Effects on Steroid 5-Alpha Reductase Gene Expression of Thai Rice Bran Extracts and Molecular Dynamics Study on SRD5A2

Round 1

Reviewer 1 Report

Authors have greatly improved their paper since the last version. In addition, they have provide answers for all the reviewer's remark. I think now it can be accepted for publication in Biology.

 

Author Response

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Reviewer 2 Report

The manuscript is well structured and implements the knowledge on the field of interest. It describes, in a correct form a lot amount of interesting and innovative results.
I suggest that it could be published in a present form.

Author Response

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Reviewer 3 Report

The paper 'Effects on Steroid 5‐alpha Reductase Genes Expression of Thai Rice Bran Extracts and Molecular Dynamics Study on SRD5A2' provides a combination of experimental and theoretical research on steroid reductase and potential inhibitory/downregulation effect of compounds from rice bran extracts.

The study is interesting and definitely should be published. However, at this stage, it requires further work and improvement. In my opinion, it is not yet ready to be published even in a journal without Impact Factor.

I provide numerous remarks and suggestions in a form of comments in the attached manuscript. Below I provide only 3 major issues

1. Language - the publication is very uneven. There are several parts that are very well written. Some however are written terribly, disqualifying it from publishing in any sensible journal. In most of the cases, I indicated these parts but the authors have to conduct an extensive edition of the text.
2. The authors discuss the twofold biological effect of their extract - a decrease of SRD5A expression level (upon treatment with extract of the model cells) and a potential decrease of the enzyme activity by inhibition (as modeled by MD). These two effects are interwoven while they are completely separate. The authors should clearly decouple these effects and discuss them separately. Furthermore, the finasteride inhibitory effect cannot be compared with tocopherol based on the binding as the former is a sacrificial inhibitor. Upon the formation of the covalent bond, the DG will skyrocket due to the addition of the NADP part.
3. MD simulation of the transmembrane enzyme iw water not in the membrane -see the structure paper:

Xiao, Q., Wang, L., Supekar, S. et al. Structure of human steroid 5α-reductase 2 with the anti-androgen drug finasteride. Nat Commun 11, 5430 (2020). https://doi.org/10.1038/s41467-020-19249-z - see the original structure paper – the enzyme is in the model membrane and ionic strength is modeled at 0.15 M NaCl. Here the protein is modeled in water which for the transmembrane enzyme is simply wrong. Perhaps these very short MD simulations did not change the structure too much one does not know. This is a methodological error. Authors should either repeat the calculation using model membrane or try to explain somehow their approach.

Finally I agree with the authors that the issue of inhibitory effects o tocopherols toward testosterone is important. While I am not suggesting running experimental at this stage the study lacks analysis with testosterone. If tocopherols were to be indeed sensible inhibitors of the SRD5A2 they should exhibit a similar range of DGbinding to really express the inhibitory effect on the substrate.

 

 

Comments for author File: Comments.pdf

Author Response

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Round 2

Reviewer 3 Report

The language of the paper was improved compared to the previous version. Now the paper reads smooth. I do not agree with the authors that presence in the literature of the papers which incorrectly model  membrane proteins justifies following such example. It is apparent to anybody working with proteins that water molecules form very strong H-bond interactions and that influences structure of the protein. However, I agree that the authors aimed at HT method for improving the results of docking... still the crude model of membrane protein with distance dependent dielectric phase that models the membrane and excludes water molecules would be feasible in such HT modelling as well. 

Reluctantly I agree for the publication... 

I also suggest to add to the supplement pdb of the docked protein-ligand complexes so it can be analysed better by the reader. 

 

 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The paper has been considerably improved since the first version.

In my opinion, one information has still to be inserted in the text before publication:

authors have precised that SRD5A2 is a transmembrane protein with the figure2 but the MD simulations have been entirely realised without a membrane model. Since the dynamical behavior of  the protein-ligand complex can be completely different in the membrane compared to solvent phase, a discussion about the results and the lack of the membrane model must be insert in the article and not only to the response to the reviewers.

In addition , the main text needs entire editing by a english writer since some sentences are confusing (i.e. p14 "The results indicated that RMSD values of the SRD5A2 backbone for all ligands oscillated between ~1 and ~3 Å . RMSD of the protein in complex with the ligand,where RMSD the values below 2 Å is considered to have a dynamic stablesystem [88]")

 

Author Response

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Reviewer 2 Report

The authors made a deep investigation of interactions between 3 isoforms of steroid 5-alpha reductase (SRD5A 1-3) and several compounds extracted from rice bran, with finasteride and dutasteride as reference enzyme inhibitors. The hypothesis behind the study is the downregulation of this family of enzymes by inhibition of steroid reduction catalysis. Downregulation has been found associated to androgenetic alopecia (AGA) prevention, since in AGA enzyme overexpression has been observed. The goal is to certify that natural compounds present in selected rice brans have an equilibrated effect on AGA, with less severe side-effects compared to synthetic steroids.

The work combines in vitro experimental studies, starting from tocopherol extraction from rice bran, with in silico studies based on routine molecular docking simulations. The latter study was limited to SRD5A2, performed for 9 extracted compounds and compared to finasteride and dutasteride reference
compounds, known to be strong SRD5A2 inhibitors. The promising tocopherol compounds (as from lowest-energy docking pose) were finally the object of a short (50 ns) MD simulation in water solvent. Alpha, beta,
and gamma tocopherol single molecules were compared with finasteride. The energy components (only last configuration obtained by MD) were analyzed by MM/PBSA routine approximations, also in water solvent.

This reviewer finds the manuscript well designed in the combination of experiments and models. Limitations due to the choice of initial structure of the enzyme (ref. 34) in the closed form (assuming NADP binds first) are now discussed. On the other hand, the bad choice of protein environment both in MD simulations and in MM/PBSA calculations has not been circumvented, neither commented. I notice that in Ref.34, both for crystallization and MD simulations the membrane environment was required. In MD simulations reported in Ref.34 the environment was POPC.

The 50-ns stability of the TM domain reported in the manuscript is a common effect of the bias of initial configuration, otherwise the crystallization solvent would be water and not the complicate recipe of Ref.34.

As a simple work-around, the short metastable MD simulations can be kept (the usage of an explicit membrane requires huge resources), but the MM/PBSA calculation might be repeated in a membrane-like environment, that is crucial especially in the electrostatic part.

This reviewer thinks that the main result of the work would not change, but for sure the MM/PBSA calculation assuming water as the environment has no scientific basis.

Author Response

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Round 2

Reviewer 2 Report

The authors did not perform the required corrections.