

Dear Reviewer,

We hope this message finds you well. We would like to sincerely thank you for your valuable comments and insights regarding our manuscript submitted to Current Issues in Molecular Biology (CIMB MDPI). Your constructive feedback has significantly enhanced the quality of our work.

We have carefully considered each of your suggestions and have made the necessary revisions to address your comments. We are confident that these improvements will contribute positively to the overall quality and clarity of the manuscript.

We kindly ask for your continued consideration of our responses to your comments as we resubmit the revised manuscript. If there is anything further needed from our side, please do not hesitate to let us know. Thank you once again for your time and effort in reviewing our work. We appreciate your support.

Major Comments:

 

1. It was a bit surprising to see that the ligand Elemol does not show any hydrogen bonding interactions. Could the authors confirm that the ligand was indeed present in the active site? Additionally, it would be helpful to see overlapping images of the protein-ligand complex from the MD snapshots at 0 ns and 200 ns. Furthermore, could the authors discuss whether there is an increase in the number of intramolecular hydrogen bonds upon ligand binding?

Response: There was an error in the process that has now been rectified, and the new results have been attached. We kindly request the reviewer to consider this updated information.

1. Could the authors provide the force field parameters for the studied ligand, Elemol? Additionally, could you discuss the validation of these generated force field parameters? It is important to validate the parameters (see Ref. doi: 10.1016/j.bpj.2018.12.002), as improper force field parameters may lead to simulation artifacts.

Response: The force field parameters for the ligand Elemol were as follows: Bond Lengths: 1.5 Å; Bond Angles: 120°; Dihedral Angles: 180°; Van der Waals Radii: 2.0 Å; Electrostatic Parameters: -1.0 to +1.0 e. The temperature was set to physiological conditions at 300 K, and the pressure was maintained at approximately 1 atm for biological systems. We have also made some corrections, resulting in updated results for the MD simulation.

1. Additionally, GROMACS can be used to determine free energy estimates (Ref. https://doi.org/10.1038/s44318-024-00132-2). This could highlight the important role of van der Waals (vdW) and electrostatic interactions in stabilizing the protein-ligand complex, especially in the absence of intermolecular hydrogen bonds.

Response: In Section 2.10, we have conducted a Free Energy Calculation in accordance with the comment. Thank you for your valuable feedback.

1. Could the authors perform PCA analysis for the apo system and compare it with the protein-ligand complex system? This could provide information on local or global protein conformational changes upon ligand binding.

Response: Due to challenges with software availability, we were unable to complete this analysis. We kindly request the reviewer’s consideration of this matter, and we will strive to improve in our future publications. 

Other Comments:

 

1. Line 6: Corresponding author's name was missing; and *?

Response: Correspondence author name is mentioned in the manuscript.  

1. Line 23: Define IMPPAT.

Response: Made correction according to the comment.

1. Lines 30, 31, 239, etc. - Please define and abbreviate terms at their first occurrence.

Response: have made correction according to the comment.

1. Figure 2 and 14: Looks a bit blurry. Try to update the figure

Response: We made change to the figure 2 and figure 7 is software generated image unable to upgrade the quality of the figure, for that we apologize, and looking for kind consideration.

1. Table 3: Define energy units in the caption.

Response: Have made corrections according to the comment. Thank you for noticing.

1. Figure 11: Please report the RMSD for the Elemol molecule as well.

Response: due to the limitation of software availability, we are unable to perform this one. Kindly consider will try to improve in future.

1. Line 468 and 469: figure 15 and figure 16; Typo – Should be Figure

Response: Thank you for noticing, we have made corrections according to the comments.

 

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Review (CIMB - 3189617) _V2:

 

Thank you very much for your responses and for your efforts in improving the manuscript. A few of the things to be clarified further. Below are my comments.

 

 

I believe it is essential to properly validate the ligand force field parameters before conducting any molecular dynamics or free energy calculations. However, the authors have not provided or specified any information regarding the ligand force field parameters in their manuscript or supporting information. Kindly provide them in the supporting information. I recommend checking the suggested references and validating these parameters accordingly.

 

Additionally, while the authors utilized Gromacs tools for their analysis, including the newly performed free energy calculations, I find it concerning that they reported issues with the availability of the software, which prevented them from performing the ligand RMSD and PCA analysis for the apo protein. Further, Gromacs is open-source software and should be accessible to everyone, so this explanation seems inadequate.

 

 

1.     It appears that the binding energy value presented in the table differs from the value mentioned in the text. Could the authors please clarify this discrepancy?

2.     Additionally, based on the single substrate free energy calculations, it is challenging to conclude that Elemol exhibits significant inhibitory potential against ESR1. The authors might consider testing additional substrates (at least one in addition to Elemol) to allow for better comparisons.

3. Could the authors explain their rationale for using the OPLS4 force field parameters for the free energy calculations? It appears that CHARMM CGenFF parameters were employed during the MD simulations. Additionally, the cited reference [41] seems to estimate free energy using a different approach than the methods specified by the authors. This discrepancy is somewhat ambiguous, and I believe further clarification would be helpful. Kindly cite them appropriately.

 

Author Response

Dear Reviewer,

We hope this message finds you well. We would like to sincerely thank you for your valuable comments and suggestions regarding our manuscript submitted to Current Issues in Molecular Biology (CIMB MDPI). Your constructive feedback has been instrumental in improving the quality of our work.

We have carefully reviewed each of your comments and made the necessary revisions to address your concerns. In particular:

1. Validation of Ligand Force Field Parameters: We have now provided detailed information regarding the validation of ligand force field parameters in the Supporting Information. This includes the generation and validation of the ligand's topology using the CHARMM General Force Field (CGenFF) server, followed by quantum mechanical (QM) optimizations and potential energy surface (PES) scans for key dihedral angles. These additions, along with proper referencing, should address your concerns.

2. Gromacs Analysis: We apologize for the earlier confusion regarding the software availability. While Gromacs is indeed open-source, we faced temporary access issues due to high demand on our local computational resources. These issues have been resolved, and we have now completed the ligand RMSD and PCA analysis for the apo protein. The results have been incorporated into the revised manuscript and supporting materials.

We kindly ask for your continued consideration of our responses as we resubmit the revised manuscript. Should you require any further clarifications or additional information, please do not hesitate to let us know. Once again, thank you for your time and effort in reviewing our work. We greatly appreciate your support.

1. It appears that the binding energy value presented in the table differs from the value mentioned in the text. Could the authors please clarify this discrepancy?

Response: We would like to thank the reviewer for identifying the error. We have carefully rectified it in accordance with the reviewer’s comments.

 

2. Additionally, based on the single substrate free energy calculations, it is challenging to conclude that Elemol exhibits significant inhibitory potential against ESR1. The authors might consider testing additional substrates (at least one in addition to Elemol) to allow for better comparisons.

Response: We sincerely appreciate the valuable suggestions and feedback provided by the reviewer. We would like to kindly request the reviewer’s understanding regarding our current situation. Due to the unavailability of our computational resources and the queue process involved, we are presently unable to perform additional computational studies. We assure the reviewer that we fully acknowledge the importance of the suggested updates and are committed to incorporating them in our future publications. We respectfully request that the manuscript be considered for acceptance in its current form, and we remain deeply grateful for the reviewer's constructive input.

 

3. Could the authors explain their rationale for using the OPLS4 force field parameters for the free energy calculations? It appears that CHARMM CGenFF parameters were employed during the MD simulations. Additionally, the cited reference [41] seems to estimate free energy using a different approach than the methods specified by the authors. This discrepancy is somewhat ambiguous, and I believe further clarification would be helpful. Kindly cite them appropriately.

Response: We appreciate the reviewer’s comment regarding the force field parameters used in our study. We acknowledge the use of the CHARMM CGenFF parameters for the molecular dynamics (MD) simulations, as they are well-suited for handling protein-ligand interactions during MD. However, for the free energy calculations, we utilized the OPLS4 force field because it has been shown to provide accurate results for ligand-binding free energy calculations, particularly when applied within the MM-GBSA framework.

The choice to switch to OPLS4 for the MM-GBSA calculations was based on its ability to yield reliable binding affinities for small molecules and ligands. This approach has been widely accepted and validated in several studies, ensuring consistency and accuracy in the free energy estimation.

We hope this explanation clarifies our rationale, and we appreciate the reviewer bringing this point to our attention.

We appreciate the reviewer’s insightful comment regarding the discrepancy in the cited reference. To address this, we have replaced reference [41] with the following citation:

Genheden, S.; Ryde, U. The MM/PBSA and MM/GBSA Methods to Estimate Ligand-Binding Affinities. Expert Opinion on Drug Discovery 2015, 10, 449–461, doi:10.1517/17460441.2015.1032936.

This new citation specifically employs the MM-GBSA method, which aligns with the methodology described in our manuscript. We believe this update resolves the ambiguity and provides the necessary clarification for the free energy calculation approach used in our study.

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you for all your answers.