Characterization of Cyclic Peptides for Antituberculosis Drug Development Targeting ClpC1

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In general, I find the article very interesting and innovative, as there aren't many studies on tuberculosis treatments. The sections are clear and well explained, and the structure of the document is well-defined. Here are some suggestions for improving the manuscript:

- In line 64, after the references, a period should be used instead of a comma, to start the next sentence with a capital letter.

- It is unclear why the ClpC1 structure of B. subtilis is shown in Figure 1b, if the focus is on this protein as a therapeutic target against M. tuberculosis.

- The title mentions cyclic peptides, but the introduction does not clearly explain what they are.  It would be advisable to explain their origin or why they are being used in these studies.

- In section 2.7, Molecular Docking, it is not accurate to say that this tool can elucidate structures. It can predict or visualize protein or molecule structures, but it does not determine them. The beginning of this paragraph should be revised. The source of the cyclic peptide structures or the software used to prepare them is not mentioned. The method used to validate the molecular docking studies is also not mentioned.

- In the methodology, the specific cyclic peptides evaluated are not mentioned, nor their source, were they purchased or synthesized? If synthesized, the synthesis method should be described. The criteria for making the modifications to the positions mentioned in the manuscript are also not described.

- Please ensure that the scientific names of the microorganisms are in italics, as in line 312 for E. coli.

- In the molecular docking results, it is stated that compound OMSB had a very high binding energy. How high was it? What is the explanation for this result? Was this study repeated?

- The discussion is too short. It could be expanded further to discuss all the results obtained and their relationships.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article is very original with a high significance of content.

The quality of presentation and overall merit are very good.

My review suggests to accept the manuscrit in the current form.

• What is the main question addressed by the research?

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. Genome mining of lab-generated, spontaneous resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test.ClpC1 is considered a valid drug target against M. tuberculosis,

(Gao WKim JAnderson JR, Akopian T, Hong S, Jin Y, Kandror O, Kim J, Lee I, Lee S, McAlpine

JBMulugeta S, Sunoqrot S, Wang Y, Yang S, Yoon T, Goldberg AL, Pauli GFSuh JFranzblau SG,

Cho S.2015.The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo. Antimicrob Agents Chemother59:.https://doi.org/10.1128/aac.04054-14)

 

• Do you consider the topic original or relevant to the field? Does it address a specific gap in the field? Please also explain why this is/ is not the case.

ClpC1 is an emerging and promising drug target against Mycobacterium tuberculosis (Mtb), as it is essential for the bacterium's survival and can be inhibited by various compounds, including natural products like ecumicin and rufomycin. These compounds bind to ClpC1, particularly its N-terminal domain (NTD), disrupting its function in protein degradation and leading to cell death. Targeting ClpC1 is particularly important for developing treatments against drug-resistant Mtb strains.

Recent studies have shown that several natural product antibiotics targeting the unfoldase N-terminal domain can impair MtbClpC1 function resulting in cell death. While the pharmacological properties of these natural product antibiotics prevent their use in the clinic, similar molecules binding to the same binding pockets can result in new drugs against Mtb (Weinhäupl, K.,

Meuret, L., Desrat, S. et al. Identification of new ClpC1-NTD binders for Mycobacterium tuberculosis drug development. Sci Rep 15, 4146 (2025). https://doi.org/10.1038/s41598-025-87535-1.).

The topic is certainly innovative, original and relevant to the sector and the work presented adds further information that aids research in this area.

 

• What does it add to the subject area compared with other published material?

The study adds further innovative elements in the evaluation process of anti-tuberculosis therapies, especially for resistant forms, with a direct target to ClpC1.

 

• Are the conclusions consistent with the evidence and arguments presented and do they address the main question posed? Please also explain why this is/is not the case.

The results presented by the authors are convincing and have solid evidence to support them.

• Are the references appropriate?

Yes, they are appropriate

• Any additional comments on the tables and figures.

None

Finally, it is recommended to eliminate the following bibliographical references, because they are considered redundant:

 

Jung I-P, Ha N-R, Kim A-R, et al (2017) Mutation analysis of the interactions between Mycobacterium tuberculosis 710

caseinolytic protease C1 (ClpC1) and ecumicin. International Journal of Biological Macromolecules 101:348–357. 711 https://doi.org/10.1016/j.ijbiomac.2017.03.126 

 

Lee H, Suh J-W (2016) Anti-tuberculosis lead molecules from natural products targeting Mycobacterium tubercu- 662

losis ClpC1. J Ind Microbiol Biotechnol 43:205–212. https://doi.org/10.1007/s10295-015-1709-3

 

Lee H, Youn I, Demissie R, et al (2023) Identification of Small Molecule Inhibitors against MMP-14 via High- 741

Throughput Screening. Bioorganic & Medicinal Chemistry 117289. https://doi.org/10.1016/j.bmc.2023.117289

 

Vaid TM, Chalmers DK, Scott DJ, Gooley PR (2020) INPHARMA-Based Determination of Ligand Binding Modes 733 at α1 -Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity. Chemistry 26:11796–11805. 734

https://doi.org/10.1002/chem.202000642

 

Ren J, Ojeda I, Patel M, et al (2019) Exploring small molecules with pan-genotypic inhibitory activities against 739

hepatitis C virus NS3/4A serine protease. Bioorganic & medicinal chemistry letters 29:2349–2353

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

You present here a very interesting study regarding the potential use of some cyclic peptides as antitubercular drugs. The subject is of great interest, considering the worldwide spread of tuberculosis, its pathogenicity and the existence of XDR and TDR forms.

The caseinolytic protein C1 represents an interesting and promising drug target for treating tuberculosis and the measurement of its ATPase activity is important for understanding drug mechanisms.

The Introduction part is well written. Though, I suggest reducing a little its length. 

Even the structures of the peptides are large, maybe you can present them in the manuscript also.

Considering that the peptides were evaluated for their antituberculosis activity, I suggest that you present the MIC values in a separate table, in order to be more easily seen and interpreted.

Also, I suggest that you present some study limitations.

The Conclusion accurately summarizes all the results obtained.

The references are up to date and in agreement with the subject presented.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors developed and optimized a sensitive luminescence-based ATPase assay for the ClpC1 protein of Mycobacterium tuberculosis. They systematically tested different ClpC1 constructs with various tags and truncations, and applied the assay to evaluate multiple ClpC1-targeting compounds. Overall, the study is well-conducted, and with minor revisions, it would be suitable for publication.

 

1. Abstract: Please include relevant numerical data in the abstract to improve clarity and impact.
2. Line 149: Ensure consistent referencing format throughout the manuscript.
3. Cite appropriate references for any methods that were developed or modified.
4. Please report the manufacturer, country, and city for all instruments used, some examples include:
   1. Line 194: JASCO 815 spectropolarimeter
   2. Line 234: Victor 3V Plate Reader (PerkinElmer)
5. Define D.I.T at its first appearance in the manuscript.
6. Define all parameters used in the equations. For example:
   1. 1: α, 𝝁_neg, 𝝁_pos
   2. 2: AC50
   3. 3: KD
7. Provide references for all reported equations.
8. Revise reference numbering; for example, after reference 29 (line 148), the next cited reference is 37 (line 283).
9. Lines 282–291 appear more suitable for the Discussion section and should be moved accordingly.
10. Line 315 refers to lane 8, which does not exist in the figure 3a; please correct.
11. Specify the fitting method used for Figures 4a, 4c, and 4d.
12. Report statistical tests and p-values for Figures 4a, 5b, 6d, and 7c.
13. Ensure that all references are cited correctly and consistently.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf