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  • Julio Cesar Armenta-Gorosave,
  • Gerson Ney Hernández-Acevedo and
  • José Luis Vique-Sánchez *
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous Reviewer 4: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript of Armenta-Gorosave et al. describes molecular docking of EXPRESS-Pick Stock small molecule screening library, in sillico and in vitro assays and proposes compounds that could be selective against SaDHQD. From the docking results, the best 16 were selected. The antimicrobial effect of the 16 acquired compounds was determined using in vitro cultures of S. aureus. The authors propose two compounds which could be developed as new antibiotics against S. aureus.

The manuscript can be published in Scientia Pharmaceutica after revision.

Authors need to add more information about DHQD to introduction. Explain their choice for DHQD as target enzyme. What about known inhibitors? Probably active molecules for other bacterial DHQD. Please add figure with inhibitors. It allow to compare compounds mentioned in the paper with described earlier.

There is no caption to Figure 1

Authors need to check Tables 1,2. Structures are beyond the borders and borders of the table 2 are beyond thepage.

Line 179 “… imipenem as a positive control”, but line 185 “…controls (vancomycin and DMSO)”

Section 3.4. MIC!!! Not CIM. Check through the whole text.

Figure 3: MIC!, add axis captions.

In the manuscript MIC was measured in µg/ml, while IC50 (what is meant CC50) are given in µM. It’s difficult to compare. Units of measurement should be the same. Please provide SI.  

After changes the manuscript can be published in Scientia Pharmaceutica.

Author Response

Reviewer 1:

The manuscript of Armenta-Gorosave et al. describes molecular docking of EXPRESS-Pick Stock small molecule screening library, in sillico and in vitro assays and proposes compounds that could be selective against SaDHQD. From the docking results, the best 16 were selected. The antimicrobial effect of the 16 acquired compounds was determined using in vitro cultures of S. aureus. The authors propose two compounds which could be developed as new antibiotics against S. aureus.

 

The manuscript can be published in Scientia Pharmaceutica after revision.

 

1.- Authors need to add more information about DHQD to introduction. Explain their choice for DHQD as target enzyme. What about known inhibitors? Probably active molecules for other bacterial DHQD. Please add figure with inhibitors. It allow to compare compounds mentioned in the paper with described earlier.

Response:   

Thank you for your comments; we have expanded the introduction to include the points you mentioned (DHQD inhibitors in other organisms) and have also added a figure and discussed other studies on DHQD in the discussion section. It is the Figure 5:

 

2.- There is no caption to Figure 1

Response:

Thank you for the comment, it was attended.   

 

Figure 1. The structure of SaDHQD (PDB:6SFH, green) and the potential site region between the amino acids: Glu35, Arg37, Arg70, Lys160, His133, Arg202, Gln225 (Blue) are shown; highlighting the surface-cavity of the proposed interaction site for the realization of molecular docking (red circle).
 

 

3.- Authors need to check Tables 1,2. Structures are beyond the borders and borders of the table 2 are beyond the page.

Response:  

Thank you for the comment, it was attended.

 4.- Line 179 “… imipenem as a positive control”, but line 185 “…controls (vancomycin and DMSO)” 

Response:   

Thank you for the comment; we have replaced imipenem instead of vancomycin, which was the positive control used in most of the assays. As well as imipenem is considered as control by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for antimicrobial susceptibility testing (AST) and MIC [1,2] [3].

 

5.- Section 3.4. MIC!!! Not CIM. Check through the whole text.

Response:   

Thank you for the comment; we have replaced CMI with MIC

 

6.- Figure 3: MIC!, add axis captions.

Response:   

Thank you for the suggestion; we have updated the figure's content to include the positive control group and added axis labels to the graph. It is the new Figure 3:

   

 7.- In the manuscript MIC was measured in µg/ml, while IC50 (what is meant CC50) are given in µM. It’s difficult to compare. Units of measurement should be the same. Please provide SI. 

Response:   

Thank you for the suggestion.

It was changed, the CC50 instead of IC50, it was used for the citotoxicity assays, and we added the equivalent for the concentration. As the reviewer recommended, now it is showing the concentrations on base od system international (SI), although, we use the concentrations to AST and MIC by EUCAST guides [1,2], which now reads:

 

…the Sa-3 compound with a CC50 of 33.1 µM (16.1 µg/mL) for C9 cells, and a CC50 of 44.7 µM (21.8 µg/mL) for PC3 cells after 24 h (Figure 4). Regarding the Sa-8 compound, after 24 h above 100 µM of concentration (51.5 µg/mL), a moderate cytotoxic effect…

 

 8.- After changes the manuscript can be published in Scientia Pharmaceutica.

Response:   

Thank you for the comment; your suggestions have improved our manuscript.

 

Thank you for your comments.

 

References

 

  1. EUCAST Version 12.0, valid from 2022-01-01 The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters; http://www.eucast.org, 2022;
  2. EUCAST Antimicrobial Susceptibility Testing EUCAST Disk Diffusion Method; http://www.eucast.org, 2025;
  3. Åhman, J.; Matuschek, E.; Kahlmeter, G. Evaluation of Ten Brands of Pre-Poured Mueller-Hinton Agar Plates for EUCAST Disc Diffusion Testing. Clinical Microbiology and Infection 2022, 28, 1499.e1-1499.e5, doi:10.1016/j.cmi.2022.05.030.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The idea behind the manuscript "Potential Ligands to 3-dehydroquinate dehydratase (SaDHQD) 2 of Staphylococcus aureus, Evaluated them by Molecular 3 Docking and In vitro Assays to Develop an Antibiotic Drug" is interesting and might have potential application in the development of S. aureus inhibitors.

But the evaluated manuscript, has some serious flaws, which will be presented.

First, the protocol of the molecular docking must be presented with more details. According to Figure 5, part of Sa-3 and Sa-8 (a ring) seems to fit to a pocket, but the rest of the molecules are oriented in different directions. How big was the search space? All that space is the catalytic site or there are some accessory sites (such as allosteric pockets)? 

Because molecular docking is known to give false positive results, a molecular dynamics study would be mandatory to evaluate the stability in time of those bound ligands.

The graphical depiction of the structures of the compounds must be made using a specialized software - for clarity, copyright issues and scientific correctness. Structures are blurry, have artifacts due to the print screen and some were flipped (such as Sa-3 and Sa-4).

Why the two compounds were tested on different concentration ranges 500-16 vs 250-8 ug/mL ? It is mandatory to use a reference antimicrobial agent as positive control.

Comments on the Quality of English Language

The manuscript must be reviewed by a person proficient in English.

Author Response

Reviewer 2:

The idea behind the manuscript "Potential Ligands to 3-dehydroquinate dehydratase (SaDHQD) of Staphylococcus aureus, Evaluated them by Molecular Docking and In vitro Assays to Develop an Antibiotic Drug" is interesting and might have potential application in the development of S. aureus inhibitors.

But the evaluated manuscript, has some serious flaws, which will be presented.

 1.- First, the protocol of the molecular docking must be presented with more details. According to Figure 5, part of Sa-3 and Sa-8 (a ring) seems to fit to a pocket, but the rest of the molecules are oriented in different directions. How big was the search space? All that space is the catalytic site or there are some accessory sites (such as allosteric pockets)? 

Response:

Thank you for the suggestion. It is described as:

Molecular docking was carried out by MOE, the region around of catalytic site in SaDHQD was used as protein target for a molecular docking [13] [14] (Figure 1), and up to 100 conformers of each molecule (from EXPRESS-Pick Stock) were used for molecular docking. The catalytic site region between the amino acids: Glu35, Arg37, Arg70, Lys160, His133, Arg202, Gln225…

 

Following the docking process, we selected the 16 best compounds by calculating the average of the 10 best ΔGvalues ​​(derived from the top 10 conformers for each compound). It is worth noting that we have developed this methodology across several molecular docking studies  [1–7]; consequently, when selecting the top compounds, we prioritized those with the most favorable average interaction scores.

We present the interaction values ​​and visualizations of the 10 studied conformers in the supplementary material (Figs. S1–S16). These figures show that all conformers interact near the catalytic site region, consistent with the initial docking setup, which targeted a potential site within the catalytic region (where Lys160 is important in the catalytic site [8,9]), that the best compounds are represented in the Figure 6, and theoretically do not interact at other locations, such as allosteric sites, although this could be investigated further in future studies.

 

 2.- Because molecular docking is known to give false positive results, a molecular dynamics study would be mandatory to evaluate the stability in time of those bound ligands.

Response:

Thank you for the suggestion. You are right; theoretical docking studies are often accompanied by molecular dynamics simulations to justify and complement the description [8] [10] [11]. However, in this study, we planned that in vitro assays were the best way to confirm our results (thereby demonstrating an antibacterial effect) and proceed with the study. Since none of the compounds would have shown an antimicrobial effect otherwise, we would have had to proceed with others; for this reason, we retained only two compounds (Sa3 and Sa8) and discarded the other 14 for this stage of experimental development.

We hope to complement the description of the interactions using molecular dynamics in the future.

 

 3.- The graphical depiction of the structures of the compounds must be made using a specialized software - for clarity, copyright issues and scientific correctness. Structures are blurry, have artifacts due to the print screen and some were flipped (such as Sa-3 and Sa-4).

Response:

Thank you for the suggestion.

We have improved the images; they were generated from molecular docking results and sourced from PubChem. We hope the reviewer agrees with the definition, layout, and clarity.

 

 4.- Why the two compounds were tested on different concentration ranges 500-16 vs 250-8 ug/mL ? It is mandatory to use a reference antimicrobial agent as positive control.

Response:

Thank you for the suggestion; we have updated the figure's content to include the positive control group and added axis labels to the graph. It is the new Figure 3. As well as imipenem is considered as control by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for antimicrobial susceptibility testing (AST) and MIC [12,13] [14].

 

       
     
 
 

Figure 3. Results of MIC assay, after 24 h of incubation. Control DMSO (Blue), Sa-3 compound (Red), and Sa-8 compound (Green). The MIC is 500 µg/mL for both compounds.
 

 

Thank you for your comments.

 

References

 

  1. Galindo-Hernández, O.; Vique-Sánchez, J.L. AXL Inhibitors Selected by Molecular Docking: Option for Reducing SARS-CoV-2 Entry into Cells. Acta Pharmaceutica 2022, 72, 329–343, doi:10.2478/acph-2022-0024.
  2. Téllez‐Valencia, A.; Hernández‐Ortiz, I.; Guadalupe López Lujano, P.; Luis Vique‐Sánchez, J. Development of Tyrosine Phosphatase 1B Inhibitors Based on Molecular Docking, Kinetics, and Toxicity Studies. ChemistrySelect 2023, 8, doi:10.1002/slct.202300549.
  3. Téllez‐Valencia, A.; Hernández‐Ortiz, I.; Guadalupe López Lujano, P.; Luis Vique‐Sánchez, J. Development of Tyrosine Phosphatase 1B Inhibitors Based on Molecular Docking, Kinetics, and Toxicity Studies. ChemistrySelect 2023, 8, doi:10.1002/slct.202300549.
  4. Trasviña-Arenas, C.H.; Ayala Medina, L.A.; Vique-Sanchez, J.L. γ-Secretase Inhibitors Selected by Molecular Docking, to Develop a New Drug Against Alzheimer’s Disease. Rep. Biochem. Mol. Biol. 2023, 12, 340–349, doi:10.61186/rbmb.12.2.340.
  5. Vique-Sánchez, J.L. Potential Inhibitors Interacting in Neuropilin-1 to Develop an Adjuvant Drug against COVID-19, by Molecular Docking. Bioorg. Med. Chem. 2021, 33, doi:10.1016/j.bmc.2021.116040.
  6. Vique-Sánchez, J.L. Potential Inhibitors Interacting in Neuropilin-1 to Develop an Adjuvant Drug against COVID-19, by Molecular Docking. Bioorg. Med. Chem. 2021, 33, 116040, doi:10.1016/j.bmc.2021.116040.
  7. Rivera‐Suárez, B.A.; García González, V.G.; Chimal‐Vega, B.; Navarro Padrón, A.C.; Galindo‐Hernández, O.; Vique‐Sánchez, J.L. Potential Ligands to Resistin Against Prostate Cancer, Evaluated by Molecular Docking and In Vitro Assays to Develop an Anticancer Drug. Chem. Biodivers. 2025, 22, doi:10.1002/cbdv.202500189.
  8. Millán-Pacheco, C.; Rios-Soto, L.; Corral-Rodríguez, N.; Sierra-Campos, E.; Valdez-Solana, M.; Téllez-Valencia, A.; Avitia-Domínguez, C. Discovery of Potential Noncovalent Inhibitors of Dehydroquinate Dehydratase from Methicillin-Resistant Staphylococcus Aureus through Computational-Driven Drug Design. Pharmaceuticals 2023, 16, 1148, doi:10.3390/ph16081148.
  9. González-Bello, C.; Tizón, L.; Lence, E.; Otero, J.M.; van Raaij, M.J.; Martinez-Guitian, M.; Beceiro, A.; Thompson, P.; Hawkins, A.R. Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of Its Active Site Covalent Adduct. J. Am. Chem. Soc. 2015, 137, 9333–9343, doi:10.1021/jacs.5b04080.
  10. Rios-Soto, L.; Téllez-Valencia, A.; Sierra-Campos, E.; Valdez-Solana, M.; Cisneros-Martínez, J.; Gómez Palacio-Gastélum, M.; Castillo-Villanueva, A.; Avitia-Domínguez, C. Finding the First Potential Inhibitors of Shikimate Kinase from Methicillin Resistant Staphylococcus Aureus through Computer-Assisted Drug Design. Molecules 2021, 26, 6736, doi:10.3390/molecules26216736.
  11. Eduardo Sanabria-Chanaga, E.; Betancourt-Conde, I.; Hernández-Campos, A.; Téllez-Valencia, A.; Castillo, R. In Silico Hit Optimization toward AKT Inhibition: Fragment-Based Approach, Molecular Docking and Molecular Dynamics Study. J. Biomol. Struct. Dyn. 2019, 37, 4301–4311, doi:10.1080/07391102.2018.1546618.
  12. EUCAST Version 12.0, valid from 2022-01-01 The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters; http://www.eucast.org, 2022;
  13. EUCAST Antimicrobial Susceptibility Testing EUCAST Disk Diffusion Method; http://www.eucast.org, 2025;
  14. Åhman, J.; Matuschek, E.; Kahlmeter, G. Evaluation of Ten Brands of Pre-Poured Mueller-Hinton Agar Plates for EUCAST Disc Diffusion Testing. Clinical Microbiology and Infection 2022, 28, 1499.e1-1499.e5, doi:10.1016/j.cmi.2022.05.030.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript addresses a relevant topic in the field of antibiotic drug discovery, focusing on the identification and evaluation of novel ligands targeting SaDHQD in Staphylococcus aureus. The study combines in silico molecular docking with in vitro antibacterial and cytotoxicity assays, aiming to propose new candidates for antibiotic development.

Some major comments to be addressed include:

  • clarify the rationale for compound selection beyond binding energy
  • discuss limitations more explicitly, including the modest antibacterial activity and cytotoxicity concerns
  • improve clarity of tables and figures
  • proofread the whole manuscript for language and formatting consistency. T The manuscript contains numerous grammatical, linguistic, and formatting errors that should be carefully corrected throughout the text.

Author Response

Reviewer 3:

This manuscript addresses a relevant topic in the field of antibiotic drug discovery, focusing on the identification and evaluation of novel ligands targeting SaDHQD in Staphylococcus aureus. The study combines in silico molecular docking with in vitro antibacterial and cytotoxicity assays, aiming to propose new candidates for antibiotic development.

Some major comments to be addressed include:

 

1.- Clarify the rationale for compound selection beyond binding energy

Response:

Thank you for the suggestion. It is described as:

Molecular docking was carried out by MOE, the region around of catalytic site in SaDHQD was used as protein target for a molecular docking [13] [14] (Figure 1), and up to 100 conformers of each molecule (from EXPRESS-Pick Stock) were used for molecular docking. The catalytic site region between the amino acids: Glu35, Arg37, Arg70, Lys160, His133, Arg202, Gln225…

 

Following the docking process, we selected the 16 best compounds by calculating the average of the 10 best ΔGvalues ​​(derived from the top 10 conformers for each compound). It is worth noting that we have developed this methodology across several molecular docking studies  [1–7]; consequently, when selecting the top compounds, we prioritized those with the most favorable average interaction scores.

We present the interaction values ​​and visualizations of the 10 studied conformers in the supplementary material (Figs. S1–S16). These figures show that all conformers interact near the catalytic site region, consistent with the initial docking setup, which targeted a potential site within the catalytic region (where Lys160 is important in the catalytic site [8,9]), that the best compounds are represented in the Figure 6.

 

2.- Discuss limitations more explicitly, including the modest antibacterial activity and cytotoxicity concerns  

Response:

Thank you for the suggestion. The comment is appropriate; the antimicrobial effect is low. We even repeated the disk diffusion assay, according to the EUCAST methodology [10,11], where Mueller-Hinton agar plates are used. We tested two concentrations, at 50 and 300 µg, and just as with the blood agar plates, the compounds Sa-3 and Sa-8 were the ones that showed a small halo (which was slightly larger at 300 µg), with a zone diameter of 8 to 10 mm for these two compounds (Figure 2). We used the established control (imipenem at 30 µg) which is required to produce a zone diameter of at least 27 mm [11]. According to EUCAST guidelines, the two drugs with the smallest inhibition zones are nitrofurantoin (at least 13 mm) and trimethoprim (at least 14 mm). Consequently, an inhibition zone of less than 10 mm suggests that Sa-3 and Sa-8 lack adequate antibacterial activity; however, these two molecules remain candidates for future consideration, particularly given their intracellular target (SaDHQD), and the potential for combination therapies.

Furthermore, we determined the MIC at 500 µg/mL for both compounds; while this value remains high, it serves as a starting point for the development of a new antibiotic, especially when considering studies identifying other targets within the shikimate pathway, such as shikimate dehydrogenase [12], and which report advances in the MIC of 12 mM [13], which is far above our results.

We recognize that this study is preliminary and that further trials are required to test the effect against resistant S. aureus isolates; however, there are currently no molecules reported experimental, only in silico [8]. These two molecules are novel (Sa-3 and Sa-8), and we have already begun to demonstrate results in vitro and they could hold the key to overcoming resistance to current treatments.

Regarding toxicity, the compound exhibits higher toxicity; however, we hope that combining it with other antibiotics will yield synergistic effects, thereby allowing for lower dosages. Furthermore, these two molecules could serve as a basis for developing derivatives aimed at enhancing their effects.

 

 

3.- Improve clarity of tables and figures

Response:  

Thank you for the suggestion; it was attended. Tables and figures were revised and improved.

 

4.- Proofread the whole manuscript for language and formatting consistency. T The manuscript contains numerous grammatical, linguistic, and formatting errors that should be carefully corrected throughout the text.

Response:  

Thank you for the suggestion; it was revised and improved by authors.

 

 

Thank you for your comments.

 

References

 

  1. Galindo-Hernández, O.; Vique-Sánchez, J.L. AXL Inhibitors Selected by Molecular Docking: Option for Reducing SARS-CoV-2 Entry into Cells. Acta Pharmaceutica 2022, 72, 329–343, doi:10.2478/acph-2022-0024.
  2. Téllez‐Valencia, A.; Hernández‐Ortiz, I.; Guadalupe López Lujano, P.; Luis Vique‐Sánchez, J. Development of Tyrosine Phosphatase 1B Inhibitors Based on Molecular Docking, Kinetics, and Toxicity Studies. ChemistrySelect 2023, 8, doi:10.1002/slct.202300549.
  3. Téllez‐Valencia, A.; Hernández‐Ortiz, I.; Guadalupe López Lujano, P.; Luis Vique‐Sánchez, J. Development of Tyrosine Phosphatase 1B Inhibitors Based on Molecular Docking, Kinetics, and Toxicity Studies. ChemistrySelect 2023, 8, doi:10.1002/slct.202300549.
  4. Trasviña-Arenas, C.H.; Ayala Medina, L.A.; Vique-Sanchez, J.L. γ-Secretase Inhibitors Selected by Molecular Docking, to Develop a New Drug Against Alzheimer’s Disease. Rep. Biochem. Mol. Biol. 2023, 12, 340–349, doi:10.61186/rbmb.12.2.340.
  5. Vique-Sánchez, J.L. Potential Inhibitors Interacting in Neuropilin-1 to Develop an Adjuvant Drug against COVID-19, by Molecular Docking. Bioorg. Med. Chem. 2021, 33, doi:10.1016/j.bmc.2021.116040.
  6. Vique-Sánchez, J.L. Potential Inhibitors Interacting in Neuropilin-1 to Develop an Adjuvant Drug against COVID-19, by Molecular Docking. Bioorg. Med. Chem. 2021, 33, 116040, doi:10.1016/j.bmc.2021.116040.
  7. Rivera‐Suárez, B.A.; García González, V.G.; Chimal‐Vega, B.; Navarro Padrón, A.C.; Galindo‐Hernández, O.; Vique‐Sánchez, J.L. Potential Ligands to Resistin Against Prostate Cancer, Evaluated by Molecular Docking and In Vitro Assays to Develop an Anticancer Drug. Chem. Biodivers. 2025, 22, doi:10.1002/cbdv.202500189.
  8. Millán-Pacheco, C.; Rios-Soto, L.; Corral-Rodríguez, N.; Sierra-Campos, E.; Valdez-Solana, M.; Téllez-Valencia, A.; Avitia-Domínguez, C. Discovery of Potential Noncovalent Inhibitors of Dehydroquinate Dehydratase from Methicillin-Resistant Staphylococcus Aureus through Computational-Driven Drug Design. Pharmaceuticals 2023, 16, 1148, doi:10.3390/ph16081148.
  9. González-Bello, C.; Tizón, L.; Lence, E.; Otero, J.M.; van Raaij, M.J.; Martinez-Guitian, M.; Beceiro, A.; Thompson, P.; Hawkins, A.R. Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of Its Active Site Covalent Adduct. J. Am. Chem. Soc. 2015, 137, 9333–9343, doi:10.1021/jacs.5b04080.
  10. EUCAST Antimicrobial Susceptibility Testing EUCAST Disk Diffusion Method; http://www.eucast.org, 2025;
  11. EUCAST Version 12.0, valid from 2022-01-01 The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters; http://www.eucast.org, 2022;
  12. Zhu, M.; Qu, J.; Deng, Q. Identification of Potential Inhibitors against Staphylococcus Aureus Shikimate Dehydrogenase through Virtual Screening and Susceptibility Test. J. Enzyme Inhib. Med. Chem. 2024, 39, doi:10.1080/14756366.2024.2301768.
  13. Corral-Rodríguez, N.F.; Moreno-Contreras, V.I.; Sierra-Campos, E.; Valdez-Solana, M.; Cisneros-Martínez, J.; Téllez-Valencia, A.; Avitia-Domínguez, C. Characterization of Natural Products as Inhibitors of Shikimate Dehydrogenase from Methicillin-Resistant Staphylococcus Aureus: Kinetic and Molecular Dynamics Simulations, and Biological Activity Studies. Biomolecules 2025, 15, 1137, doi:10.3390/biom15081137.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript is scientifically interesting, particularly because it combines large-scale virtual screening of approximately 500000 compounds with experimental antibacterial evaluation. However, in its current form, the study remains too preliminary to support several of the conclusions of the authors  and I therefore recommend major revision.

The principal concern is the lack of experimental evidence demonstrating that SaDHQD is the actual intracellular target of Sa-3 and Sa-8. Docking only predicts possible binding, whereas the biological experiments demonstrate growth inhibition but not direct inhibition of SaDHQD. Ideally, the authors should perform a biochemical assay using recombinant SaDHQD to determine enzymatic IC50 values, Ki values and the inhibition mechanism. If such experiments cannot be provided, all claims describing Sa-3 and Sa-8 as specific SaDHQD inhibitors should be highly moderated

A second major limitation is that the antibacterial evaluation deals with  only on the methicillin-susceptible S. aureus ATCC 25923 strain, despite the strong emphasis on MRSA and antimicrobial resistance throughout the manuscript. Sa-3 and Sa-8 should therefore be tested against at least one or two reference MRSA strains and, ideally, several clinical isolates. MICs should be determined using a standardized CLSI or EUCAST-compliant broth microdilution method.Thus, the MIC methodology itself also requires clarification and standardization. The initial selection of Sa-3 and Sa-8 based on disk diffusion is another weakness. Since chemically diverse compounds may diffuse very differently through agar, disk diffusion can underestimate the activity of poorly diffusible molecules. Determining MIC values for all 16 compounds, or at least for a larger subset, would provide a more reliable comparison and would also allow evaluation of the relationship between docking score and antibacterial potency.

The docking results are also somewhat overinterpreted since the scores of the 16 compounds are very close and  cannot reliably distinguish their true binding affinities. The docking protocol coud be also better validated...

The selectivity of the compounds deserves more careful analysis. MIC values should be converted to molar units and compared directly with mammalian-cell IC50 values to calculate selectivity indices.

Finally, the manuscript contains a relatively high number of self-citations, several of which refer to previous studies by the authors on unrelated therapeutic targets such as  cancer, Alzheimer disease and SARS-CoV2. Thus, the citations in the part focusing on the previous research activities of the authors do not appear necessary for interpreting the present SaDHQD study. The authors should revise the reference list

Author Response

 Reviewer 4:

The manuscript is scientifically interesting, particularly because it combines large-scale virtual screening of approximately 500000 compounds with experimental antibacterial evaluation. However, in its current form, the study remains too preliminary to support several of the conclusions of the authors  and I therefore recommend major revision.

 

1.- The principal concern is the lack of experimental evidence demonstrating that SaDHQD is the actual intracellular target of Sa-3 and Sa-8. Docking only predicts possible binding, whereas the biological experiments demonstrate growth inhibition but not direct inhibition of SaDHQD. Ideally, the authors should perform a biochemical assay using recombinant SaDHQD to determine enzymatic IC50 values, Ki values and the inhibition mechanism. If such experiments cannot be provided, all claims describing Sa-3 and Sa-8 as specific SaDHQD inhibitors should be highly moderated

Response:

Thank you for the suggestion. The comment is appropriate; we focus on determining the antimicrobial effect. by MIC and disk diffusion assay, according to the EUCAST methodology [1,2]. So, it is necessary to demonstrate the antibacterial effect and the selectivity regarding SaDHQD; however, we have already made progress toward conducting assays with recombinant protein (relating to in silico results with in vitro results). It is worth mentioning, that we have experience to demonstrate the selectivity of compounds with in vitro assays (recombinant proteins) [3] [4] [5] [6], even preliminary in vivo results [7]  [8], surely, the next phase is to evaluate with recombinant protein or, strain resistant to methicillin.

We recognize that this study is preliminary and that further trials are required to determine the selectivity of Sa-3 and Sa-8 compounds against SaDHQD, by recombinant protein assays. We mentioned it in the discussion and will take this suggestion to evaluate this soon using, by enzyme activity or fluorescence or ELISA assays. We will seek to collaborate with researchers who have access to these or other trials, and to expand upon the results of Sa-3 and Sa-8.

 

2.- A second major limitation is that the antibacterial evaluation deals with  only on the methicillin-susceptible S. aureus ATCC 25923 strain, despite the strong emphasis on MRSA and antimicrobial resistance throughout the manuscript. Sa-3 and Sa-8 should therefore be tested against at least one or two reference MRSA strains and, ideally, several clinical isolates. MICs should be determined using a standardized CLSI or EUCAST-compliant broth microdilution method.Thus, the MIC methodology itself also requires clarification and standardization. The initial selection of Sa-3 and Sa-8 based on disk diffusion is another weakness. Since chemically diverse compounds may diffuse very differently through agar, disk diffusion can underestimate the activity of poorly diffusible molecules. Determining MIC values for all 16 compounds, or at least for a larger subset, would provide a more reliable comparison and would also allow evaluation of the relationship between docking score and antibacterial potency.

Response:

Thank you for the suggestion. The comment is appropriate; the antimicrobial effect is low. We even repeated the disk diffusion assay, according to the EUCAST methodology [1,2], where Mueller-Hinton agar plates are used. We tested two concentrations, at 50 and 300 µg, and just as with the blood agar plates, the compounds Sa-3 and Sa-8 were the ones that showed a small halo (which was slightly larger at 300 µg), with a zone diameter of 8 to 10 mm for these two compounds (Figure 2). We used the established control (imipenem at 30 µg) which is required to produce a zone diameter of at least 27 mm [2]. According to EUCAST guidelines, the two drugs with the smallest inhibition zones are nitrofurantoin (at least 13 mm) and trimethoprim (at least 14 mm). Consequently, an inhibition zone of less than 10 mm suggests that Sa-3 and Sa-8 lack adequate antibacterial activity; however, these two molecules remain candidates for future consideration, particularly given their intracellular target (SaDHQD), and the potential for combination therapies.

Furthermore, we determined the MIC to be 500 µg/mL for both compounds; while this value remains high, it serves as a starting point for the development of a new antibiotic, especially when considering studies identifying other targets within the shikimate pathway, such as shikimate dehydrogenase [9], and which report advances in the MIC of 12 mM [10], which is far above our results.

We recognize that this study is preliminary and that further trials are required to test the effect against resistant S. aureus isolates; however, there are currently no molecules reported experimental, only in silico [11]. These two molecules are novel (Sa-3 and Sa-8), and we have already begun to demonstrate results in vitro and they could hold the key to overcoming resistance to current treatments.

Regarding toxicity, the compound exhibits higher toxicity; however, we hope that combining it with other antibiotics will yield synergistic effects, thereby allowing for lower dosages. Furthermore, these two molecules could serve as a basis for developing derivatives aimed at enhancing their effects.

 

We have updated the figure's content to include the positive control group and added axis labels to the graph. It is the new Figure 3. As well as imipenem is considered as control by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for antimicrobial susceptibility testing (AST) and MIC [1,2] [12].

 

 

       
     
 
 

Figure 3. Results of MIC assay, after 24 h of incubation. Control DMSO (Blue), Sa-3 compound (Red), and Sa-8 compound (Green). The MIC is 500 µg/mL for both compounds.
 

 

 3.- The docking results are also somewhat overinterpreted since the scores of the 16 compounds are very close and  cannot reliably distinguish their true binding affinities. The docking protocol could be also better validated...

Response:

Thank you for the suggestion. It is described as:

Molecular docking was carried out by MOE, the region around of catalytic site in SaDHQD was used as protein target for a molecular docking [13] [14] (Figure 1), and up to 100 conformers of each molecule (from EXPRESS-Pick Stock) were used for molecular docking. The catalytic site region between the amino acids: Glu35, Arg37, Arg70, Lys160, His133, Arg202, Gln225…

 

Following the docking process, we selected the 16 best compounds by calculating the average of the 10 best ΔGvalues ​​(derived from the top 10 conformers for each compound). It is worth noting that we have developed this methodology across several molecular docking studies  [3,4,13–17]; consequently, when selecting the top compounds, we prioritized those with the most favorable average interaction scores.

We present the interaction values ​​and visualizations of the 10 studied conformers in the supplementary material (Figs. S1–S16). These figures show that all conformers interact near the catalytic site region—consistent with the initial docking setup, which targeted a potential site within the catalytic region (where Lys160 is important in the catalytic site [11,18])—, that the best compounds are represented in the Figure 5.

 

 

 

 4.- The selectivity of the compounds deserves more careful analysis. MIC values should be converted to molar units and compared directly with mammalian-cell IC50 values to calculate selectivity indices.

Thank you for the suggestion.

The selection of compounds is a careful, theoretically justified process; as previously mentioned, there have been advances in new drug development, proceeding from a chemical library (considering potential sites and target), to the selection of the best compounds [3,4,13–17]; consequently, when selecting the top compounds, we prioritized those with the most favorable average interaction scores. It is worth mentioning that we have experience to demonstrate the selectivity of compounds with its target, by in vitro assays (recombinant proteins) [3] [4] [5] [6], even preliminary in vivo results [7]  [8].

We present the interaction values, main interactions of each compound-SaDHQD, ​​and visualizations of the 10 conformers studied in the supplementary material. These figures show that all conformers interact near the catalytic site region, consistent with the initial docking setup, which targeted a potential site within the catalytic region (where Lys160 is important in the catalytic site [11,18]).

 

In addition, it was changed, the CC50 instead of IC50, it was used for the citotoxicity assays, and we added the equivalent for the concentration. Now it is showing the concentrations on base od system international (SI), although, we use the concentrations to AST and MIC by EUCAST guides [1,2], which now reads:

 

…the Sa-3 compound with a CC50 of 33.1 µM (16.1 µg/mL) for C9 cells, and a CC50 of 44.7 µM (21.8 µg/mL) for PC3 cells after 24 h (Figure 4). Regarding the Sa-8 compound, after 24 h above 100 µM of concentration (51.5 µg/mL), a moderate cytotoxic effect…

 

5.- Finally, the manuscript contains a relatively high number of self-citations, several of which refer to previous studies by the authors on unrelated therapeutic targets such as  cancer, Alzheimer disease and SARS-CoV2. Thus, the citations in the part focusing on the previous research activities of the authors do not appear necessary for interpreting the present SaDHQD study. The authors should revise the reference list

Response:

Thank you for the suggestion; the reviewer is correct. In the manuscript, we do reference our previous work, but all of it relates to molecular docking, the methodology, and the experimental results. We believe these self-citations demonstrate to readers that our research group has experience in drug development.

 

 Thank you for your comments.

 

References

 

  1. EUCAST Antimicrobial Susceptibility Testing EUCAST Disk Diffusion Method; http://www.eucast.org, 2025;
  2. EUCAST Version 12.0, valid from 2022-01-01 The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters; http://www.eucast.org, 2022;
  3. Téllez‐Valencia, A.; Hernández‐Ortiz, I.; Guadalupe López Lujano, P.; Luis Vique‐Sánchez, J. Development of Tyrosine Phosphatase 1B Inhibitors Based on Molecular Docking, Kinetics, and Toxicity Studies. ChemistrySelect 2023, 8, doi:10.1002/slct.202300549.
  4. Rivera‐Suárez, B.A.; García González, V.G.; Chimal‐Vega, B.; Navarro Padrón, A.C.; Galindo‐Hernández, O.; Vique‐Sánchez, J.L. Potential Ligands to Resistin Against Prostate Cancer, Evaluated by Molecular Docking and In Vitro Assays to Develop an Anticancer Drug. Chem. Biodivers. 2025, 22, doi:10.1002/cbdv.202500189.
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  7. García-González, V.G.; Morales, A.B.O.; Navarro Padrón, A.C.; Chimal-Vega, B.; Sánchez-Alavez, M.; Serafín-Higuera, I.R.; Galindo-Hernández, O.; Téllez-Valencia, A.; Vique-Sánchez, J.L. Advances in the Development of Protein Tyrosine Phosphatase 1B Inhibitor, Evaluated by in Vitro and in Vivo Assays. Results Chem. 2025, 16, 102465, doi:10.1016/j.rechem.2025.102465.
  8. Vique‐Sánchez, J.L.; López Lujano, P.G.; Rodríguez Fonseca, K.A.; Benítez‐Cardoza, C.G. Compounds Interacting with Cholecystokinin as Potential Drugs Against Excessive Weight Gain and Obesity. ChemistrySelect 2023, 8, doi:10.1002/slct.202300196.
  9. Zhu, M.; Qu, J.; Deng, Q. Identification of Potential Inhibitors against Staphylococcus Aureus Shikimate Dehydrogenase through Virtual Screening and Susceptibility Test. J. Enzyme Inhib. Med. Chem. 2024, 39, doi:10.1080/14756366.2024.2301768.
  10. Corral-Rodríguez, N.F.; Moreno-Contreras, V.I.; Sierra-Campos, E.; Valdez-Solana, M.; Cisneros-Martínez, J.; Téllez-Valencia, A.; Avitia-Domínguez, C. Characterization of Natural Products as Inhibitors of Shikimate Dehydrogenase from Methicillin-Resistant Staphylococcus Aureus: Kinetic and Molecular Dynamics Simulations, and Biological Activity Studies. Biomolecules 2025, 15, 1137, doi:10.3390/biom15081137.
  11. Millán-Pacheco, C.; Rios-Soto, L.; Corral-Rodríguez, N.; Sierra-Campos, E.; Valdez-Solana, M.; Téllez-Valencia, A.; Avitia-Domínguez, C. Discovery of Potential Noncovalent Inhibitors of Dehydroquinate Dehydratase from Methicillin-Resistant Staphylococcus Aureus through Computational-Driven Drug Design. Pharmaceuticals 2023, 16, 1148, doi:10.3390/ph16081148.
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  13. Galindo-Hernández, O.; Vique-Sánchez, J.L. AXL Inhibitors Selected by Molecular Docking: Option for Reducing SARS-CoV-2 Entry into Cells. Acta Pharmaceutica 2022, 72, 329–343, doi:10.2478/acph-2022-0024.
  14. Téllez‐Valencia, A.; Hernández‐Ortiz, I.; Guadalupe López Lujano, P.; Luis Vique‐Sánchez, J. Development of Tyrosine Phosphatase 1B Inhibitors Based on Molecular Docking, Kinetics, and Toxicity Studies. ChemistrySelect 2023, 8, doi:10.1002/slct.202300549.
  15. Trasviña-Arenas, C.H.; Ayala Medina, L.A.; Vique-Sanchez, J.L. γ-Secretase Inhibitors Selected by Molecular Docking, to Develop a New Drug Against Alzheimer’s Disease. Rep. Biochem. Mol. Biol. 2023, 12, 340–349, doi:10.61186/rbmb.12.2.340.
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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors made most of the requests

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript can be accepted in its present form.

Reviewer 4 Report

Comments and Suggestions for Authors

accept as it is