Date: 19.08. 2024

To

Editor-in-Chief

Plos One

Subject: Submission of the revised manuscript

Dear Sir,

We have carried out essential revisions of our manuscript entitled “Structure-based Virtual Screening of Trachyspermum ammi Metabolites Targeting Acetylcholinesterase for Alzheimer's Disease Treatment” suggested by you and the reviewers and would like to submit the 1st-revised version. We believe these comments/suggestions have significantly improved our manuscript. Please find enclosed the rebuttal letter where we have addressed all questions and comments to the reviewer, which are indicated as yellow highlights in the manuscript. Please be noted that all authors consented and approved the revised version of the manuscript. We believe the updated version will be worthy of publishing in your journal.

Thanking you in advance for considering our work, I remain,

Sincerely,

Dr. Sumon Ganguli

Professor

Department of Applied Chemistry and Chemical Engineering, Faculty of Science,

University of Chittagong, Chittagong, 4331, Bangladesh

Email: sumonganguli@cu.ac.bd

(Corresponding author on behalf of all authors) 

Author Responses to the Reviewers’ Comments

Reviewer #1:

Reviewer’s comment: Alzheimer's disease (AD) has seen a rapid global increase, highlighting the need for new treatments due to the side effects of current anti-acetylcholinesterase (AChE) drugs. This study identified potent anti-AChE compounds from 150 bioactive phytochemicals in Trachyspermum ammi using structure-based drug design and in-silico tools. Compounds were screened for drug-likeness, bioavailability, and toxicity, followed by molecular docking and 100-nanosecond molecular dynamics simulations to assess stability. Viridifloral, 2-Methyl-3-glucosyloxy-5-isopropyl phenol, Alpha-Curcumene, and Sterol showed high AChE affinity and stable interactions. These candidates also met Lipinski’s rule and ADMET criteria, with DFT optimization confirming favorable reactivity and stability. The study proposes these compounds as potential novel AChE inhibitors for AD treatment, warranting further in-vitro and in-vivo validation for efficacy and safety. Although the study provided interesting insights, further revisions are recommended.

The author(s) response: We would like to express our sincere gratitude for your insightful comments and suggestions. We have carefully reviewed and addressed each point raised. Specifically, we have made revisions to the molecular docking analysis by re-evaluating the binding interactions, which has enhanced the accuracy and interpretation of the docking results within the manuscript. In addition, to further improve the discussion section, we have expanded our analysis on the implications of the identified compounds in Alzheimer's disease treatment, which contributes to a more comprehensive understanding of their potential therapeutic benefits.

We also conducted a thorough review of the entire manuscript, making minor edits to enhance clarity, consistency, and overall flow. These revisions include refining the language for better readability and ensuring that all data presented is precise and well-organized. We believe these revisions have significantly strengthened our study and more effectively highlight the potential of the identified compounds—Viridifloral, 2-Methyl-3-glucosyloxy-5-isopropyl phenol, Alpha-Curcumene, and Sterol—as novel AChE inhibitors for Alzheimer's disease treatment.

We are grateful for your constructive feedback and hope that the revised manuscript meets your expectations. Thank you once again for your invaluable input, which has undoubtedly contributed to the improvement of our work. We look forward to your further assessment and hope for a favorable review.

Reviewer’s comment: The present study lacks depth, focusing solely on phytochemicals targeting AChE. AD is a complex pathology primarily due to misfolded proteins (Beta-amyloid and tau). Targeting AChE alone does not suffice for publication, as dysregulation of this enzyme is not AD-specific. It is crucial to explore the in silico activities of the top-ranked ligands against beta-amyloid and tau.

The author(s) response: Thank you for your valuable and insightful feedback. We fully acknowledge the complexity of Alzheimer’s disease (AD) and agree that targeting a single pathway, such as acetylcholinesterase (AChE), may not address the multifaceted nature of the disease. As you rightly pointed out, AD is influenced by various factors, including the aggregation of misfolded proteins like amyloid β (Aβ) and tau, alongside cholinergic hypothesis, oxidative stress, and inflammation.

However, we believe that according to cholinergic hypothesis targeting AChE remains relevant, especially given the current limitations in our understanding of AD pathogenesis. While the amyloid hypothesis has been a cornerstone of AD research for decades, recent findings indicate that Aβ might act as an early trigger rather than the sole driver of the disease. This suggests that while Aβ is necessary in the early stages, it might not be sufficient to sustain the pathogenic cascade in later stages of AD1. However, we would like to highlight the challenges associated with targeting tau and Aβ, as evidenced by the numerous clinical trials that have not yet succeeded. For instance, tau-targeting strategies, such as inhibiting tau aggregation, employing tau-based vaccinations, and stabilizing microtubules, have faced significant obstacles. The tau aggregation inhibitor TRx0237 failed to demonstrate therapeutic benefits in phase III trials2, and the only passive tau vaccine that reached phase III, intravenous immunoglobulin (IVIG), did not meet the primary endpoints in patients with mild-to-moderate AD3. Additionally, other tau-targeting approaches, like modulating kinases and phosphatases, have primarily been explored in preclinical studies, facing substantial challenges due to the incomplete understanding of AD pathology, the lack of robust biomarkers, and the difficulties associated with drug delivery across the blood-brain barrier4.

Given this context, our study focused on phytochemicals targeting AChE, not to dismiss the significance of Aβ and tau, but rather to explore an area that still holds therapeutic promise, particularly for symptomatic relief. AChE inhibitors, although offering only symptomatic benefits, remain the most widely used and available treatment for AD. They provide a crucial, if limited, therapeutic option for patients, giving them and their caregivers some measure of hope.

In summary, while our current study is focused on AChE inhibition, we agree that a multi-target approach, including Aβ and tau, would provide a more robust therapeutic strategy against AD. We will take this into account for future work and appreciate your guidance in helping us strengthen our research direction.

Thank you once again for your constructive feedback, which has significantly contributed to improving our manuscript.

Reference:

1. Musiek, E. S. & Holtzman, D. M. Three dimensions of the amyloid hypothesis: time, space and ‘wingmen’. Nat Neurosci 18, 800–806 (2015).

2. Gauthier, S. et al. Efficacy and safety of tau-aggregation inhibitor therapy in patients with mild or moderate Alzheimer’s disease: a randomised, controlled, double-blind, parallel-arm, phase 3 trial. The Lancet 388, 2873–2884 (2016).

3. Li, C. & Götz, J. Tau-based therapies in neurodegeneration: opportunities and challenges. Nat Rev Drug Discov 16, 863–883 (2017).

4. Du, X., Wang, X. & Geng, M. Alzheimer’s disease hypothesis and related therapies. Transl Neurodegener 7, 2 (2018).

Reviewer’s comment: Provide the rationale for limiting molecular dynamics simulations to only 100 ns

The author(s) response: Thank you for your insightful comment regarding the duration of the molecular dynamics (MD) simulations. We acknowledge the importance of an appropriate simulation time in ensuring the reliability of the results. The decision to limit the simulations to 100 nanoseconds (ns) was based on a thorough review of similar studies within the field. Many recent publications, particularly those investigating the dynamics of protein-ligand interactions, have successfully employed 100 ns simulations to capture significant conformational changes and interactions within the system.

However, we have now extended the MD simulations to 200 ns for all complexes as a further validation step. We have updated all the analyses, calculations, figures and tables accordingly. This extended duration allowed us to capture longer-term dynamics and potential rare events that may not be evident in shorter simulations. The results from the 200 ns simulations reinforced the findings from the initial 100 ns simulations, confirming the stability and reliability of the system's behavior over a more extended period. We have included these additional results in the revised manuscript, highlighting that the conclusions drawn from the 100 ns simulations are robust and consistent even with the extended simulation times. We trust that this addresses your concern and believe that the additional data further strengthens the study. Please feel free to reach out if further clarification or additional information is required. Thank you once again for your valuable feedback.

Reviewer’s comment: Cite the importance of other phytochemicals from endemic plants shown to inhibit AChE in vitro (DOI: 10.1021/acsomega.1c00137).

The author(s) response: Thank you. We have included the reference along with a discussion on the significance of the phytochemicals from endemic plants.

Reviewer’s comment: Explain why an in vitro enzymatic confirmatory assay was not performed against AChE, as it is a simple microplate assay.

The author(s) response: Thank you for your insightful question. Our study primarily focused on utilizing in-silico approaches to predict the inhibitory potential of phytochemicals against AChE. Given our background as researchers from Bangladesh, we face significant resource constraints, including the lack of funding, which limits our ability to perform certain experimental validations such as in vitro enzymatic assays. These computational methods were prioritized as they allow for the initial screening of a large number of compounds, enabling us to identify potential candidates for further investigation despite our limitations. We recognize that in vitro assays are a valuable and straightforward method for confirming bioactivity, and we aim to incorporate these confirmatory assays in future studies as resources permit. We hope this explanation clarifies our approach, and we appreciate your understanding of the challenges we face. Thank you once again for your valuable feedback.

Reviewer’s comment: What were the reference drugs used as controls for the molecular docking?

The author(s) response: Thank you for your concern regarding the selection of the reference drug as a control. We chose galantamine as the reference drug in our molecular docking and molecular dynamics simulations for several reasons. Galantamine is a well-established, FDA-approved acetylcholinesterase (AChE) inhibitor used in the treatment of Alzheimer's disease. Its efficacy and safety have been extensively validated, making it a reliable benchmark for evaluating the inhibitory potential of other compounds. Furthermore, galantamine is a phytochemical-based drug, specifically an alkaloid extracted from the bulbs and flowers of various plants such as Galanthus nivalis (snowdrop) and Leucojum aestivum (summer snowflake). This natural origin aligns with the focus of our study on phytochemicals, providing a relevant and appropriate comparison for the other compounds investigated. The choice of galantamine also allows us to directly compare the potential of novel phytochemicals against a clinically relevant standard, thereby enhancing the relevance and translational value of our findings.

Reviewer’s comment: Discuss the limitations of the present study.

The author(s) response: Thank you very much for the comment. We acknowledge that our study has certain limitations, which were largely influenced by the strategic focus and resource constraints that guided our research. These limitations, however, do not undermine the validity of our findings but rather point to areas where future work could expand and refine the study's conclusions.

First, the study was conducted with a relatively limited sample size. This decision was made due to constraints related to sample availability and computational resources. While the selected sample size was sufficient for the initial exploration and provided valuable insights, we recognize that a larger sample size would enhance the generalizability of the results. Future studies could address this limitation by incorporating more extensive and diverse datasets, which would allow for broader validation of the findings across different populations and conditions.

Secondly, our reliance on in silico methods, such as molecular docking and molecular dynamics simulations, without extensive experimental validation, was a deliberate choice driven by resource limitations, including the lack of funding and access to laboratory facilities. These computational approaches were chosen for their ability to efficiently screen a large number of compounds and identify potential candidates for further investigation. While these methods are powerful, we acknowledge that they may not fully capture the complexities of biological systems. To address this, future research could incorporate in vitro and in vivo experiments to validate the computational predictions, thereby ensuring that the identified compounds have the desired biological activity in real-world scenarios.

Additionally, the modeling process involved certain assumptions necessary to simplify the complex biological systems under study. These assumptions were made to make the analysis feasible within the scope of our resources and time constraints. However, we recognize that such simplifications might overlook some of the nuances of these systems. Future studies could refine these models by incorporating more detailed biological data and utilizing more advanced modeling techniques, thereby reducing the reliance on assumptions and providing a more accurate representation of the biological processes involved.

Lastly, the study's focus on specific pathways and a narrow set of parameters was a strategic decision to maintain a clear and manageable research scope. While this focus allowed us to generate specific and actionable insights, it may have limited the exploration of other potentially relevant factors. Future research could expand the scope to include a broader range of variables or pathways, offering a more comprehensive understanding of the biological systems in question.

We believe that acknowledging these limitations and outlining potential future directions not only enhances the transparency of our study but also demonstrates our commitment to continuous improvement. We appreciate your insights, which have guided us in reflecting on these aspects of our work. Thank you once again for your valuable comment. 

Reviewer #2:

Reviewer’s comment: In the present manuscript, authors have explored “Structure-based Virtual Screening of Trachyspermum ammi Metabolites Targeting Acetylcholinesterase for Alzheimer's Disease Treatment". The study lacks a specific hypothesis being tested.

The author(s) response: Thank you very much for your constructive feedback. We appreciate your point regarding the need for a clear hypothesis in our manuscript. The central hypothesis of our study is that metabolites derived from Trachyspermum ammi have the potential to inhibit acetylcholinesterase (AChE) and could therefore contribute to the treatment of Alzheimer’s disease (AD). This hypothesis guided our structure-based virtual screening approach, aiming to identify specific compounds from T. ammi that interact effectively with AChE.

To address your concern, we have revised the Introduction to explicitly state this hypothesis, ensuring that the study's objective is clearly conveyed. We believe this clarification will strengthen the manuscript and align it more closely with the expectations for hypothesis-driven research.

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