Natural Products Targeting PAD4 in NETosis: Structural and Mechanistic Insights into Direct and Indirect Inhibition

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors The author edited an extensive analysis of PAD4 biological involvement in NETosis and the pharmacological strategies to target PAD4 in inflammatory and cancer diseases. This review partially overlaps with other reviews written on this theme, but it is more focused on the potential role of natural products in combating PAD4-related diseases. The review is well written and accompanied by explanatory figures and tables, I have only a few minor points: -in my opinion, it is better to mention figures and table into the text rather than at the end of the paragraphs. For example figure 2 could be cited in paragraph 2 to allow understanding PAD4 structural features with the aid of the figure -in paragraph 2 it should be specified that PAD4 dimerizes. This point is breafly cited in paragraph 3.5, but paragraph 2, being centred on PAD4 structural features, sould contain this information. Moreover, Ca2+ binding sites of the protein should be included. Among natural and synthetic compounds, are there studies oriented toward targeting Ca2+ binding sites? This point should be addressed. If Ca2+ interaction is required for protein activation, molecules targeting Ca2+ binding sites could interfere with PAD4 biological outcomes. If there are experimental or computational studies on this, add a paragraph to describe them and add Ca2+ binding sites to figure 2. If it is not the case, specify that there is a lack of studies related to this aspect. -in figure 2 (panels B and C) are the images extrapolated from Vina? These docking studies are taken from another work, or were they made by the author? Please specify and cite appropriate literature. If the docking is made by the author, specify  the protocol used to run the calculations in the caption. Moreover, the different colours of the residues should be explained. -several enzymatic assays are cited in paragraph 3 and Table1, It can confuse those who are not in the field. I suggest adding a paragraph to briefly introduce the various techniques cited. Moreover, specify technical acronyms such as Ki and Kis. -the same argument is valid for computational tools, which should be briefly introduced   -specify that PADI4 is the gene related to the PAD4 protein, the first time it is cited -in table 4 specify in the caption why for some compounds the PAD4- inhibitory pathway is considered as "highly possible" -a lot of acronyms are not specified, I suggest adding a list of abbreviations at the end of the manuscript

Author Response

The author edited an extensive analysis of PAD4 biological involvement in NETosis and the pharmacological strategies to target PAD4 in inflammatory and cancer diseases. This review partially overlaps with other reviews written on this theme, but it is more focused on the potential role of natural products in combating PAD4-related diseases. The review is well written and accompanied by explanatory figures and tables, I have only a few minor points: -in my opinion, it is better to mention figures and table into the text rather than at the end of the paragraphs.

For example figure 2 could be cited in paragraph 2 to allow understanding PAD4 structural features with the aid of the figure -in paragraph 2.

⟶ I have revised the manuscript by citing all figures and tables at appropriate positions within the main text.

it should be specified that PAD4 dimerizes. This point is breafly cited in paragraph 3.5, but paragraph 2, being centred on PAD4 structural features, sould contain this information.

Moreover, Ca2+ binding sites of the protein should be included. Among natural and synthetic compounds, are there studies oriented toward targeting Ca2+ binding sites? This point should be addressed. If Ca2+ interaction is required for protein activation, molecules targeting Ca2+ binding sites could interfere with PAD4 biological outcomes. If there are experimental or computational studies on this, add a paragraph to describe them and add Ca2+ binding sites to figure 2. If it is not the case, specify that there is a lack of studies related to this aspect.

⟶ I have revised Section 2.1 to explicitly describe PAD4 dimerization and incorporated detailed information on Ca²⁺-binding sites, including their structural roles and relevance to enzymatic activation. In addition, I have addressed the lack of studies targeting Ca²⁺-binding sites and updated Figure 2 accordingly.

 

-in figure 2 (panels B and C) are the images extrapolated from Vina? These docking studies are taken from another work, or were they made by the author? Please specify and cite appropriate literature. If the docking is made by the author, specify the protocol used to run the calculations in the caption. Moreover, the different colours of the residues should be explained.

⟶ To visualize the binding modes of representative inhibitors, molecular docking analyses of GSK484 and JBI-589 were performed using the CB-Dock2 platform (https://cadd.labshare.cn/cb-dock2/), which integrates cavity detection and AutoDock Vi-na-based docking. The docking was conducted using the crystal structure of PAD4 (PDB: 3B1U), and the resulting binding poses were selected based on predicted binding affinity and cavity matching. Two-dimensional interaction diagrams were subsequently generat-ed using Discovery Studio 2024 Client to illustrate residue-level interactions within the U-shaped active-site tunnel (Figure 2B).

 

-several enzymatic assays are cited in paragraph 3 and Table1, It can confuse those who are not in the field. I suggest adding a paragraph to briefly introduce the various techniques cited. Moreover, specify technical acronyms such as Ki and Kis. -the same argument is valid for computational tools, which should be briefly introduced  

⟶ I have added explanatory notes to Table 1 to clarify the enzymatic assays, computational methods, and technical terms such as IC₅₀, Ki, and Kis.

 

-specify that PADI4 is the gene related to the PAD4 protein, the first time it is cited -in table 4 specify in the caption why for some compounds the PAD4- inhibitory pathway is considered as "highly possible"

⟶ I have clarified that PAD4 is encoded by the PADI4 gene and added an explanation for the term “highly possible” in the Table 4 caption.

 

-a lot of acronyms are not specified, I suggest adding a list of abbreviations at the end of the manuscript.

⟶ I have added a list of abbreviations.

Reviewer 2 Report

Comments and Suggestions for Authors

 

The manuscript reviews the inhibition mechanisms of PAD4 by both synthetic and natural products. Through structure-based analysis, Moon systematically compared the binding characteristics and interaction networks of synthetic and natural inhibitors with PAD4 and classified the inhibitor binding modes into four categories: (i) active-site–directed inhibitors, (ii) mixed and active-site–adjacent inhibitors, (iii) allosteric and hybrid modulators, and (iv) functionally validated PAD4 binders. Overall, this article provides a structural and mechanistic overview of natural products that target PAD4 and regulate NETosis, and it offers a promising perspective on safer and more effective therapeutic strategies for PAD4-associated diseases based on natural inhibitors.

Additionally, some minor points should be addressed, as outlined below:

 

1, Figure 1: Some of the abbreviations are not explained either in the main text or in the figure legend. For example, in panel B, what does “DC” stand for? In panel C, does “ECM” refer to the extracellular matrix? In addition, several other features of cancer cells are depicted in the figure but are not labeled; please label these as well. Finally, please explain all protein abbreviations in the figure for clarity.

 

2, Figure 2B: The meaning of the different residue colors is not clear. In addition, hydrogen bonds and hydrophobic interactions are not clearly depicted. Please clarify the color scheme and clearly indicate hydrogen bonding and hydrophobic interactions in the figure. Also, specify the software used to generate this figure in the legend.

 

3, Figure 3: There is a question mark in the figure. Please clarify what this question mark indicates in the context of the schematic.

 

4, Line 421: The abbreviation “MM-GBSA” is used for the first time without being defined. Please provide the full term when it first appears in the text.

 

 

5, Some statements in the manuscript are not clearly supported by references. For example, when the authors summarize the binding categories of PAD4 inhibitors, several statements should be accompanied by appropriate citations.

In particular, the statement, “In addition to experimentally validated PAD4 inhibitors derived from natural products, a growing number of studies have applied in silico approaches to identify natural compounds with the potential to bind PAD4” (line 419), appears to lack a supporting reference.

Similarly, the following passage should be supported by relevant citations:

“Despite differences in compound origin and computational workflows, in silico studies converge on several common features of PAD4 ligand engagement. Predicted natural product binders are consistently localized to regions overlapping with or adjacent to the catalytic cavity, particularly the substrate-recognition groove and Ca²⁺-responsive active-site environment. Across studies, docking poses frequently involve residues such as Asp350 and His471, which are implicated in substrate positioning and catalysis. In contrast, direct engagement of the catalytic cysteine Cys645 is rarely predicted, supporting a model in which these compounds interact with PAD4 through non-covalent, reversible binding modes. Such interactions are expected to modulate substrate accessibility or local active-site geometry rather than induce irreversible enzymatic inactivation.”

Please review the entire manuscript and add appropriate supporting references where needed.

 

6, In this study, the authors also performed molecular docking analyses. For example, “Two-dimensional interaction diagrams showing the predicted binding modes of representative synthetic PAD4 inhibitors docked using CB-Dock2 (https://cadd.labshare.cn/cb-dock2/).” However, there is no description of the docking methodology or basic parameters used. Please provide a concise description of the docking methods and key parameters.

Author Response

1, Figure 1: Some of the abbreviations are not explained either in the main text or in the figure legend. For example, in panel B, what does “DC” stand for? In panel C, does “ECM” refer to the extracellular matrix? In addition, several other features of cancer cells are depicted in the figure but are not labeled; please label these as well. Finally, please explain all protein abbreviations in the figure for clarity. ⟶ All abbreviations have been clarified and compiled into a comprehensive list, which has been added at the end of the manuscript.     2, Figure 2B: The meaning of the different residue colors is not clear. In addition, hydrogen bonds and hydrophobic interactions are not clearly depicted. Please clarify the color scheme and clearly indicate hydrogen bonding and hydrophobic interactions in the figure. Also, specify the software used to generate this figure in the legend. ⟶ Figure 2B has been revised to clarify the color scheme and interaction types, and the software used has been specified in the manuscript.     3, Figure 3: There is a question mark in the figure. Please clarify what this question mark indicates in the context of the schematic. ⟶ A clarification regarding the question mark has been added to the Figure 3 legend.     4, Line 421: The abbreviation “MM-GBSA” is used for the first time without being defined. Please provide the full term when it first appears in the text. ⟶ The full term for MM-GBSA has been added.     5, Some statements in the manuscript are not clearly supported by references. For example, when the authors summarize the binding categories of PAD4 inhibitors, several statements should be accompanied by appropriate citations.   In particular, the statement, “In addition to experimentally validated PAD4 inhibitors derived from natural products, a growing number of studies have applied in silico approaches to identify natural compounds with the potential to bind PAD4” (line 419), appears to lack a supporting reference.   Similarly, the following passage should be supported by relevant citations:   “Despite differences in compound origin and computational workflows, in silico studies converge on several common features of PAD4 ligand engagement. Predicted natural product binders are consistently localized to regions overlapping with or adjacent to the catalytic cavity, particularly the substrate-recognition groove and Ca²⁺-responsive active-site environment. Across studies, docking poses frequently involve residues such as Asp350 and His471, which are implicated in substrate positioning and catalysis. In contrast, direct engagement of the catalytic cysteine Cys645 is rarely predicted, supporting a model in which these compounds interact with PAD4 through non-covalent, reversible binding modes. Such interactions are expected to modulate substrate accessibility or local active-site geometry rather than induce irreversible enzymatic inactivation.”   Please review the entire manuscript and add appropriate supporting references where needed. ⟶ Appropriate supporting references have been added.     6, In this study, the authors also performed molecular docking analyses. For example, “Two-dimensional interaction diagrams showing the predicted binding modes of representative synthetic PAD4 inhibitors docked using CB-Dock2 (https://cadd.labshare.cn/cb-dock2/).” However, there is no description of the docking methodology or basic parameters used. Please provide a concise description of the docking methods and key parameters.

⟶ A concise description of the docking methodology and key parameters has been added to the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This review manuscript summarizes current knowledge on PAD4, an isozyme of peptidyl arginine deiminase, with emphasis on natural modulators of its activity in cells. This enzyme regulates histone citrullination, chromatin dynamics, and neutrophil extracellular trap formation. This involvement is associated with PAD4 role in inflammatory and immune diseases. Accordingly, PAD4 is a popular target in searches for various synthetic and natural modulators of its activity. There were more than fifty reviews on this enzyme published during the preceding five years.

The manuscript is well structured and clearly written. Importantly, the author has his/her own publications in the field of PAD4 studies, qualifying him/her as an expert in the field. Approximately one third of the 80 references are to publications of the last three years. Altogether, this review will a worthy guide for both beginners and experts in the field.

 

I have only minor remarks:

1. Already in the Abstract, the author classifies PAD4 modulators as active-site-directed, mixed and active-site-adjacent, allosteric, and functionally validated. I believe that the last category considers a different aspect and belongs to different classification (functionally validated and theoretically predicted or putative). A third classification implicitly used is direct (interacting with PAD4 directly) and indirect (acting via interacting cellular pathways). These three independent classifications should be considered separately but are sometimes messed in the manuscript.
2. Five Ca2+-binding sites in PAD4 are mentioned. Some more details are clearly needed for a full description.
3. Section 2 title does not seem optimal.
4. Lines 154-155: this is tautology, because chemical complementarity is determined by active site architecture, how else?
5. Section 3 title: please see remark 1.
6. Line 355: Earlier Ki/Kd’s are shown in uM and mM.
7. Line 521: Consider replacing “PAD4” with “cellular PAD4 activity”.
8. Table 1 shows IC50/Ki values in uM, whereas Tables 2 and 3 show free energies of interactions. For easy comparison, consider also showing in Tables 2 and 3 Kd values calculated from the free energy values (for instance, in parentheses).

Author Response

1. Already in the Abstract, the author classifies PAD4 modulators as active-site-directed, mixed and active-site-adjacent, allosteric, and functionally validated. I believe that the last category considers a different aspect and belongs to different classification (functionally validated and theoretically predicted or putative). A third classification implicitly used is direct (interacting with PAD4 directly) and indirect (acting via interacting cellular pathways). These three independent classifications should be considered separately but are sometimes messed in the manuscript.

⟶ A clarifying statement has been added at the beginning of Section 3 to explicitly distinguish the independent classification criteria for PAD4 modulators.

 

 

2. Five Ca2+-binding sites in PAD4 are mentioned. Some more details are clearly needed for a full description.

⟶ Additional details regarding the five Ca²⁺-binding sites in PAD4 have been included in the manuscript.

 

3. Section 2 title does not seem optimal.

⟶ The title of Section 2 has been revised to “Structural Basis of PAD4 and Its Implications for Inhibitor Development.”

 

4. Lines 154-155: this is tautology, because chemical complementarity is determined by active site architecture, how else?

⟶ The sentence has been revised to remove redundancy.

 

5. Section 3 title: please see remark 1.

⟶ The title and content of Section 3 have been revised accordingly.

 

6. Line 355: Earlier Ki/Kd’s are shown in uM and mM.

⟶ The units have been standardized throughout the manuscript.

 

7. Line 521: Consider replacing “PAD4” with “cellular PAD4 activity”.

⟶ The term has been revised to “cellular PAD4 activity” to improve clarity.

 

8. Table 1 shows IC50/Ki values in uM, whereas Tables 2 and 3 show free energies of interactions. For easy comparison, consider also showing in Tables 2 and 3 Kd values calculated from the free energy values (for instance, in parentheses).

⟶ Revised Tables 2 and 3 now include Kd values calculated from the docking-derived binding free energies (shown in parentheses), and the IC50/Ki values in Table 1 have been converted from mM to μM for consistency.