Review History


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Summary

  • The initial submission of this article was received on September 27th, 2019 and was peer-reviewed by 2 reviewers and the Academic Editor.
  • The Academic Editor made their initial decision on November 4th, 2019.
  • The first revision was submitted on June 9th, 2020 and was reviewed by 1 reviewer and the Academic Editor.
  • A further revision was submitted on August 17th, 2020 and was reviewed by the Academic Editor.
  • The article was Accepted by the Academic Editor on August 18th, 2020.

Version 0.3 (accepted)

· Aug 18, 2020 · Academic Editor

Accept

Thank you for carefully responding to the reviewers' concerns.

Version 0.2

· Jun 24, 2020 · Academic Editor

Minor Revisions

Please address the remaining questions and suggestions raised by the reviewer. Thank you.

Reviewer 1 ·

Basic reporting

The writing is much better in this revision, but there are still some grammar and/or typo issues. I have tried to note most of them below.
1. Figure 1B, 1D, 2B, and 2D need explanations of the color scheme being used for the electrostatic potential surfaces.
2. Some of the residue labels in Figure 2A and 2C are not easily readable, especially the D49.A because of the red text over red VDW atoms.
3. Line 159-160 – Add the word ‘for’ between the words ‘enhances affinity’ and ‘sulfonamides’
4. Line 270 – ‘that’ should be ‘than’
5. Line 277 – delete the word it’s
6. Line 293 – delete the word ‘for’
7. Figure 4F appears to be missing some data. The graph does not fully extend all the way to 400 ns.
8. Why are only 400 ns of the simulation data shown in Figure 4? Based on lines 292-293, I assume the first 100 ns of the simulation are not shown, I just do not understand the reasoning behind this omission.
9. For consistency, all the graphs in Figure 4 should have the same range for the y-axis.
10. The authors should be consistent with how they identify specific amino acids. For example, in lines 132, 135, 383, 444, 471, and several times in lines 409-415, the authors use one-letter codes and residue number (e.g. Y48). Contrarily, on lines 339-344, the authors use all-caps 3-letter codes with residue number (e.g. TYR48). This same inconsistency is found in the figures.
11. Lines 349 – ‘and’ should be ‘are’
12. Figure 9 is probably best represented as a table instead.
13. The resolution of the graphs in Figures 10 and 11 is poor and should be improved prior to publication.
14. Line 359 – The authors refer to their energy decomposition as ‘Pairwise’, which would suggest the energies correspond to the interaction energy between two different residues. However, the data appears to show a ‘per-residue’ decomposition since the energies are reported as the energy of a single residue, and not it paired with another residue. This should be clarified by the authors.
15. Line 373 – enzyme should be enzymes
16. Line 388 – IC50 needs to have the 50 appropriately subscripted
17. Line 402 – “…formation 4 hydrogen…” should be “…formation of four hydrogen…”
18. Lines 445 – remove ‘an’
19. Lines 463 – Plasmodium should be italicized

Experimental design

Overall, the experimental design is very straightforward and reasonable. And the methods are explained well by the authors. I have only one main criticism of the experimental design, and that is concerning the details of the MM-PBSA calculation, as noted below.

1. For MM-PBSA calculations, why were exactly 625 snapshots chosen? And why only 625 snapshots? And why randomly? And how were the frames “randomly” chosen? And why did these snapshots only come from 200-300 ns of the trajectory (as described on line 319).

Validity of the findings

Most of the conclusions drawn from the current data meets the journals standards. However, I found one area where conclusions are drawn that may be missing part of the explanation based on the data presented.

1. Lines 331-332 – The authors mention the importance of the desolvation free energy term contributes to the observed enhanced binding for pTPI over hTPI, but the authors neglect to mention that the ΔEvdW term also contributes significantly (Table 2), and possibly more so than the desolvation free energy.

Additional comments

This study is interesting and has the potential to be intriguing to many people in the field. I believe the research has high impact capability. The authors have done a fantastic job of responding to my previous comments. I am very impressed with the amount of work the authors put into the revisions and the paper is significantly better than the previous version. I applaud their work, especially knowing it is being performed at a teaching undergraduate university (I also work at a PUI). I have only minimal comments/suggestions for the current version, and once those are addressed, I emphatically support the publication of this manuscript. Great work!

Version 0.1 (original submission)

· Nov 4, 2019 · Academic Editor

Major Revisions

The reviewers have identified several critical issues with your manuscript that need to be addressed for further consideration for publication in PJPC. Most notably, I would like to see the concerns of Reviewer #2 addressed regarding the relevancy of the sulfonamides studied. One way to do this would be to include similar computational studies of known inhibitors of Pf TPI for comparison. Even better would be inclusion of experimental data.

Reviewer 1 ·

Basic reporting

The hypothesis for the study is not clear based only on the introduction. The authors should revise the last paragraph of the into to include a clear hypothesis and/or a summary of main results/conclusions.
The English is unclear in certain places. I have tried to note most of them below.
1. Line 52. you should change curing to treating
2. Line 67. put parentheses around Figure 1B
3. Line 67. Delete the semicolon
4. Line 79. There is a typo or missing word or two in this sentence because it does not currently make sense as it is.
5. Line 64. Add the PDB codes after listing the structure of hTPI and pTPI.
6. Line 81. Delete ". This is" to combine the two sentences
7. Line 83. change it to it's
8. Line 113. Add 'the' after "The docking scores for "
9. Line 118. I believe B3LY0 should be B3LYP for the level of theory
10. Line 124. This sentence is missing a verb.
11. Line 126. remove the comma
12. Line 153. Delete the comma
13. Line 169-173. Equations 4 and 6 are never referenced in the text of the manuscript.
14. Line 171. You skipped Equation 5. There is an Eqn 4 and 6, but no Eqn 5.
15. Line 177. This sentence is incomplete. Or maybe the word 'that' needs to be deleted. It is unclear to the reader.
16. Line 182. Delete the word represents
17. Line 187. Delete the semicolon and change 'and' to 'an' following where that semicolon is currently.
18. Line 196. The I for ionic strength needs to be capitalized.
19. Line 197. It should be “water, respectively) and…”
20. Line 229. (Figure 3, right panel) should just be (Figure 3B)
21. Line 256. kJ/mol does not need to be italicized
22. Line 264. “more restrain” is not grammatically correct, even if I’m reading it the way the authors intended
23. Line 268. “thus repulsion the ligand sulfaE” – this doesn’t make sense and is likely a typo or incorrect form of the word repulsion
24. Line 323. Remove the ‘and’ after the parentheses
25. Lines 517-521. This reference is not correct. At the very least, no year is listed. The issue and volume may also be incorrect, but maybe it’s just a new reference that is online only currently?
26. Line 391. The use of 'buy' in this sentence is not correct.
27. Figures 1 and 2. The amino acids being explicitly displayed in Figures 1 and 2 should be labelled accordingly.

Experimental design

I have ordered the flawed sections of the experimental design below by priority (1 being highest priority).
1. The authors should run simulations of the apo (i.e. no ligand) pTPI and hTPI. Several comments in the text (see lines 225-227, 236-237, 245-247), make conclusions on what happens to the structure of TPI when the ligand binds. However, since the authors did not simulate the apo TPI structures, they do not have a set of simulations to compare with their ligand-bound simulations. Thus, the conclusions based on those findings cannot be validated.
2. Why do the authors only run 100 ns for each simulation? With the size of the systems, the authors could easily run each to at least 1000 ns. The RMSD plots (Figure 3) suggest that none of the simulations have reached a stable structural conformation with the bound ligand. Instead, the RMSD values continue to increase as the simulation progresses up until the end (100 ns). This suggests the protein-ligand complexes have not reached a stable structure (i.e. converged). Thus, the authors should extend their simulations well beyond 100 ns until the structures reach a stable conformation that the authors can trust and verify.
3. The authors should perform a hydrogen bonding analysis on their simulations. On multiple occasions (Lines 361 and 349) they mention the stabilizing interactions as polar and/or hydrogen bonds, but the authors do not support these statements with the corresponding analysis. The authors should report percent occupancies and lifetimes for any hydrogen bonds relevant to protein-ligand stability.
4. Why was sulfaE chosen as the representative ligand to test? The authors should make this clear. And if there is no obvious reason, the reviewers should run additional simulations to include the other two ligands mentioned from the previous work.
5. It is unclear how many frames/snapshots were used for MM-PBSA calculations for each simulation. Line 210 says that 20 snapshots were used. from each simulation. However, line 217 says that about 2000 conformations were used for MM-PBSA (is this per simulation or total across the four simulations?). If only 20 snapshots were used per simulation, that's only 80 snapshots. So where did the 2000 come from? In my experience, a minimum of 200 frames must be analyzed to get reasonable results from MM-PBSA.
6. At the very least, they authors should explain why they chose not to include entropic calculations in their methods. On lines 338-340, the authors mention the potential effect of entropy on ligand affinity. The authors should calculate the entropy of the systems using snapshots from their simulations to support their conclusion here.
7. Line 143 states that 6000 water molecules were added for each simulation. Was it precisely 6000, or is that an approximation? If it is not exact, the authors should clarify.
8. Line 192. The Poisson-Boltzmann equation. Did the authors use the linear or non-linear form of the PB equation? And why? The authors should justify the reasoning for their choice.

Validity of the findings

Most of the conclusions drawn from the current data meets the journals standards. However, there are certain areas where conclusions are drawn and/or statements are made that do not have data to back up the claims. These areas have all been highlighted in the comments on the Experimental Design, but I will restate them here for completeness.
1. I am skeptical of the validity of one of the simulations and/or the MM-PBSA results. Specifically, the simulation with sulfaE bound to the hTPI dimer interface. The MM-PBSA results state the average binding free energy is +35.86 kJ/mol. This value suggests the ligand and protein are incompatible and are energetically unfavorable for binding. If that is the case, it would be expected that the ligand should dissociate from the binding pocket of the dimer interface to mitigate this unfavorable bound state. However, the authors make no note or mention of the dynamics of this simulation, or the physical meaning of this energetic unfavorability. The polar solvation term is clearly the leading cause of this energetic instability. However, the authors do not explain why the polar solvation would tend to be so positive for these complexes, and especially so for the hTPI dimer interface simulation. Such a high positive binding free energy suggests 1) something is fundamentally wrong with the simulation, or 2) the MM-PBSA calculation did not use the proper variables to accurately represent the complex. Without seeing the simulations up close, it's difficult to suggest which of these may be the culprit of these poor results. Either way, I'm highly skeptical of these energies as they are being reported.
2. Several comments in the text (see lines 225-227, 236-237, 245-247), make conclusions on what happens to the structure of TPI when the ligand binds. However, since the authors did not simulate the apo TPI structures, they do not have a set of simulations to compare with their ligand-bound simulations. Thus, the conclusions based on those findings cannot be validated.
3. The RMSD plots (Figure 3) suggest that none of the simulations have reached a stable structural conformation with the bound ligand. Instead, the RMSD values continue to increase as the simulation progresses up until the end (100 ns). This suggests the protein-ligand complexes have not reached a stable structure (i.e. converged). Thus, the authors should extend their simulations well beyond 100 ns until the structures reach a stable conformation that the authors can trust and verify.
4. On multiple occasions (Lines 361 and 349) mention the stabilizing interactions as polar and/or hydrogen bonds, but the authors do not support these statements with the corresponding analysis. The authors should report percent occupancies and lifetimes for any hydrogen bonds relevant to protein-ligand stability.
5. On lines 338-340, the authors mention the potential effect of entropy on ligand affinity. The authors should calculate the entropy of the systems using snapshots from their simulations to support their conclusion here.

Additional comments

This study is interesting and has the potential to be intriguing to many people in the field. I believe the research has high impact capability. However, I believe the manuscript currently is not ready to be accepted because it has a number of flaws. The experimental design is lacking many important steps that would help make the project complete. Additionally, several of the conclusions are stated without proper evidence to support them. Hopefully, the suggestions I have given seem reasonable and you are able to perform the necessary revisions to complete the manuscript.

Reviewer 2 ·

Basic reporting

1. Professional English is used throughout the manuscript. There are some grammatical and formatting errors in the manuscript.

2. The citations are sufficient and the information presented in the introduction is broad and somewhat vague.

Experimental design

1. The research is within the aims and scope of the journal.

2. The research question is well-defined but it is not particularly meaningful, and it does not fill any knowledge gap as far as I know.

3. The methods are sufficiently described.

Validity of the findings

The focus of the work is to explore the interactions between pTPI/hTPI and sulfonamides using docking and MD simulations. There is no experimental evidence that 'sulfonamides' in general and in particular the 'antimalarial sulfonamides' used in the work can inhibit or bind to TPI. Without such premise, using modeling to explore interactions quite frankly does not contribute any new useful knowledge or hypothesis.

If the compounds were being consider as potential inhibitors of TPI, it would be better to use modeling and/or experimental tools to compare them with known inhibitors and non-inhibitors.

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