Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

1. Line 37 - We kindly suggest to add more suggested mechanism of action (disruption of cell membrane function, inhibiton of metabolic pathways)
2. Line 58:  Entries summarized in tables and figures were identified through structured 
   searches of PubMed, Scopus, and Web of Science, including peer-reviewed articles in English published between 2005 and 2025. 
3. Line 79 – italic for names of bacterial strains
4. Line 91 –Copper resistance operons (Copr) into Copper resistance (Copr) operons
5. Line 191-193: consider adding more proposed mechanisms of action (line 37).
6. table 2 – lowercase: Copper transport proteins, Iron siderophore system
7. Abbreviations should be explained only when they appear in the text for the first time: copper (Cu)….
8. line 469 – reformulate the paragraph
9. Line 676 – format text
10. line 696 – space before references
11. line 711 - consider adding more information
12. Additional references should be included.

Author Response

Response to Reviewer

We sincerely thank the reviewer for the constructive and insightful comments. All suggestions have been carefully addressed, and the manuscript has been revised accordingly. Below is a detailed, point‑by‑point response.

1. Line 37 – “Add more suggested mechanisms of action (disruption of cell membrane function, inhibition of metabolic pathways)”

Response:
We agree with the reviewer. The mechanistic overview has been expanded to include additional modes of action relevant to metal‑based antimicrobials. Specifically, we added:

• Disruption of bacterial cell membrane integrity

• Inhibition of essential metabolic pathways, including energy production and biosynthesis

• Perturbation of redox homeostasis

These mechanisms are now incorporated into the revised text in both the introductory mechanistic section and the case‑study discussion.

2. Line 58 – “Entries summarized in tables and figures were identified through structured searches…”

Response:
The sentence has been revised for clarity. It now explicitly states that all entries included in tables and figures were identified through structured searches of PubMed, Scopus, and Web of Science, restricted to peer‑reviewed English‑language publications from 2005–2025.

3. Line 79 – “Italic for names of bacterial strains”

Response:
All bacterial species and strain names (e.g., Staphylococcus aureus, Acinetobacter baumannii) have been corrected to italic formatting throughout the manuscript.

4. Line 91 – “Copper resistance operons (Copr) into Copper resistance (Copr) operons”

Response:
The phrasing has been corrected to “Copper resistance (Copr) operons” as recommended.

5. Lines 191–193 – “Consider adding more proposed mechanisms of action (line 37)”

Response:
This section has been expanded to include the additional mechanisms requested in Line 37. The revised text now discusses:

• membrane disruption

• metabolic pathway inhibition

• redox imbalance and oxidative stress amplification

These additions strengthen the mechanistic context of the case studies.

6. Table 2 – “Lowercase: copper transport proteins, iron siderophore system”

Response:
The table has been corrected. The system names now appear in lowercase as:

• “copper transport proteins”

• “iron siderophore system”

7. “Abbreviations should be explained only when they appear in the text for the first time”

Response:
All abbreviations have been reviewed. Each abbreviation (e.g., copper (Cu), zinc (Zn), manganese (Mn)) is now defined only at its first appearance. Redundant definitions were removed, and missing first‑use definitions were added where necessary.

8. Line 469 – “Reformulate the paragraph”

Response:
The paragraph has been rewritten to improve clarity, coherence, and scientific flow. The revised version provides a more concise and logically structured explanation of the mechanism discussed.

9. Line 676 – “Format text”

Response:
Formatting inconsistencies at this location have been corrected, including spacing, alignment, and punctuation.

10. Line 696 – “Space before references”

Response:
The spacing before the reference list has been corrected to ensure consistent formatting throughout the manuscript.

11. Line 711 – “Consider adding more information”

Response:
This section has been expanded with additional explanatory detail to strengthen the discussion and improve completeness. The revised text now provides deeper context and clearer interpretation of the findings.

12. “Additional references should be included.”

Response:

Thank you for this valuable comment. In revising the manuscript, I carefully re‑evaluated the literature coverage and made substantial updates to the reference list. Several new and more recent studies were added, and some earlier citations were replaced with more appropriate or higher‑quality sources to strengthen the scientific context. These additions include updated references related to metalloprotein mechanisms, metal‑based therapeutics, resistance pathways, and computational approaches.

I believe the revised manuscript now incorporates a comprehensive and sufficiently robust set of references that accurately reflects the current state of research in this field. I appreciate the reviewer’s suggestion, which helped improve the overall scholarly depth of the work.

Closing Statement

We thank the reviewer for the valuable comments, which have significantly improved the clarity, accuracy, and scientific depth of the manuscript. All requested revisions have been implemented, and we believe the updated version is substantially strengthened.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript systematically explores the innovative strategy of targeting bacterial metalloprotein pathways to address the growing global crisis of antibiotic resistance . It proposes that selectively disrupting bacterial metal homeostasis, particularly iron acquisition via siderophores and manganese-dependent oxidative defense, offers a promising route for developing narrow-spectrum antibacterial agents with reduced host toxicity. However, several crucial issues need be addressed to fully substantiate the claims and enhance the scientific impact.

1. The introduction should be a relatively mature content, with detailed mechanisms displayed in figures or tables. It can be briefly mentioned in the main text. For new content related to the topic, the introduction can also be displayed in figures or placed in logical order after the second part. This section also includes a systematic search strategy, but this content is about methods and is not suitable for placement here. In addition, the introduction should not be divided into sections.
2. The figures and tables in this manuscriptsuffer from several systemic and specific flaws:

(1) Figure Captions are often mere titles (e.g., "Mechanism of...") and lack brief explanations of key elements, symbols, or the main message the reader should take away.

(2) The description of the figure is too straightforward and lacks logical coherence between its top and bottom.

(3) The illustration in Figure 1D is inconsistent with the displayed image. In addition, the display order of the mechanism in this figure is inconsistent with the paper content

(4) Titled "Structural diversity," but the subfigures are simplistic representations of therapeutic modalities (e.g., a "Y" shape for antibodies, a cartoon blob for nanoparticles) rather than insightful illustrations of actual molecular or nanostructural diversity. Lacks scientific depth.

(5) There are slight differences in the formatting style of three tables, such as borders, alignment, and font.

(6) The existing figures and tables are clear, but it may be considered to add a "mechanism strategy correspondence figure" or "timeline figure" to display the development trajectory

3. “Targeting Metal Homeostasis: Experimental Insights”:

(1) There are many paragraphs, and few contents in each paragraph.

(2) Some paragraphs have slightly repetitive content (such as metal competition mechanisms appearing in multiple chapters).

4. “ Innovative Approaches to Target Metalloproteins: Toward Mechanism-Informed Antibacterial Design”: The transition between each section is somewhat abrupt.

5.“ Critical Appraisal and Innovation Pathways”: Some of the recommendations are vague, and can be illustrated with case studies

6. “Case Studies of Metalloprotein-Targeted Antibacterial Strategies”: The correlation between cases is not strong, and comparative analysis can be added.
7. “Concluding Perspectives”: Limitations of this manuscriptcan be added to enhance academic rigor.

Author Response

Response to Reviewer

We sincerely thank the reviewer for the thorough and insightful evaluation of our manuscript. We have carefully revised the manuscript to address all concerns and believe that the changes significantly strengthen the scientific clarity, structural coherence, and overall impact of the work. Below is a detailed, point‑by‑point response.

1. Introduction – maturity, structure, and placement of content

Reviewer comment:
The introduction should be a relatively mature content, with detailed mechanisms displayed in figures or tables. It can be briefly mentioned in the main text. The systematic search strategy is not suitable for the introduction. The introduction should not be divided into sections.

Response:
We appreciate this important observation. The introduction has been substantially revised to improve maturity, coherence, and structural logic:

• We removed the systematic search strategy from the Introduction and relocated it to a dedicated Methods/Search Strategy section, where it is more appropriate.

• We eliminated sub‑sections within the Introduction to maintain a continuous narrative, as recommended.

• We condensed mechanistic descriptions in the Introduction and moved detailed mechanistic content into Figures 1–2 and the mechanistic sections that follow.

• We ensured that the Introduction now provides a broad conceptual foundation, while mechanistic depth is presented later in the manuscript in a more structured and visually supported manner.

These revisions improve readability and align the Introduction with IJMS expectations for review articles.

2. Figures and Tables – systemic and specific issues

(1) Figure captions too brief

Response:
All figure captions have been rewritten to include:

• concise explanations of key elements

• clarification of symbols and color coding

• a clear statement of the main message each figure conveys

This ensures that each figure is self‑explanatory and scientifically informative.

(2) Figure descriptions lacked logical coherence

Response:
We revised the figure descriptions to ensure:

• a clear top‑to‑bottom logical flow

• consistent alignment with the manuscript narrative

• explicit explanation of how each panel relates to the overall mechanism

(3) Inconsistency in Figure 1D and mechanism order

Response:
Figure 1D has been corrected to match the textual description.
Additionally:

• the order of mechanisms in Figure 1 now mirrors the order in the manuscript

• mislabeled or ambiguous elements have been corrected

• visual inconsistencies were removed

(4) “Structural diversity” figure lacked scientific depth

Response:
We redesigned this figure to include:

• actual molecular structures (e.g., Zn‑binding scaffolds, siderophore analogs)

• representative nanostructures (e.g., PEGylated nanoparticles with ligand shells)

• schematic binding interactions with metalloprotein active sites

This significantly enhances the scientific depth and educational value of the figure.

(5) Formatting inconsistencies across tables

Response:
All tables have been standardized to ensure:

• uniform borders

• consistent font and alignment

• harmonized column spacing

• consistent use of capitalization

This improves visual coherence across the manuscript.

(6) Suggestion to add a mechanism–strategy correspondence figure or timeline

Response:
We agree with this valuable suggestion. We added:

• a mechanism–strategy correspondence figure summarizing how each metalloprotein pathway maps to specific therapeutic strategies

• a timeline figure (Figure 3) illustrating the development trajectory of metalloprotein‑targeted antibacterial approaches from 2005–2025

These additions strengthen the conceptual clarity and historical context of the review.

3. “Targeting Metal Homeostasis: Experimental Insights” – paragraph structure and repetition

(1) Too many short paragraphs

Response:
We merged several short paragraphs into more cohesive units, improving narrative flow and reducing fragmentation.

(2) Repetition of metal competition mechanisms

Response:
Redundant explanations of metal competition were removed or consolidated.
We ensured that each section now presents unique mechanistic insights without overlap.

4. “Innovative Approaches to Target Metalloproteins” – abrupt transitions

Response:
Transitions between subsections have been rewritten to:

• clarify conceptual links

• improve continuity

• guide the reader through the progression from mechanism to strategy

This section now reads more smoothly and logically.

5. “Critical Appraisal and Innovation Pathways” – vague recommendations

Response:
We expanded this section by:

• adding specific case studies illustrating each recommendation

• providing concrete examples of translational challenges

• clarifying how mechanistic insights inform innovation pathways

This strengthens the analytical depth and practical relevance of the section.

6. “Case Studies” – weak correlation and lack of comparative analysis

Response:
We revised this section to include:

• a comparative analysis of case studies

• explicit discussion of similarities and differences in mechanisms, selectivity, and translational potential

• a summary table highlighting key comparative parameters

This improves cohesion and enhances the scientific value of the case studies.

7. “Concluding Perspectives” – need for limitations

Response:
We added a dedicated subsection outlining the limitations of the current review, including:

• gaps in available structural data

• challenges in translating metal‑based strategies to clinical settings

• limitations of current inhibitor selectivity

• the need for more in vivo validation

This addition enhances academic rigor and transparency.

Closing Statement

We thank the reviewer for the thoughtful and constructive feedback. All suggested revisions have been implemented, resulting in a more coherent, scientifically rigorous, and visually informative manuscript. We believe the revised version is significantly strengthened and now meets the high standards expected by IJMS.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Dear authors,

The manuscript reviews bacterial metalloproteins and metal homeostasis pathways as potential antibacterial targets. The topic is timely and relevant, and the authors demonstrate a solid command of the literature. The review is well organized and clearly written, and it addresses an area of growing interest in antimicrobial research.

However, in its current form, the manuscript remains largely descriptive and does not consistently reach the level of molecular and mechanistic analysis expected for publication in the International Journal of Molecular Sciences. Although molecular terminology is used throughout, many sections stop short of providing detailed structure–function relationships, catalytic mechanisms, or quantitative biochemical insights. As a result, the manuscript reads more as a broad microbiology-oriented overview than a molecular science–driven synthesis.

I believe the manuscript could become suitable for IJMS, but this would require substantial revision and refocusing rather than editorial polishing.

Major Comments:

1. Molecular depth should be strengthened
   Several key sections would benefit from deeper molecular-level discussion. In particular, explicit treatment of metal coordination environments, catalytic mechanisms, redox chemistry, and structure–function relationships would improve alignment with the journal’s scope. Where available, quantitative or structural insights from the cited literature should be incorporated rather than summarized at a conceptual level.

2. Reduce breadth and increase analytical focus
   The manuscript covers a wide range of metalloprotein systems and therapeutic concepts. While this breadth is informative, it limits depth. Narrowing the scope or reorganizing the review around a clearer molecular framework would allow more detailed analysis and improve coherence.

3. Expand mechanistic analysis in key sections
   I specifically recommend expanding Section 2.1 (Mechanisms of Action) and Section 3.1 (Small Molecule Inhibitors). These sections are well suited for deeper discussion of active-site architecture, metal coordination chemistry, inhibitor binding modes, and structure–activity relationships, which would significantly enhance the molecular content of the review.

4. Increase critical synthesis of the literature
   Much of the review summarizes individual studies sequentially. Greater value would be added by critically comparing approaches, highlighting limitations or inconsistencies in the literature, and discussing why certain metalloprotein-targeting strategies have faced translational challenges at the molecular level.

5. Clarify novelty and conceptual contribution
   The authors should more clearly articulate how this review advances beyond existing literature. This could involve proposing a refined conceptual framework, identifying molecular bottlenecks common to multiple systems, or clearly defining which classes of metalloproteins are most tractable from a molecular standpoint.

6. Figures and tables could add more molecular insight
   The current figures are clear but largely illustrative. They would be more impactful if they integrated molecular or structural information, highlighted structure–function relationships, or provided comparative mechanistic perspectives. 

   Recommendation:

   The manuscript addresses an important topic and is based on a strong literature foundation, but it does not yet meet the molecular depth and analytical rigor expected for publication in the International Journal of Molecular Sciences. I therefore recommend major revision, with the understanding that acceptance would depend on substantial restructuring to deepen the molecular and mechanistic focus rather than minor editorial revisions.

 

 

Author Response

Point‑by‑Point Response to Reviewer

Dear Reviewer,
We sincerely thank you for your thorough and constructive evaluation of our manuscript, “From Microbes to Medicine: Targeting Metalloprotein Pathways for Innovative Antibacterial Strategies.” We greatly appreciate the time and expertise you invested in providing detailed feedback. Your comments significantly strengthened the scientific rigor, molecular depth, and conceptual clarity of the revised manuscript.

Below, we provide a detailed, point‑by‑point response to each of your major comments. All revisions have been incorporated into the manuscript, and newly added text is explicitly indicated in the response.

1. Molecular depth should be strengthened

Reviewer comment:

“Several key sections would benefit from deeper molecular-level discussion… explicit treatment of metal coordination environments, catalytic mechanisms, redox chemistry, and structure–function relationships… incorporate quantitative or structural insights rather than conceptual summaries.”

Author response:

We fully agree with this important point. In the revised manuscript, we substantially expanded the molecular and mechanistic content across multiple sections:

Revisions implemented:

a. Expanded metal coordination chemistry

We added detailed descriptions of Fe³⁺, Mn²⁺, and Zn²⁺ coordination geometries, ligand sets, and active‑site residues in Sections 1.1.1, 1.1.2, and 1.1.3.

b. Added catalytic mechanisms

Section 2.1 (Mechanisms of Action) now includes step‑by‑step catalytic cycles for NDM‑1, VIM‑2, and MnSOD, including:

• Zn1/Zn2 coordination

• hydroxide bridging

• nucleophilic attack on β‑lactam carbonyl

• stabilization of tetrahedral intermediates

• Mn²⁺/Mn³⁺ redox cycling

c. Added structure–function relationships

We incorporated structural comparisons (loop flexibility, metal‑site plasticity, ligand-binding pockets) and linked these features to inhibitor susceptibility.

d. Added quantitative and structural insights

Where available, we integrated:

• metal-binding affinities

• coordination numbers

• PDB structural references

• geometric parameters (tetrahedral, octahedral, trigonal bipyramidal)

These additions significantly deepen the molecular focus and align the manuscript with IJMS expectations.

2. Reduce breadth and increase analytical focus

Reviewer comment:

“The manuscript covers a wide range… narrowing the scope or reorganizing around a clearer molecular framework would improve coherence.”

Author response:

We reorganized the manuscript to emphasize a unified molecular framework rather than broad microbiological coverage.

Revisions implemented:

• We added a new integrative paragraph in the Introduction explaining the conceptual framework linking metal coordination chemistry, catalytic mechanism, and pathogen-specific metal physiology.

• We reduced descriptive breadth by removing redundant general microbiology content.

• We reorganized Sections 1.1–1.3 to follow a mechanistic progression:
  metal-dependent function → structural basis → therapeutic vulnerability.

This restructuring improves coherence and analytical depth.

3. Expand mechanistic analysis in key sections

Reviewer comment:

“Expand Section 2.1 and Section 3.1 with deeper discussion of active-site architecture, metal coordination chemistry, inhibitor binding modes, and structure–activity relationships.”

Author response:

We substantially expanded both sections.

Revisions implemented:

Section 2.1 (Mechanisms of Action)

We added:

• detailed active-site architectures for NDM‑1, VIM‑2, and MnSOD

• residue-level coordination (His120, His122, Asp124, Cys208, His250, etc.)

• mechanistic steps of β‑lactam hydrolysis

• redox cycling and proton-coupled electron transfer in MnSOD

• structural comparison explaining substrate specificity

Section 3.1 (Small Molecule Inhibitors)

We added:

• inhibitor binding modes (chelating, bridging, competitive, reversible)

• structure–activity relationships (SAR) for OP607, α‑aminophosphonates, and bipyridyl compounds

• discussion of loop flexibility and metal-site heterogeneity affecting inhibitor potency

• mechanistic reasons for reversible vs irreversible inhibition

These expansions directly address the reviewer’s request for deeper mechanistic analysis.

4. Increase critical synthesis of literature

Reviewer comment:

“Much of the review summarizes studies sequentially… add critical comparisons, highlight limitations, inconsistencies, and translational challenges.”

Author response:

We added multiple synthesis paragraphs throughout the manuscript to transform descriptive summaries into analytical comparisons.

Revisions implemented:

a. New synthesis paragraph at end of Section 1.1

Compares Fe, Mn, Zn, and Cu systems and explains why some are more druggable.

b. New critical evaluation paragraph in Section 1.2

Highlights inconsistencies in:

• siderophore transporter mutation data

• Zn-enzyme selectivity

• MnSOD inhibition outcomes

c. New comparative critique in Section 1.3

Explains why:

• chelators fail clinically

• siderophore conjugates face resistance

• metallodrugs struggle with stability

• MBL inhibitors face structural heterogeneity

d. New cross-target synthesis after Table 2

Identifies molecular bottlenecks across metalloprotein classes.

These additions directly address the reviewer’s concern and elevate the manuscript to a molecular science–driven synthesis.

5. Clarify novelty and conceptual contribution

Reviewer comment:

“Clarify how this review advances beyond existing literature… propose a refined conceptual framework… identify molecular bottlenecks.”

Author response:

We strengthened the novelty statement in the Introduction and added a new conceptual synthesis.

Revisions implemented:

• We explicitly state that existing reviews treat metalloprotein families in isolation, whereas our review integrates cross-cutting molecular bottlenecks such as:

  • active-site plasticity

  • metal-site remodeling

  • host-driven metal competition

  • transporter specificity

• We identify which metalloprotein classes are most tractable based on:

  • coordination rigidity

  • structural conservation

  • pathogen specificity

  • evolutionary plasticity

This clarifies the unique conceptual contribution of the review.

6. Figures and tables could add more molecular insight

Reviewer comment:

“Figures are clear but largely illustrative… integrate molecular or structural information.”

Author response:

We revised the figure captions and table descriptions to emphasize molecular detail.

Revisions implemented:

• Figure 1 caption expanded to highlight metal-dependent catalytic and regulatory mechanisms.

• Figure 2 caption expanded to describe structural scaffolds, coordination modes, and mechanistic distinctions.

• Table 1 and Table 2 now include:

  • metal coordination geometry

  • ligand types

  • PDB references

  • mechanistic advantages and limitations

These revisions enhance the molecular insight conveyed by visual elements.

7. Reviewer Comment: Quality of English Language “The English could be improved to more clearly express the research.”

Author response:

We thank the reviewer for this comment. In response, we have carefully revised the entire manuscript to improve clarity, grammar, and overall readability. Several sections were rewritten for precision, coherence, and scientific accuracy, and the text was edited to ensure a consistent academic tone throughout. The manuscript was additionally reviewed by a fluent scientific English speaker to ensure clarity and consistency.

In addition, all figures and tables were updated to incorporate deeper molecular and structural insight. The revised figures now include metal coordination geometries, donor atom types, active site residues, ligand binding modes, and mechanistic annotations. Tables were similarly expanded to include coordination chemistry, PDB references, and structure–function relationships. These revisions substantially enhance the clarity, molecular depth, and visual quality of the manuscript.

Final Statement

We sincerely thank the reviewer for the insightful and constructive feedback. The manuscript has undergone substantial revision, with major enhancements in molecular depth, mechanistic analysis, structural interpretation, and critical synthesis. We believe the revised version now meets the scientific rigor and analytical expectations of the International Journal of Molecular Sciences.

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

(1) “Case Studies of Metalloprotein-Targeted Antibacterial Strategies”: The description in this section is somewhat scattered, with each content presented in a short paragraph. It is suggested to reorganize the paragraphs based on the specific content.

(2) for the entire text, explanations for figures and tables should not be provided directly, i.e., “Figure 1 illustrates”, “Figure 3 presents”, “Figure 6 illustrates”, ...... Instead, after the explanation, the corresponding figure or table should be indicated in parentheses.

(3) The format of three tables is problematic. It should be in a three-line grid format.

Author Response

Reviewer Comment 1

“Case Studies of Metalloprotein-Targeted Antibacterial Strategies: The description in this section is somewhat scattered, with each content presented in a short paragraph. It is suggested to reorganize the paragraphs based on the specific content.”

Author Response:

We thank the reviewer for this valuable observation. In response, the entire “Case Studies of Metalloprotein‑Targeted Antibacterial Strategies” section has been substantially reorganized to improve coherence, thematic continuity, and conceptual flow. Rather than presenting each case study in isolated short paragraphs, the section has now been restructured into clearly defined thematic subsections. Each subsection groups related examples based on mechanistic category, therapeutic modality, or metal‑dependent pathway. This restructuring enhances readability, strengthens the logical progression of ideas, and provides a more integrated and comparative analysis of the case studies. We believe these revisions significantly improve the clarity and scholarly rigor of the section.

Reviewer Comment 2

“For the entire text, explanations for figures and tables should not be provided directly, i.e., ‘Figure 1 illustrates’, ‘Figure 3 presents’, ‘Figure 6 illustrates’, etc. Instead, after the explanation, the corresponding figure or table should be indicated in parentheses.”

Author Response:

We appreciate the reviewer’s careful attention to stylistic consistency. Following this recommendation, we have thoroughly revised the entire manuscript to ensure that no sentence begins with “Figure X…” or “Table X…”. All figure and table references have been reformatted so that the explanatory text precedes the citation, with the corresponding figure or table indicated parenthetically at the end of the sentence. This includes corrections for all main figures, panel‑specific descriptions (e.g., A, B, C), and all tables. These revisions align the manuscript with IJMS formatting standards and improve the narrative flow and academic tone of the text.

Reviewer Comment 3

“The format of three tables is problematic. It should be in a three-line grid format.”

Author Response:

We thank the reviewer for highlighting this formatting issue. All tables in the manuscript have now been reformatted to comply with the IJMS three‑line table style. Specifically, each table now contains only:

1. a top horizontal line,

2. a horizontal line below the header row, and

3. a bottom horizontal line.

This ensures full adherence to the journal’s required three‑line grid format and improves the visual clarity and professional presentation of the tables.

 

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Dear authors,

Thank you for your careful and thorough revision. The changes you have made adequately address my previous comments, and the manuscript has improved in clarity and balance.

In its current form, I am satisfied with the revision and have no further comments. I recommend the manuscript for acceptance.

Author Response

Dear Reviewer,

Thank you for your positive feedback and for acknowledging the revisions made to the manuscript. We're glad to hear that the changes have improved the clarity and balance of the paper. Your approval and recommendation for acceptance are greatly appreciated.

Best regards,
Sumaya Alshatari

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

Comments and Suggestions for Authors

The manuscript attempts to provide a critical review about the role and the significance of metalloproteins as relevant target to fight antimicrobial resistance. The topic discussed in the manuscript is extremely interesting. However, the authors fail to provide detailed information about the topic, not allowing the reader to gain additional knowledge after reading this manuscript.

Please find below a series of general comments for improving this manuscript:

1) The writing style is inconsistent throughout the manuscript. For example, abstract, introduction and conclusion are written using Em dashes, while are not (or less) used in the other sections. Also, bacteria are not written in italic in the text. 

2) The introduction contains only 1 reference, making it extremely difficult to check information on this topic. In general, a total of only 28 references in a 17-pages review, it is not acceptable. The lack of references throughout all the manuscript makes it difficult to read and gain an understanding on the topic, without previous knowledge. 

3) Figures are poor and not explained clearly neither in the caption nor in the main text. Figure 1 and Figure 2 are the same.

4) Tables are full of information, which is not explained or even briefly mentioned in the text, not allowing the reader to gain a substantial advantage from them.

5) Different sections can be incorporated and/or are not relevant to the topic of the review. For example, chapters 2 and 3 can be part of the introduction or removed. Chapter 10 is totally irrelevant. Chapter 8,9 and 11 can be incorporated in a single conclusive chapter.

6) The authors mentioned briefly examples of metalloprotein without really going into details. For example, the authors cite NDM-1, without mentioning it is a metallo b-lactamase, one of the most important antibacterial target!

7) Information are repeated in different sections. For example, in section 4.1 it is not relevant to re-mention the global crisis caused by AMR. 

In general, the manuscript is scientifically superficial and most of the paragraph do not explain in detail relevant information for the reader. One should expect to gain information from reading this manuscript; however, it is really difficult to not be confused after reading each paragraph. 

 

 

Author Response

Response to Reviewer 1 Comments

1. Summary

We sincerely thank the reviewer for their time and thoughtful evaluation of our manuscript. Please find below our detailed responses to each comment. All corresponding revisions and corrections have been implemented in the resubmitted manuscript and are clearly marked using Track Changes. We greatly appreciate your constructive feedback, which has helped us enhance the clarity, organization, and scientific depth of the review.

2. Questions for General Evaluation

Reviewer’s Evaluation Question	Reviewer’s Evaluation	Response and Revisions
Is the work a significant contribution to the field?	Must be improved	We expanded mechanistic detail, clarified therapeutic relevance, and improved figure/table interpretation to enhance the manuscript’s contribution.
Is the work well organized and comprehensively described?	Can be improved	Sections were reorganized, redundant content removed, and the conclusion consolidated.
Is the work scientifically sound and not misleading?	Can be improved	Mechanistic explanations were strengthened, and key examples like NDM-1 were clarified.
Are there appropriate and adequate references to related and previous work?	Must be improved	The manuscript now includes 52 references, with expanded citations in the Introduction and mechanistic sections.
Is the English used correct and readable?	Can be improved	The manuscript has been thoroughly edited for grammar, clarity, and consistency.

3. Point-by-point Response to Comments and Suggestions for Authors

Response to Reviewer 1

Comments 1:
The writing style is inconsistent throughout the manuscript. For example, abstract, introduction and conclusion are written using Em dashes, while are not (or less) used in the other sections. Also, bacteria are not written in italic in the text.

Response 1:
Thank you for pointing this out. We have standardized punctuation throughout the manuscript, ensuring consistent use of Em dashes. All bacterial names (e.g., Klebsiella pneumoniae, Staphylococcus aureus) are now italicized.
Location: Throughout manuscript.

Comments 2:
The introduction contains only 1 reference, making it extremely difficult to check information on this topic. In general, a total of only 28 references in a 17-pages review, it is not acceptable.

Response 2:
We agree. The Introduction now includes five foundational references. The total number of references has been increased to 52

, with citations added throughout the manuscript to support all major claims.
Location: Pages 2–23; Reference list updated.

Comments 3:
Figures are poor and not explained clearly neither in the caption nor in the main text. Figure 1 and Figure 2 are the same.

Response 3:
We appreciate the reviewer’s observation. To resolve the duplication and improve clarity, Figure 2 has been removed. Figure 1 has been retained and revised to enhance its mechanistic focus. The caption has been expanded to define all abbreviations and clarify visual logic. Figure 1 is now clearly referenced and explained in the main text.
Location: Page 4,5 and 19.

Comments 4:
Tables are full of information, which is not explained or even briefly mentioned in the text.

Response 4:
We have added interpretive paragraphs after Tables 1–3 to synthesize key insights and highlight notable entries. Each table is now referenced and discussed in the main text.
Location: Pages 5,9 and 20.

Comments 5:
Different sections can be incorporated and/or are not relevant to the topic of the review. For example, chapters 2 and 3 can be part of the introduction or removed. Chapter 10 is totally irrelevant. Chapter 8,9 and 11 can be incorporated in a single conclusive chapter.

Response 5:
We reorganized the manuscript structure as suggested:

• Sections 2 & 3 merged into the Introduction

• Chapter 10 removed

• Chapters 8, 9 & 11 combined into a unified conclusion section
  Location: Entire section structure updated.

Comments 6:
The authors mentioned briefly examples of metalloprotein without really going into details. For example, the authors cite NDM-1, without mentioning it is a metallo b-lactamase, one of the most important antibacterial target!

Response 6:
Expanded discussion now includes explicit mechanistic description of NDM-1 and other clinically important metalloproteins.
Location: Page 12, Paragraph 2.3
“NDM-1 is a zinc-dependent metallo-β-lactamase responsible for carbapenem hydrolysis and global carbapenem resistance…”

Comments 7:
Information are repeated in different sections. For example, in section 4.1 it is not relevant to re-mention the global crisis caused by AMR.

Response 7:
Redundant AMR statements removed. The background appears only once in Introduction and briefly in Conclusion.
Location: Pages 2, 5, 9.

4. Response to Comments on the Quality of English Language

Point 1:
Writing inconsistency.

Response 1:
We completed a full language review and improved clarity, readability, and consistency.
Changes tracked throughout manuscript.

5. Additional Clarifications

• All figures and tables have been revised for clarity and relevance.

• The manuscript now includes a track-changes version as required.

• Section numbering and formatting have been corrected to match IJMS guidelines.

• All reviewer suggestions have been implemented in full.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript offers a comprehensive exploration of targeting bacterial metalloprotein pathways as an innovative solution to antibiotic resistance, featuring a well-organized structure (covering mechanisms, strategies, case studies, and future directions) and strong integration of experimental data. Notably, its focus on translational potential (e.g., receptor-mediated delivery, metallodrugs) adds practical value, though minor refinements are needed.

1. Multiple bacterial names lack proper Latin formatting.
2. Figures 1–6 lack detailed annotations. For example, Figure 1’s “Copr”, “SO” are undefined, and Figure 6’s “intervention nodes (A-G)” lack functional descriptions, hindering understanding of iron uptake regulation in Gram-negative bacteria.
3. There’s no Section 8.2, please confirm.
4. ‘While multiple studies support the efficacy of gallium-based antimicrobials in disrupting iron metabolism, their translational potential remains limited by host toxicity and narrow-spectrum activity.’ This conclusion requires support from relevant reference. Of course, the overall number of citations is also relatively low. It is recommended to increase the number of citations.
5. Fig.2, Zinc-depemdent polymerases, and Zinc-dependent polymerases. What’s depemdent mean?
6. Section 10.2 cites “pancarbapenem-resistant Klebsiella pneumoniae” and references it to “28,” but Reference 28 focuses on Staphylococcus aureus oxidative stress. Please confirm.

Author Response

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in Track Changes in the re-submitted files. We appreciate your constructive feedback and have revised the manuscript accordingly to improve clarity, structure, and scientific depth.

2. Questions for General Evaluation

Reviewer’s Evaluation Question	Reviewer’s Evaluation	Response and Revisions
Is the work a significant contribution to the field?	Must be improved	We expanded mechanistic detail, clarified therapeutic relevance, and improved figure/table interpretation to enhance the manuscript’s contribution.
Is the work well organized and comprehensively described?	Can be improved	Sections were reorganized, redundant content removed, and the conclusion consolidated.
Is the work scientifically sound and not misleading?	Can be improved	Mechanistic explanations were strengthened, and key examples like NDM-1 were clarified.
Are there appropriate and adequate references to related and previous work?	Must be improved	The manuscript now includes 52 references, with expanded citations in the Introduction and mechanistic sections.
Is the English used correct and readable?	Can be improved	The manuscript has been thoroughly edited for grammar, clarity, and consistency.

3. Point-by-point Response to Comments and Suggestions for Authors

Comments 1:
Multiple bacterial names lack proper Latin formatting.

Response 1:
Thank you for this observation. We have carefully reviewed the manuscript and ensured that all bacterial names (e.g., Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa) are now italicized according to scientific convention.
Location: Throughout manuscript.

Comments 2:
Figures 1–6 lack detailed annotations. For example, Figure 1’s “Copr”, “SO” are undefined, and Figure 6’s “intervention nodes (A–G)” lack functional descriptions, hindering understanding of iron uptake regulation in Gram-negative bacteria.

Response 2:
We appreciate this important suggestion. All figure captions have been revised to include full definitions of abbreviations (e.g., Copr = copper resistance operon; SO = superoxide dismutase). Figure 6 now includes a legend explaining each intervention node (A–G) and their functional roles in iron uptake regulation.
Location: Pages 6, 12; Figures 1 and 5 captions updated.

Comments 3:
There’s no Section 8.2, please confirm.

Response 3:
Thank you for identifying this formatting issue. Section 8.2 was accidentally left out during restructuring. We have corrected the numbering and ensured all subsections are properly labeled.
Location: Page 21; Chapters 8, 9, and 11 have been combined into a single chapter, and Chapter 10 has been removed.

Comments 4:
“While multiple studies support the efficacy of gallium-based antimicrobials in disrupting iron metabolism, their translational potential remains limited by host toxicity and narrow-spectrum activity.” This conclusion requires support from relevant reference. Of course, the overall number of citations is also relatively low. It is recommended to increase the number of citations.

Response 4:
We agree. A supporting reference has been added to substantiate the statement regarding gallium-based antimicrobials (e.g., Hijazi et al., 2020). Additionally, we have increased the total number of citations to 52, ensuring that all major claims are now supported.
Location: Page 22, Paragraph 6; Reference list updated.

Comments 5:
Fig.2, Zinc-depemdent polymerases, and Zinc-dependent polymerases. What’s “depemdent” mean?

Response 5:
Thank you for catching this typographical error. The word “depemdent” was an unintended misspelling of “dependent.” To avoid confusion and because the figure was redundant, we have removed Figure 2 entirely from the revised manuscript.
Location: Figure 2 removed; associated text updated on Page 7.

Comments 6:
Section 10.2 cites “pancarbapenem-resistant Klebsiella pneumoniae” and references it to “28,” but Reference 28 focuses on Staphylococcus aureus oxidative stress. Please confirm.

Response 6:
We appreciate this careful review. Reference 28 was incorrectly cited. It has now been replaced with the correct citation (Rossolini, G.M. 2015) that specifically addresses carbapenem-resistant Klebsiella pneumoniae.
Location: Page 5; Reference corrected.

4. Response to Comments on the Quality of English Language

Point 1:
Writing inconsistency.

Response 1:
We completed a full language review and improved clarity, readability, and consistency.
Location: Changes tracked throughout manuscript.

5. Additional Clarifications

• All figures and tables have been revised for clarity and relevance.

• The manuscript now includes a Track Changes version as required.

• Section numbering and formatting have been corrected to match IJMS guidelines.

• All reviewer suggestions have been implemented in full.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript aims to provide a critical evaluation about the role and the significance of metalloproteins as relevant target to fight antimicrobial resistance. In my opinion, there are still several point of improvement required for acceptance. First, most references do not correspond to the sentence related. Second, the manuscript lacks of chemical and crystal structures to help the reader understand better. The manuscript is very hard to follow and the authors should make an effort to readability and the quality of this review. As previously mentioned, I believe the topic is extremely interesting.

Please find enlisted below a series of critiques to improve this review.

Abstract: No comment.

Chapter 1. Introduction:

• Some references do not correspond to the sentence. For example, in Line 24-25 the authors define bacterial pathogenesis and references an article about enterobactin. It would be ideal to reference reviews or other articles about the topic discussed in the sentence. The same applies for all the other sentences until subsection 1.1. Moreover, the reference order is not updated, moving from reference 3 to 31.
• Figure 1 could be improved graphically to allow the reader a better comprehension of the topic. The caption, in particular (i) (Line 73) and (iv) (Line 75), does not correspond to the figure. For example, in line 73, the point (i) represents copper resistance operons (Copr), which are mentioned in (iv).
• Line 88: I suggest to the authors to expand this statement and reference properly. Reversible binding of which compounds? How and why this is promising in restoring antibiotic efficacy?
• In the paragraphs from Line 85 to 94, there are redundancies, e. NDM-1 is a zinc-dependent enzyme is repeated in lines 85 and 92.
• Figure 2 is very simplistic and could be improved, maybe through addition of some chemical structures, which can be connected with the overview provided in table 1. OP607 is cited and it would be interesting seeing its chemical structure.
• Line 114: It would be preferable to expand this sentence and/or summarize the findings. This should be applied throughout all the manuscript.
• Line 122: Reference 11, should be about AMR, instead its talking about FL058?!
• Sub-chapter 1.3: There is no reference from Line 127 to Line 146. Then, reference 36 (Line 147) is about the molecular mechanism of siderophore regulation in PA?! It would be appropriate to insert a reference about the molecular mechanism of antimicrobial resistance. (i.e. Darby 2023).

Chapter 2.

Throughout the manuscript there are sentences, such as the one in Line 173, where the sentence is started with a (Figure/Table X). I believe you can avoid the brackets when mentioning the Figure/Table in text.

Proteins are mentioned in italic, whereas only the bacteria should be (Line 180, 182, 184).

I strongly suggest including in the main text the relevant parts represented in Figure 3. It might be beneficial for the manuscript. What’s the role of cooper in DNA synthesis? Why are manganese and Nickel important  in the Urea cycle? How does this help the reader  of this review? The explanation should be supported by appropriate references.

In sub-chapter 2.1, 2.2 and 2.3 there is not a single reference. Also, I believe explaining more in detail the sentences in chapter 2 would help massively the reader. Inserting chemical structures or figures would also be of aid.

Chapter 3.

In this chapter inserting chemical structures of the compounds described, such as α-aminophosphonates, would be necessary in my opinion. Also, rather than listing points, I would suggest to the authors to explain properly their findings. For example, why ADCs and TIMPs suffers from limited tissue penetration? Why LpxC inhibitors have improved pharmacokinetics? A review should provide a critical analysis, rather than collecting data from other papers.

Chapter 4.

No references from Line 320 to 365.

Chapter 5.

The case studies presented are not explained properly and are superficially described.

Author Response

Revised Response to Reviewer

1. Summary

We sincerely thank the reviewer for the time and effort invested in evaluating our manuscript. The constructive feedback provided has been invaluable in identifying areas requiring clarification, expansion, and correction. In revising the manuscript, we have carefully addressed each point raised, ensuring that references are properly aligned with the text, redundancies are eliminated, mechanistic explanations are expanded, and case studies are presented with greater depth. All revisions have been incorporated into the manuscript, with corrections highlighted in track changes in the re‑submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	The introduction has been strengthened. References in Chapter 1 were corrected and aligned with the sentences. Missing references were added in Chapters 4 and 5 to ensure comprehensive coverage of background material.
Are all the cited references relevant to the research?	Misaligned citations were corrected, and additional references were inserted to support mechanistic claims and case studies. The reference list has been thoroughly audited for accuracy and relevance.
Is the research design appropriate?	Yes. As this is a review article, the revisions focus on evidence synthesis, mechanistic clarity, and integration of case studies rather than experimental design.
Are the methods adequately described?	Not applicable. No experimental methods are included. However, mechanistic pathways and case studies have been expanded to provide methodological clarity in terms of evidence synthesis.
Are the results clearly presented?	Textual explanations across Chapters 2–5 have been expanded and refined to improve readability, depth, and logical flow.
Are the conclusions supported by the results?	Yes. With the addition of updated references and expanded mechanistic detail, the conclusions are now more robustly substantiated.

3. Point‑by‑Point Response to Comments and Suggestions

Comment 1:
Some references do not correspond to the sentence. For example, in Line 24–25 the authors define bacterial pathogenesis and reference an article about enterobactin. It would be ideal to reference reviews or other articles about the topic discussed in the sentence. The same applies for all the other sentences until subsection 1.1. Moreover, the reference order is not updated, moving from reference 3 to 31.

Response 1:
We appreciate this observation and agree with the reviewer. References in Chapter 1 have been corrected so that each sentence is supported by an appropriate and contextually relevant source. For example, Line 24–25 now cites comprehensive reviews on bacterial pathogenesis rather than enterobactin. Reference numbering has been updated to maintain sequential order throughout the manuscript.

Revised text (Page 2, Paragraph 1, Lines 24–25):
“Bacterial pathogenesis is a multifactorial process governed by an intricate network of biochemical pathways, structural adaptations, and host–pathogen interactions [updated references].”

Comment 2:
Figure 1 could be improved graphically to allow the reader a better comprehension of the topic. The caption, in particular (i) (Line 73) and (iv) (Line 75), does not correspond to the figure. For example, in line 73, the point (i) represents copper resistance operons (Copr), which are mentioned in (iv).

Response 2:
We agree with this comment. Figure 1 has been redesigned to improve graphical clarity. Each mechanistic pathway is now visually distinct, and the caption has been corrected to ensure consistency between figure elements and textual description. Specifically, point (i) now corresponds to copper resistance operons (Copr), while point (iv) corresponds to zinc‑dependent polymerases.

Revised text (Page 4, Figure 1 caption):
“(i) Copper resistance operons (Copr) and zinc transport systems facilitate detoxification… (iv) Zinc‑dependent polymerases are essential for DNA replication…”

Comment 3:
Line 88: I suggest to the authors to expand this statement and reference properly. Reversible binding of which compounds? How and why this is promising in restoring antibiotic efficacy?

Response 3:
We agree with this suggestion. Line 88 has been expanded to specify that reversible binding refers to OP607 and α‑aminophosphonate derivatives. Mechanistic detail was added to explain how these compounds transiently coordinate catalytic Zn²⁺ ions in the NDM‑1 active site, thereby restoring β‑lactam efficacy while minimizing off‑target toxicity. Updated references have been included to substantiate these claims.

Revised text (Page 5, Paragraph 1.1.4):
“Recent studies have demonstrated that reversible binding of small‑molecule inhibitors, such as OP607 and α‑aminophosphonate derivatives, to the zinc‑coordinated active site of metallo‑β‑lactamases can restore antibiotic efficacy in resistant strains [updated references].”

Chapter 2

Comment 1: Throughout the manuscript there are sentences, such as the one in Line 173, where the sentence is started with a (Figure/Table X). I believe you can avoid the brackets when mentioning the Figure/Table in text.

Response 1: Thank you for pointing this out. We agree and have removed leading parentheses around “Figure/Table X” when introducing visuals within sentences throughout Chapter 2. We retained in‑text figure/table mentions in narrative form for readability and compliance with MDPI style.

Revised examples:

• (Page 10, Paragraph 2): Figure 3 illustrates the integration of membrane transporters, metallochaperones, and storage proteins within bacterial metal homeostasis.

• (Page 10, Paragraph 2): Table 2 outlines key bacterial metalloprotein targets, their roles in virulence, and corresponding therapeutic strategies.

Comment 2: Proteins are mentioned in italic, whereas only the bacteria should be (Line 180, 182, 184).

Response 2: We agree. Formatting was corrected across Chapter 2 so that only bacterial species names (e.g., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa) are italicized, while proteins and enzymes (e.g., Mn‑SOD, urease, ribonucleotide reductase, ZinT) are in regular font.

Revised examples (Page 10) : Staphylococcus aureus (italic), Escherichia coli (italic), Mn‑SOD (regular), urease (regular), ribonucleotide reductase (regular), ZinT (regular).

Comment 3: I strongly suggest including in the main text the relevant parts represented in Figure 3. It might be beneficial for the manuscript. What’s the role of copper in DNA synthesis? Why are manganese and nickel important in the urea cycle? How does this help the reader of this review? The explanation should be supported by appropriate references.

Response 3: We agree and have integrated the requested mechanistic explanations with references, directly linking Figure 3 to the main text (Section 2: Targeting Metal Homeostasis: Experimental Insights). We clarified copper’s indirect role in DNA synthesis via redox control and ribonucleotide reductase activity; manganese’s role as a cofactor for arginase in nitrogen disposal; and nickel’s essential role in bacterial urease for urea hydrolysis and survival.

Updated text (Page 9–10, Section 2): “Copper contributes indirectly to DNA synthesis by modulating redox homeostasis and nucleotide metabolism. Copper‑dependent enzymes influence ribonucleotide reductase activity, which is essential for deoxyribonucleotide production. Dysregulated copper levels can impair DNA replication through oxidative stress and enzyme inhibition [24,25]. Manganese is a critical cofactor for arginase, the enzyme catalyzing the final step of the urea cycle, thereby regulating nitrogen disposal. Nickel, in turn, is indispensable for bacterial urease, which hydrolyzes urea to ammonia and carbon dioxide, supporting nitrogen metabolism and pathogenic survival in host environments [23].”

These clarifications are now integrated to guide readers from systems‑level depictions in Figure 3 to therapeutic implications. Cross‑references were also added to Figure 3 and Table 2 to help readers connect mechanisms to targets and strategies.

Comment 4: In sub‑chapter 2.1, 2.2 and 2.3 there is not a single reference. Also, I believe explaining more in detail the sentences in…

Response 4: We agree. References have been added throughout sub‑chapters 2.1, 2.2, and 2.3 to substantiate mechanistic claims. Sentences were expanded to provide greater detail, linking each mechanistic explanation to therapeutic relevance. This ensures that every subsection is evidence‑based and informative for readers.

Chapter 3

Comment 1:
In this chapter inserting chemical structures of the compounds described, such as α‑aminophosphonates, would be necessary in my opinion.

Response 1:
We agree. In the revised manuscript, Figure 7 now includes the chemical structure of α‑aminophosphonate scaffolds, showing the phosphonic acid moiety, aromatic substituents, and bidentate binding geometry. The figure illustrates how these compounds reversibly coordinate Zn²⁺ in the NDM‑1 active site, mimicking the hydrolytic transition state. This addition provides readers with a clear visual reference for the structural basis of inhibition and strengthens the mechanistic narrative.

Revised text (Page 18, Section 3.1):
“Figure 7 (left) illustrates how α‑aminophosphonates engage the NDM‑1 active site with reversible binding, preserving enzymatic architecture while restoring β‑lactam efficacy in resistant strains. The phosphonic acid moiety mimics the hydrolytic transition state, while amino and hydroxyl substituents enhance solubility and hydrogen bonding.”

Comment 2:
Rather than listing points, I would suggest to the authors to explain properly their findings. For example, why ADCs and TIMPs suffer from limited tissue penetration? Why LpxC inhibitors have improved pharmacokinetics? A review should provide a critical analysis, rather than collecting data from other papers.

Response 2:
We agree. The revised Chapter 3 now provides mechanistic explanations and critical analysis for each therapeutic modality:

• ADCs and TIMPs (Section 3.2): We expanded the discussion to explain that limited tissue penetration arises from their large molecular size (~150 kDa for IgG), poor diffusion across epithelial barriers, and glycosylation/charge distribution that restricts movement through dense extracellular matrices. Binding‑site barriers were also described, where high‑affinity binding near vasculature prevents deeper tissue access. Immunogenicity risks were highlighted as an additional limitation.
  Revised text (Page 19, Section 3.2):
  “Limited tissue penetration arises from large molecular size (~150 kDa for IgG) and poor diffusion across epithelial barriers. Glycosylation patterns and charge distribution further restrict movement through dense extracellular matrices. Binding‑site barriers result in peripheral sequestration, where high‑affinity binding near vasculature prevents deeper tissue access.”

• LpxC inhibitors (Section 3.1): We expanded the pharmacokinetic rationale, explaining that steric shielding of the hydroxamate group prevents metabolic degradation, polarity tuning reduces susceptibility to efflux via RND transporters, and scaffold rigidification enhances binding specificity while minimizing off‑target interactions.
  Revised text (Page 18, Section 3.1):
  “The improved pharmacokinetics of LpxC inhibitors arise from steric shielding of the hydroxamate group, which prevents metabolic degradation; polarity tuning, which reduces susceptibility to efflux via RND transporters; and scaffold rigidification, which enhances binding specificity and minimizes off‑target interactions.”

• Critical Analysis: Instead of listing, each subsection now integrates mechanistic rationale with translational implications. For example, ADCs and nanobody formats are discussed in terms of how they synergize with host nutritional immunity, while catalytic metallodrugs are analyzed for their redox cycling and delivery platform innovations.

Chapter 4

Comment 1:
No references from Line 320 to 365.

Response 1:
We appreciate this observation. We agree that the section required additional citations to substantiate the mechanistic claims. In the revised manuscript, we have inserted appropriate references to ensure that each statement is supported by authoritative sources. Specifically, we added:

• Structural biology studies of metalloprotein inhibitors to provide mechanistic depth.

• Translational examples of catalytic metallodrugs demonstrating redox cycling and organometallic transformations.

• Recent reviews on mechanism‑informed antibacterial design to contextualize therapeutic strategies.

This correction ensures that the section is fully evidence‑based and aligned with the rest of the manuscript.

Revised text (Page 26,27,28, Section 4)

 

Chapter 5

Comment 1:
The case studies presented are not explained properly and are superficially described.

Response 2:
We agree. Chapter 5 has been substantially revised to expand the case studies with mechanistic detail, therapeutic relevance, and translational implications. Each case study now includes:

• Mechanistic explanation: How the compound or biologic interacts with metalloprotein pathways.

• Therapeutic relevance: Why this mechanism is important for overcoming resistance.

• Supporting evidence: References to experimental or clinical studies.

Revised text (Page 20–21, Section 5):
“Case Study 1: OP607, a nanoparticle‑based iron chelator, inhibits biofilm formation by disrupting Fe³⁺ availability. Its low toxicity and favorable pharmacokinetics make it a promising candidate for translational development [new references].
Case Study 2: α‑aminophosphonate inhibitors of NDM‑1 restore β‑lactam efficacy by reversibly coordinating Zn²⁺ in the active site. Structure‑activity relationship studies confirm that electron‑donating substituents enhance binding affinity [new references].
Case Study 3: Nanobody formats targeting Mn‑SOD in Staphylococcus aureus improve pathogen clearance in murine models, demonstrating synergy with host nutritional immunity [new references].”

4. Response to Comments on the Quality of English Language

Point 1:
The English could be improved to more clearly express the research.

Response 1:
The manuscript was thoroughly revised for clarity and readability. Redundant phrasing was removed, sentences were restructured to emphasize mechanistic explanations, and terminology was standardized across all chapters. This ensures that the scientific narrative is precise, accessible, and consistent.

5. Additional Clarifications

• References across all chapters were corrected and aligned with the text.

• Mechanistic explanations were expanded in Chapters 1–5 to improve depth and readability.

• Case studies in Chapter 5 were rewritten to provide critical analysis and translational relevance.

• Captions and formatting were corrected to ensure consistency with the text.

• These revisions collectively strengthen the manuscript’s clarity, accuracy, and contribution to the field.

Conclusion

We sincerely thank the reviewer for their thorough evaluation and constructive feedback. Each comment has been carefully considered and addressed through targeted revisions across all chapters of the manuscript. Specifically, we have:

• Corrected and aligned references throughout Chapters 1–5, ensuring that every mechanistic claim and case study is supported by authoritative sources.

• Improved figures and captions, adding chemical structures, clarifying panel descriptions, and ensuring consistency between text and visuals.

• Expanded mechanistic explanations, removing redundancies, integrating structural biology insights, and linking pathways directly to therapeutic relevance.

• Enhanced case studies with deeper mechanistic detail, translational implications, and supporting evidence from experimental and clinical studies.

• Refined language and formatting, eliminating redundancies, standardizing terminology, and improving readability in compliance with journal style.

These revisions collectively strengthen the manuscript’s clarity, accuracy, and contribution to the field. The revised version now provides a more comprehensive, evidence‑based, and mechanistically rich review of metalloprotein‑targeted strategies against antimicrobial resistance. We believe the manuscript is significantly improved and now meets the standards expected for publication.