Development of DHODH inhibitors incorporating virtual screening, pharmacophore modeling, fragment-based optimization methods, ADMET, molecular docking, molecular dynamics, PCA analysis, and free energy landscape

Qu Wang; Yu Hao Xu; Heng Jiang; Biao Deng; Zhu Liang

doi:10.1371/journal.pone.0342461

Peer Review History

Original SubmissionJune 10, 2025
4 Jul 2025 Decision Letter - Peter Mbugua Njogu, Editor Dear Dr. Liang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Address all comments, suggestions and revisions recommended by our reviewers. ============================== Please submit your revised manuscript by Aug 18 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Peter Mbugua Njogu, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. 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Thank you for stating the following financial disclosure: “This project was supported by Zhanjiang Competitive Science and Technology Project (2021A05076); Clinica Research Program, Affiliated Hospital of Guangdong Medical University (LCYJ2018B001); Clinical Research Program, Affiliated Hospital of Guangdong Medical University (LCYJ2021A004); Cohort Study Program of Affiliated Hospital of Guangdong Medical University (LCYJ2022DL003); General Project of Guangdong Provincial Bureau of Traditional Chinese Medicine (2022107).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. We notice that your supplementary figures are uploaded with the file type 'Other'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. Additional Editor Comments: Review the reviewers' comment and address each as widely and deeply as possible. Additionally: 1. Identify Compounds 65 and 66 by chemical name. 2. What was the basis for the selection of the 6,140 molecules for screening? 3. Is the crystal structure of DHODH complexed with MEDS433 available? Explain why this model was the most appropriate for this research work. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: No ****** Reviewer #1: Comments to the authors 1. The abstract should be revised to improve clarity and include specific numerical data such as screening size, docking scores, binding energies, and the final selected compound. 2. The details on the fragment replacement strategy should be included. It should specify which fragments were replaced in each lead compound and how the seven potent candidates were modified. 3. Include the significance of the selection of the SPC water model in molecular dynamics simulations. 4. True positive (TP) and false negative (FN) values are not reported in the pharmacophore model validation; these should be included to substantiate model performance. 5. Table 5 is not appropriate as compound names are missing. The manuscript contains numerous grammatical issues and inconsistencies in tense and requires thorough language editing for clarity. e.g., "we utilised virtual screening..." should be changed to "Virtual screening was employed..." 6. Discuss the limitations or future directions of the study in the manuscript. 7. Supplementary data not provided Reviewer #2: Dear Editor, I commend the authors for submitting the manuscript "Development of DHODH inhibitors incorporating virtual screening, pharmacophore modeling, fragment-based optimization methods, ADMET, molecular docking, molecular dynamics, PCA analysis, and free energy landscape" (ID PONE-D-25-31041). This study presents a computational perspective on drug design of DHODH inhibitors. I have identified several significant weaknesses in the manuscript that must be addressed before it can be considered for publication in PLOS ONE. The manuscript is unsuitable for publication in its current form. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: report-PONE-D-25-31041.pdf https://doi.org/10.1371/journal.pone.0342461.r001
Revision 1
5 Aug 2025 Author Response 1. Identify Compounds 65 and 66 by chemical name. Response: Thank you for the constructive comments. We have already referred to the chemical names of compounds 65�[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl 3-(4-{[(2S)-2-hydroxypropyl]oxy}phenyl) and 66�[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl 4-(4-{[(2S)-2-hydroxypropyl]oxy}phenyl), but for the sake of conciseness in the subsequent description, we still choose to use compounds 65 and 66. 2. What was the basis for the selection of the 6,140 molecules for screening? Response: Thank you for the constructive comments. This FDA data is provided by relevant manufacturers and is a database of 6,140 compounds established taking into account the reliability of reusing old drugs. 3. Is the crystal structure of DHODH complexed with MEDS433 available? Explain why this model was the most appropriate for this research work. Response: Thank you for the constructive comments. This structure is available. Additionally, the structure selected in this article is based on published literature and has been cited in the main text. We have made all the term substitutions in the manuscript. “The X-ray structure of DHODH complexed with the 2-Hydroxypyrazolo[1,5-a] pyridine inhibitor, MEDS433, was chosen as the structural model for DHODH (PDB code: 6FMD). Additionally, the docking active sites were determined concerning the study previously published by Galati S et al(1). (Lines 62-65) 4. The abstract should be revised to improve clarity and include specific numerical data such as screening size, docking scores, binding energies, and the final selected compound. Response: Thank you for the constructive comments. We have rewritten the description of the abstract. (Lines 9-21) 5. The details on the fragment replacement strategy should be included. It should specify which fragments were replaced in each lead compound and how the seven potent candidates were modified. Response: Thank you for the constructive comments. We have made all the term substitutions in the manuscript. We have already provided a detailed description of the replacement options for FBDD in the method section. (Lines 107-113) 6. Include the significance of the selection of the SPC water model in molecular dynamics simulations. Response: Thank you for the constructive comments. Our view on the SPC water model is as follows: Although the SPC water model is a simplified model, it can reasonably describe the core properties of water and meet the basic requirements of most molecular dynamics simulations. Structural characterization. The calculation results of the radial distribution function (RDF) of the SPC/E model show that the SPC model can reveal the short-range ordered characteristics of the hydrogen bond network between water molecules through the RDF peak positions of atomic pairs such as O-O and O-H (for example, the first peak position of O-O in liquid water reflects the tight packing under the action of hydrogen bonds). And the structural differences between liquid and solid (ice) (short-range order vs. long-range order) provide a basis for understanding the microstructure of condensed matter. 7. True positive (TP) and false negative (FN) values are not reported in the pharmacophore model validation; these should be included to substantiate model performance. Response: Thank you for the constructive comments. We conducted machine learning model calculations for the true positives and false negatives of the pharmacophore and presented them in the methods and results sections of this paper. (Lines 90-94) 8. Table 5 is not appropriate as compound names are missing. The manuscript contains numerous grammatical issues and inconsistencies in tense and requires thorough language editing for clarity. e.g., "we utilised virtual screening..." should be changed to "Virtual screening was employed..." Response: Thank you for the constructive comments. We have completed the information in the table and made corrections to the inappropriate expressions in the text. 9. Discuss the limitations or future directions of the study in the manuscript. Response: Thank you for the constructive comments. In the discussion section, we re-examined the limitations of this research method and the directions that can be experimentally verified subsequently. (Lines 477-486) 10. Supplementary data not provided Response: Thank you for the constructive comments. We have refined the supplementary data. Major Points 11) On Abstract: Provide more quantitative results and analysis. No crucial result is presented. Response: Thank you for the constructive comments. We have rewritten the summary and described the key data in it. (Lines 9-21) 12) On Introduction: According to the authors “Numerous dihydroorotate dehydrogenase inhibitors (DHODH) have advanced to clinical trials to treat various oncological conditions”; however, this claim is not supported by any references. Appropriate citations should be provided to substantiate this statement. The introduction should present the primary goal, as well as the tools used to achieve it. Response: Thank you for the constructive comments. We have already refined the relevant references for this sentence in the introduction. 13 On Materials and Methods: 3.1 Most critical point: The selected PDB structure (6FMD) contains FMN as a cofactor. However, it is unclear how this cofactor was treated in the classical simulations, including both molecular docking and molecular dynamics. The authors should clarify whether FMN was retained, removed, or parameterized, and how its presence may have influenced the results. Response: Thank you for the constructive comments. For the FMN structure and others, we had already cleaned and removed water from the 6FMD structure on PyMOL before the docking. Therefore, we do not need the FMN structure. (Lines 63-65) 13.2 Proper reference for Discovery Studio. I suggest other programs for ADMET (e.g. SwissADME). Response: Thank you for the constructive comments. We have already cited relevant literature for the ADMET section of Discovery Studio. (Lines 115-118) 13.3 The authors should be more rigorous in describing the methodological procedures adopted. Some essential pieces of information are missing, such as the coordinates of the centre of mass and the size of the search box used in the molecular docking simulations; It’s not clear how were docking procedures validated, so I recommend use a consensual docking procedure; How were pKa values computed for computational simulations (e.g. PROPKA method)? Response: Thank you for the constructive comments. We have described the docking operations we performed more precisely in the method, including the size and coordinates of the docking box. Additionally, we introduced Autodock Vina for the verification of the docking. We also conducted verifications for the Pka values and summarized them in the table. (Lines 125-129) and (Lines 184-192) 13.4 To run MD of each system in triplicate (at least 200 ns each). The authors should explain how the selected ensemble from molecular dynamics (MD) simulations was chosen for binding free energy (BEF) analysis and whether it is statistically robust. Response: Thank you for the constructive comments. We re-conducted three 200ns dynamic simulations and carried out numerous analyses on these six models. Meanwhile, FEL and others were also recalculated. (Lines 503-539) 13.5 I recommend including key analyses such as molecular fingerprints, followed by residual decomposition analysis; Response: Thank you for the constructive comments. For the stability analysis of the docking model and the specific analysis of residues, we have conducted analyses such as the residue analysis of RMSF and G-mmpbsa, and all the above analyses have been carried out three times. (Lines 503-539) 13.6 Include DUH (crystal inhibitor) as a reference in all simulations. Response: Thank you for the constructive comments. We believe that the binding energy performance of the known inhibitors in precise docking is not good and is much higher than that of potential compounds. Therefore, we think it is not necessary to introduce the positive control complex group selected in this paper into the kinetic simulation. (Lines 503-539) Minor Points 14) To include PDB and input files as supplementary material. Response: Thank you for the constructive comments. We have completed the above requirements in the supplementary materials. 15) English review Response: Thank you for the constructive comments. We have polished and revised the description of the article. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0342461.r002
13 Sep 2025 Decision Letter - Peter Mbugua Njogu, Editor Dear Dr. Liang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. A few major issues remain as highlighted by our reviewers: Provide the systematic names of lead Compounds 65 and 66 in the body text. Address to the fullest extent the technical concerns raised by Reviewer 2. In doing this, make reference to the Review comments provided in the first review of this manuscript. In particular, explain how you handled the role of flavin mononucleotide as an essential cofactor in the enzymatic activity of DHODH. This is important since the PDB structure utilized in this study PDB structure (6FMD) contains FMN as a cofactor. Why would the computational simulations not require FMN and how did this affect the results? plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Peter Mbugua Njogu, Ph.D. Academic Editor PLOS ONE Journal Requirements: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: (No Response) Reviewer #2: No ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: No ****** Reviewer #1: The manuscript Title "Development of DHODH inhibitors incorporating virtual screening, pharmacophore modeling, fragment-based optimization methods, ADMET, molecular docking, molecular dynamics, PCA analysis, and free energy landscape". The authors addressed all the comments in scientific way with clear explanation. So, I recommended this manuscript for acceptance 1. Chemical structures and systematic names of lead compounds especially. 65 and 66 are absent from main tables and figures that reducing transparency and hindering external validation. Author is instructed that Please provide 2D structures and systematic names directly in the main manuscript, not just in reviewer responses or supporting files. Reviewer #2: Dear Editor, In the revised version of the manuscript entitled “Development of DHODH inhibitors incorporating virtual screening, pharmacophore modeling, fragment-based optimization methods, ADMET, molecular docking, molecular dynamics, PCA analysis, and free energy landscape” (ID: PONE-D-25-31041R1), the authors have only partially addressed my previous concerns. Unfortunately, the most critical issue remains unresolved. I have also identified several additional weaknesses that must be carefully addressed before the manuscript can be considered for publication in PLOS ONE. In its current form, the manuscript remains unsuitable for publication. See PDF file with details. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: report-PONE-D-25-31041-R1.pdf https://doi.org/10.1371/journal.pone.0342461.r003
Revision 2
30 Oct 2025 Author Response Reviewers' comments: Reviewer #(Remaeks to the Author): 1. Identify Compounds 65 and 66 by chemical name. Response: Thank you for the constructive comments. We have already referred to the chemical names of compounds 65�[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl 3-(4-{[(2S)-2-hydroxypropyl]oxy}phenyl) and 66�[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl 4-(4-{[(2S)-2-hydroxypropyl]oxy}phenyl), We have already added the corresponding chemical names in the main text. 2. What was the basis for the selection of the 6,140 molecules for screening? Response: Thank you for the constructive comments. This FDA data is provided by relevant manufacturers and is a database of 6,140 compounds established taking into account the reliability of reusing old drugs. 3. Is the crystal structure of DHODH complexed with MEDS433 available? Explain why this model was the most appropriate for this research work. Response: Thank you for the constructive comments. This structure is available. Additionally, the structure selected in this article is based on published literature and has been cited in the main text. We have made all the term substitutions in the manuscript. “The X-ray structure of DHODH complexed with the 2-Hydroxypyrazolo[1,5-a] pyridine inhibitor, MEDS433, was chosen as the structural model for DHODH (PDB code: 6FMD). Additionally, the docking active sites were determined concerning the study previously published by Galati S et al(1). (Lines 62-65) 4. The abstract should be revised to improve clarity and include specific numerical data such as screening size, docking scores, binding energies, and the final selected compound. Response: Thank you for the constructive comments. We have rewritten the description of the abstract. (Lines 9-21) 5. The details on the fragment replacement strategy should be included. It should specify which fragments were replaced in each lead compound and how the seven potent candidates were modified. Response: Thank you for the constructive comments. We have made all the term substitutions in the manuscript. We have already provided a detailed description of the replacement options for FBDD in the method section. (Lines 107-113) 6. Include the significance of the selection of the SPC water model in molecular dynamics simulations. Response: Thank you for the constructive comments. Our view on the SPC water model is as follows: Although the SPC water model is a simplified model, it can reasonably describe the core properties of water and meet the basic requirements of most molecular dynamics simulations. Structural characterization. The calculation results of the radial distribution function (RDF) of the SPC/E model show that the SPC model can reveal the short-range ordered characteristics of the hydrogen bond network between water molecules through the RDF peak positions of atomic pairs such as O-O and O-H (for example, the first peak position of O-O in liquid water reflects the tight packing under the action of hydrogen bonds). And the structural differences between liquid and solid (ice) (short-range order vs. long-range order) provide a basis for understanding the microstructure of condensed matter. 7. True positive (TP) and false negative (FN) values are not reported in the pharmacophore model validation; these should be included to substantiate model performance. Response: Thank you for the constructive comments. We conducted machine learning model calculations for the true positives and false negatives of the pharmacophore and presented them in the methods and results sections of this paper. (Lines 90-94) 8. Table 5 is not appropriate as compound names are missing. The manuscript contains numerous grammatical issues and inconsistencies in tense and requires thorough language editing for clarity. e.g., "we utilised virtual screening..." should be changed to "Virtual screening was employed..." Response: Thank you for the constructive comments. We have completed the information in the table and made corrections to the inappropriate expressions in the text. 9. Discuss the limitations or future directions of the study in the manuscript. Response: Thank you for the constructive comments. In the discussion section, we re-examined the limitations of this research method and the directions that can be experimentally verified subsequently. (Lines 477-486) 10. Supplementary data not provided Response: Thank you for the constructive comments. We have refined the supplementary data. Major Points 11) On Abstract: Provide more quantitative results and analysis. No crucial result is presented. Response: Thank you for the constructive comments. We have rewritten the summary and described the key data in it. (Lines 9-21) 12) On Introduction: According to the authors “Numerous dihydroorotate dehydrogenase inhibitors (DHODH) have advanced to clinical trials to treat various oncological conditions”; however, this claim is not supported by any references. Appropriate citations should be provided to substantiate this statement. The introduction should present the primary goal, as well as the tools used to achieve it. Response: Thank you for the constructive comments. We have already refined the relevant references for this sentence in the introduction. 13 On Materials and Methods: 3.1 Most critical point: The selected PDB structure (6FMD) contains FMN as a cofactor. However, it is unclear how this cofactor was treated in the classical simulations, including both molecular docking and molecular dynamics. The authors should clarify whether FMN was retained, removed, or parameterized, and how its presence may have influenced the results. Response: Thank you for the constructive comments. For the FMN , we preserved the structure and described the specific processing operations in the text, as well as recalculated the subsequent simulations. 13.2 Proper reference for Discovery Studio. I suggest other programs for ADMET (e.g. SwissADME). Response: Thank you for the constructive comments. We have already cited relevant literature for the ADMET section of Discovery Studio. (Lines 115-118) 13.3 The authors should be more rigorous in describing the methodological procedures adopted. Some essential pieces of information are missing, such as the coordinates of the centre of mass and the size of the search box used in the molecular docking simulations; It’s not clear how were docking procedures validated, so I recommend use a consensual docking procedure; How were pKa values computed for computational simulations (e.g. PROPKA method)? Response: Thank you for the constructive comments. We have described the docking operations we performed more precisely in the method, including the size and coordinates of the docking box. Additionally, we introduced Autodock Vina for the verification of the docking. We also conducted verifications for the Pka values and summarized them in the table. (Lines 125-129) and (Lines 184-192) 13.4 To run MD of each system in triplicate (at least 200 ns each). The authors should explain how the selected ensemble from molecular dynamics (MD) simulations was chosen for binding free energy (BEF) analysis and whether it is statistically robust. Response: Thank you for the constructive comments. We reconducted three 200ns dynamic simulations and carried out numerous analyses on these six models. Meanwhile, FEL and others were also recalculated. (Lines 503-539) 13.5 I recommend including key analyses such as molecular fingerprints, followed by residual decomposition analysis; Response: Thank you for the constructive comments. For the stability analysis of the docking model and the specific analysis of residues, we have conducted analyses such as the residue analysis of RMSF and G-mmpbsa, and all the above analyses have been carried out three times. (Lines 503-539) 13.6 Include DUH (crystal inhibitor) as a reference in all simulations. Response: Thank you for the constructive comments. We added the simulation results of DUH (crystal inhibitor) in the simulation operations such as molecular docking and kinetic simulation (Lines Minor Points 14) To include PDB and input files as supplementary material. Response: Thank you for the constructive comments. We have completed the above requirements in the supplementary materials. 15) English review Response: Thank you for the constructive comments. We have polished and revised the description of the article. Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0342461.r004
26 Jan 2026 Decision Letter - Peter Mbugua Njogu, Editor Development of DHODH inhibitors incorporating virtual screening, pharmacophore modeling, fragment-based optimization methods, ADMET, molecular docking, molecular dynamics, PCA analysis, and free energy landscape PONE-D-25-31041R2 Dear Dr. Liang, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Peter Mbugua Njogu, Ph.D. Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0342461.r005
Formally Accepted
Acceptance Letter - Peter Mbugua Njogu, Editor PONE-D-25-31041R2 PLOS One Dear Dr. Liang, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Peter Mbugua Njogu Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0342461.r006

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