New Chemical Scaffold with Antimicrobial Activity Identified in a Screening of Industrial Photoactive Compounds

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I recommend major revisions. The study presents intriguing findings, but several important shortcomings limit the strength of the conclusions. The key issues are outlined below:

1. Mechanism Working Very Insufficient. The link to RNase Y is intriguing, but the evidence is correlative and insufficient to support the authors' hypothesis that rny mutations confer tolerance via altered mRNA stability/degradasome function.
2. Gram-Negative Intrinsic Resistance Insufficiently ExploredTesting a limited set of efflux mutants (ΔemrE, ΔmdrA, ΔacrB and combinations) is inadequate to conclude efflux is not involved. Gram-negatives have redundant/complex efflux systems. No permeabilization experiments (e.g., polymyxin B nonapeptide, EDTA, or outer membrane disruptors) to test uptake barriers. No assessment of porin expression or OM permeability differences. The alternative hypothesis (Gram-positive-specific target) is plausible but unsupported. Larger panels of effusion mutants or chemical/genetic permeabilization experiments should be performed. It would be better if larger panels of effusion mutants or chemical/genetic permeabilization experiments were performed.

3. The tert-butyl, carboxyl, and thiophene moieties appear essential based on the data, yet follow-up analogs show no significant potency improvements. Structural alerts are acknowledged but not addressed experimentally. Moreover, no meaningful ADME data are provided, which is a notable gap for compounds at this stage.
4. MIC and MBC values rely heavily on the MTT assay, where colored compounds may interfere with readout despite the inclusion of agar controls. To confirm results, standard broth microdilution methods combined with OD measurement or alternative viability indicators (e.g., resazurin) should be included. Mutation frequency data (7 × 10⁻⁷) are reported only for MSSA; no data or explanation is provided for why no mutants were recovered from MRSA despite its lower MIC. Whole-genome sequencing of 17 mutants highlights rny mutations, but it is unclear whether other recurrent mutations were present. If relevant, a full summary of mutations should be included in the main text .Many figures and tables lack positive controls for the assays, reducing confidence in the results.
5. NMR spectra of newly synthesized compounds can be provided. In the NMR data, the proton/carbon to which each signal belongs should be indicated. The NMR data of some compounds do not support the structures. All spectra should be carefully re-reviewed, with particular attention to proton integration and signal assignments.

Author Response

Comments and Suggestions for Authors

I recommend major revisions. The study presents intriguing findings, but several important shortcomings limit the strength of the conclusions.

Answer.- We thank the reviewer for the thorough evaluation of our manuscript and their constructive comments. We have revised the manuscript to clarify conclusions, and moderate interpretations where appropriate. Point-by-point responses are provided below.

Comment 1: Mechanism Working Very Insufficient. The link to RNase Y is intriguing, but the evidence is correlative and insufficient to support the authors' hypothesis that rny mutations confer tolerance via altered mRNA stability/degradasome function.

Answer.- We identified mutations in rny in 10/17 mutants and demonstrated that engineering them back into the wild-type background were sufficient to confer reduced susceptibility to Compound 18.

We agree, however, that our data do not directly demonstrate that altered mRNA stability is the precise molecular mechanism for the phenotype. Accordingly, we have revised the manuscript to clearly distinguish between genetic causality and downstream mechanistic interpretation. We now present altered RNA processing as a hypothesis based on RNase Y function and avoid overstating mechanistic conclusions. [Discussion, paragraph starting in line 264]

Comment 2: Gram-Negative Intrinsic Resistance Insufficiently Explored. Testing a limited set of efflux mutants (ΔemrE, ΔmdrA, ΔacrB and combinations) is inadequate to conclude efflux is not involved. Gram-negatives have redundant/complex efflux systems. No permeabilization experiments (e.g., polymyxin B nonapeptide, EDTA, or outer membrane disruptors) to test uptake barriers. No assessment of porin expression or OM permeability differences. The alternative hypothesis (Gram-positive-specific target) is plausible but unsupported. Larger panels of effusion mutants or chemical/genetic permeabilization experiments should be performed. It would be better if larger panels of effusion mutants or chemical/genetic permeabilization experiments were performed.

Answer.- Gram-negative intrinsic resistance is multifactorial and often involves redundant efflux systems and permeability barriers. We have modified the Results [line 128] and Discussion [line 292] sections to avoid overinterpretation and to clarify that while the deletion of these efflux systems did not restore susceptibility, additional efflux pumps cannot be excluded.

Comment 3: The tert-butyl, carboxyl, and thiophene moieties appear essential based on the data, yet follow-up analogs show no significant potency improvements. Structural alerts are acknowledged but not addressed experimentally. Moreover, no meaningful ADME data are provided, which is a notable gap for compounds at this stage.

Answer.- We thank the reviewer for this valuable comment. The additional derivatives synthesized in this study (Compounds 40–47) were intended to explore preliminary structure–activity relationships within the 4H-pyran-4-ylidene scaffold rather than to optimize antimicrobial potency. These compounds allowed us to confirm key structural features required for activity, namely the presence of tert-butyl substituents, a carboxyl group, and a five-membered heterocycle.

Although these analogs did not significantly improve potency, they helped define the basic pharmacophoric elements of the scaffold. The structural alerts associated with these groups were discussed to acknowledge potential medicinal chemistry liabilities; however, experimentally addressing them would require the design of additional analogs, which was beyond the scope of this work focused on scaffold identification.

We have revised the SAR section to clarify that the current analysis represents preliminary pharmacophore identification rather than a full optimization. We have moderated statements suggesting that specific groups are definitely required and clarified that the chemical space remains limited [line 206 and line 214].

Comment 4: MIC and MBC values rely heavily on the MTT assay, where colored compounds may interfere with readout despite the inclusion of agar controls. To confirm results, standard broth microdilution methods combined with OD measurement or alternative viability indicators (e.g., resazurin) should be included. Mutation frequency data (7 × 10⁻⁷) are reported only for MSSA; no data or explanation is provided for why no mutants were recovered from MRSA despite its lower MIC. Whole-genome sequencing of 17 mutants highlights rny mutations, but it is unclear whether other recurrent mutations were present. If relevant, a full summary of mutations should be included in the main text . Many figures and tables lack positive controls for the assays, reducing confidence in the results.

Answer.- We appreciate the reviewer’s concern regarding colorimetric interference in assays relying on optical readouts. We evaluated the absorbance of the compound panel at the MTT detection wavelength (580 nm). Thirteen compounds (03, 05, 06, 13, 14, 22, 23, 24, 37, 38, 40, 41, and 44) exhibited intrinsic absorbance corresponding to 10-60% of S. aureus positive growth control readout. Notably, compounds 05 and 13 were identified as active against C. diphteriaedespite the potential interference with the MTT assay readout, indicating that intrinsic coloration did not generate suspicious activity calls. The full absorbance dataset has now been included in the Supporting Information for transparency [Tables S5 and S6]. Regarding MBC determinations, these were performed by plating aliquots from treated cultures onto agar and assessing colony formation following incubation. The use of resazurin provided an objective readout that is independent on visual interpretation. We attempted mutant isolation in MRSA on multiple independent occasions. While a small number of colonies were recovered, none exhibited stable MIC increases upon retesting. We have now clarified this in the Results section [line 167].

Regarding whole-genome sequencing, Table S4 summarizes all single nucleotide polymorphisms identified in the study. No short insertions or deletions were detected in any of the analyzed mutants. Structural variants or large genomic rearrangements were not assessed, as these cannot be reliably resolved using short-read sequencing.

Finally, we appreciate the reviewer’s suggestion regarding the inclusion of additional positive controls. All antimicrobial susceptibility assays were performed using standardized broth microdilution methodology under established laboratory conditions. Appropriate internal controls were included in each experiment, including growth controls treated with DMSO, sterility controls, and independent biological replicates, which allowed clear validation of assay performance. Notably, the S. aureus strains (ATCC 29213 – MSSA and ATCC 43300 – MRSA) used in this study are routinely handled in our laboratory, and MIC determinations for reference antibiotics oxacillin (0.12 µg/mL and 4 µg/mL, respectively; the latter consistent with the breakpoints defined for resistance) and rifampicin (0.006 µg/mL) consistently match the values reported in CLSI M100.

We have explicitly included this information in the manuscript:

“For S. aureus MSSA and MRSA dose-response assays, performance was validated by MIC determinations for reference antibiotics oxacillin (0.12 µg/mL and 4 µg/mL, respectively; the latter consistent with the breakpoints defined for resistance) and rifampicin (0.006 µg/mL), which matched the values reported in CLSI M100” [line 562].

When the activity of 4H-pyran-4-ylidenes was characterized across multiple strains, at least one of them had been previously characterized for  susceptibility to these compounds using standardized methodology, thereby providing an internal reference to validate assay performance. Given that the objective of these experiments was comparative evaluation of the tested compounds under consistent conditions, we believe that the implemented controls were sufficient to ensure data reliability. However, we acknowledge that the inclusion of reference antibiotics may provide additional benchmarking, and we will consider incorporating such controls in future studies.

Comment 5: NMR spectra of newly synthesized compounds can be provided. In the NMR data, the proton/carbon to which each signal belongs should be indicated. The NMR data of some compounds do not support the structures. All spectra should be carefully re-reviewed, with particular attention to proton integration and signal assignments.

Answer.- We thank the reviewer for this remark. Following the query, spectra have been revised and carefully re-examined to verify proton integrations and signal assignments. For the newly synthesized compounds, copies of the NMR spectra together with those of IR and MS have been incorporated into the Supporting Information file. All available characterization data for the newly synthesized compounds have been included in the Supporting Information. Assignment of the protons has been indicated. In some cases, complete characterization could not be obtained due to the very low solubility of certain compounds, which prevented acquisition of reliable ¹³C NMR spectra.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript describes the identification of 4H-pyran-4-ylidene derivatives as a novel antimicrobial scaffold obtained through screening of a photoactive industrial compound library. The study addresses an important and timely issue given the global rise in antimicrobial resistance and the urgent need for new chemical scaffolds. While the work is potentially valuable, several concerns should be addressed to improve the scientific rigor, clarity, and completeness of the manuscript. Therefore, major revision is recommended.

1. Keywords - should be arranged alphabetically.
2. Abstract - The abstract contains first-person expressions (e.g., "we found," "we demonstrate"). The journal style typically favors an objective scientific tone; therefore, the authors are encouraged to use passive voice consistently throughout the abstract and other sections of the manuscript.
3. References - The reference section should be carefully revised to ensure full compliance with the journal's author guidelines.
4. Introduction -
   • A detailed clarification is expected regarding the rationale for selecting photoactive molecular materials as a discovery strategy rather than employing conventional drug design approaches. The authors should explicitly justify this choice and explain the conceptual or mechanistic advantages of this strategy in the context of antimicrobial drug development.
   • The title of the manuscript emphasizes the screening of “industrial photoactive compounds” as the central strategy of the study; however, the introduction does not adequately define or contextualize this concept. The introduction does not explain what photoactive compounds are, describe their relevant properties, or provide a clear rationale for selecting them for antimicrobial screening. As this represents the main novelty of the work, the authors should include a concise background section that defines photoactive compounds and justifies this strategy, supported by appropriate references, to ensure alignment between the title and the introduction.
5. Results -
   • The manuscript lacks primary spectral characterization data for the synthesized compounds. The authors should provide copies of the ¹H-NMR, ¹³C-NMR, MS, FTIR, and any other relevant spectra in the SI. Additionally, compound purity has not been reported. HPLC chromatograms for all synthesized compounds should be included in the SI file.
   • The manuscript does not clearly specify the origin or preparation of the tested compounds (such as 2, 11, 18, 19, and 27). The authors should explicitly state whether these compounds were synthesized in-house, commercially purchased, or obtained from another source. Moreover, the corresponding spectral characterization data for these compounds are not provided in the SI file. The authors should therefore clarify the source of each compound and include appropriate characterization and purity data prior to biological evaluation.
   • The manuscript states that subculturing MSSA following exposure to Compound 18 resulted in an increased MIC (25 µM to 100 µM). However, no experimental data supporting this claim are cited in section 2.2. The corresponding MIC determination data, including the number of replicates and variability, should be provided.
   • The statement, "selectivity indices ranging between 3.4 and 70" should be reported with precise calculated values (e.g., 4.16 and 74.26). In addition, the authors should briefly discuss the relevance and interpretation of the selectivity index in the context of antimicrobial drug development.
   • In the SAR section, the rationale for designing and synthesizing compounds 40-47 is not sufficiently explained. The authors should clearly state the structural hypothesis or pharmacological reasoning that guided the design of these derivatives
6. Discussion -
   • The SAR analysis, particularly Figure 5, requires more detailed discussion. The authors should explicitly address:

• The proposed pharmacophore features
• The influence of substituent position and electronic properties
• Any steric effects observed
• Correlations between structural modifications and antibacterial potency
  • The concluding section should be better aligned with the title and overall objective of the study. The authors should explicitly state whether the original hypothesis—namely, that screening industrial photoactive compounds could yield novel antimicrobial scaffolds—was supported by the findings.

7. Supplementary file - Title of this file should match with that of the manuscript.
8. Figure - Figures 2 and 4 are blur.
9. Abbreviations - All abbreviations should be defined at first mention and used consistently throughout the manuscript. The authors should revise this throughout.

Author Response

Comments and Suggestions for Authors

The manuscript describes the identification of 4H-pyran-4-ylidene derivatives as a novel antimicrobial scaffold obtained through screening of a photoactive industrial compound library. The study addresses an important and timely issue given the global rise in antimicrobial resistance and the urgent need for new chemical scaffolds. While the work is potentially valuable, several concerns should be addressed to improve the scientific rigor, clarity, and completeness of the manuscript. Therefore, major revision is recommended.

We sincerely thank the reviewer for the thoughtful evaluation of our manuscript. We are grateful for the constructive comments and suggestions provided, which have helped us to improve the rigor, clarity and completeness of the manuscript. We have addressed all concerns raised and revised the manuscript accordingly.

Comment 1: Keywords - should be arranged alphabetically.

Answer.- Keywords have been reordered alphabetically in accordance with the guidelines.

Comment 2: Abstract - The abstract contains first-person expressions (e.g., "we found," "we demonstrate"). The journal style typically favors an objective scientific tone; therefore, the authors are encouraged to use passive voice consistently throughout the abstract and other sections of the manuscript.

Answer.- The abstract has been revised to remove first-person expressions and now uses a passive-voice style consistent with journal conventions.

Comment 3: References - The reference section should be carefully revised to ensure full compliance with the journal's author guidelines.

Answer.- The reference list has been reviewed to ensure full compliance with the journal’s formatting guidelines.

Comment 4: Introduction

• A detailed clarification is expected regarding the rationale for selecting photoactive molecular materials as a discovery strategy rather than employing conventional drug design approaches. The authors should explicitly justify this choice and explain the conceptual or mechanistic advantages of this strategy in the context of antimicrobial drug development.

Answer.- We thank the reviewer for this helpful suggestion. The rationale for screening photoactive molecular materials was to explore an underrepresented chemical space that is not typically covered by conventional drug discovery libraries. Photoactive materials, originally developed for applications such as solar energy conversion, frequently contain extended conjugated systems and unique substitution patterns that differ from those found in traditional medicinal chemistry collections. We therefore reasoned that this chemical diversity could provide access to novel antimicrobial scaffolds and decided to explore them. Our observations are described in this manuscript.

• The title of the manuscript emphasizes the screening of “industrial photoactive compounds” as the central strategy of the study; however, the introduction does not adequately define or contextualize this concept. The introduction does not explain what photoactive compounds are, describe their relevant properties, or provide a clear rationale for selecting them for antimicrobial screening. As this represents the main novelty of the work, the authors should include a concise background section that defines photoactive compounds and justifies this strategy, supported by appropriate references, to ensure alignment between the title and the introduction.

Answer.- We thank the reviewer for this helpful suggestion. We have modified in this sense the last part of the introduction to better define the nature of the photoactive compounds used in this study and to provide additional background on their properties and applications [line 93].

Comment 5: Results

• The manuscript lacks primary spectral characterization data for the synthesized compounds. The authors should provide copies of the ¹H-NMR, ¹³C-NMR, MS, FTIR, and any other relevant spectra in the SI. Additionally, compound purity has not been reported. HPLC chromatograms for all synthesized compounds should be included in the SI file.

Answer.- Following this query and that of referee 1, copies of the NMR spectra together with those of IR and MS have been incorporated into the Supporting Information file for the newly synthesized compounds. Compounds were purified by medium pressure chromatography and their purity (>95%) was verified by NMR and mass spectrometry, as indicated in the Supporting Information.

• The manuscript does not clearly specify the origin or preparation of the tested compounds (such as 2, 11, 18, 19, and 27). The authors should explicitly state whether these compounds were synthesized in-house, commercially purchased, or obtained from another source. Moreover, the corresponding spectral characterization data for these compounds are not provided in the SI file. The authors should therefore clarify the source of each compound and include appropriate characterization and purity data prior to biological evaluation.

Answer.- We thank the reviewer for raising this important point. The origin of the compounds tested in this study is indicated in Table S1 of the Supporting Information, where all compounds are identified either as newly synthesized in this work (“synthesized in-house”) or accompanied by their corresponding bibliographic reference if they were previously reported.

In particular, compounds shown in Figure 1 that have been previously described are:

• Compounds 02, 11, 18 and 19 reported in: Andrés-Castán, J. M.; Andreu, R.; Villacampa, B.; Orduna, J.; Franco, S. Solar Energy 2019, 193, 74–84.
• Compound 27 reported in: Pérez-Tejada, R.; Martínez de Baroja, N.; Franco, S.; Pellejà, L.; Orduna, J.; Andreu, R.; Garín, J. Dyes and Pigments 2015, 123, 293–303.

For the sake of clarity, we have now explicitly indicated this information in the manuscript. Specifically:

• Page 2, line 85: the following sentence has been added: “See Table S1 for the chemical structure of compounds 01–39, together with the indication of whether the compound is newly synthesized in this work or previously reported.”
• Figure 1 caption: references corresponding to compounds 02, 11, 18, 19, and 27 have been included.
• Page 7, line 182: a similar clarification has been added in the section describing compounds 40–47: “See Table S1 for the chemical structure of compounds 40–47, together with the indication of whether the compound is newly synthesized in this work or previously reported.”

Regarding the spectral characterization data, these have been incorporated into the Supporting Information as explained in our response to the previous comment.

• The manuscript states that subculturing MSSA following exposure to Compound 18 resulted in an increased MIC (25 µM to 100 µM). However, no experimental data supporting this claim are cited in section 2.2. The corresponding MIC determination data, including the number of replicates and variability, should be provided.

Answer.- Following exposure to 25 µM Compound 18, MSSA cells were diluted 1:1000 into fresh compound-free medium and allowed to grow overnight. This was done twice. After this passage, MIC values were determined by broth microdilution as described in the Methods section. Each biological replicate was tested in technical duplicates alongside the parental strain. Under these conditions, the post-exposure population consistently exhibited an MIC increase from 25 µM to 100 µM for Compound 18. The methodological details have been clarified in the Methods section of the revised manuscript.

• The statement, "selectivity indices ranging between 3.4 and 70" should be reported with precise calculated values (e.g., 4.16 and 74.26). In addition, the authors should briefly discuss the relevance and interpretation of the selectivity index in the context of antimicrobial drug development.

Answer.- The selectivity indices have been revised to report the precise calculated values. An additional sentence has been included in the discussion [line 430] to clarify interpretation and relevance in the context of early-stage antimicrobial drug development.

• In the SAR section, the rationale for designing and synthesizing compounds 40-47 is not sufficiently explained. The authors should clearly state the structural hypothesis or pharmacological reasoning that guided the design of these derivatives.

Answer.- The rationale behind the design of compounds 40–47 has now been clarified in the SAR section (line 275). These derivatives were synthesized to probe the structural features suggested by the preliminary screening results, particularly the role of the tert-butyl substituents, the presence of the carboxyl group, and the influence of different heterocyclic or bulky substituents on antimicrobial activity. For the sake of clarity, this explanation has now been incorporated into the manuscript.

Page 5, Section 2.5 (Structure–activity relationships): the following sentence has been added before introducing compounds 40–47: "Based on these preliminary observations, a small set of additional derivatives (Compounds 40–47) was designed and synthesized to probe the structural requirements of the scaffold, particularly the role of the tert-butyl substituents, the presence of the carboxyl group, and the influence of different heterocyclic or bulky substituents on antimicrobial activity".

Comment 6: Discussion

• The SAR analysis, particularly Figure 5, requires more detailed discussion. The authors should explicitly address:
  • The proposed pharmacophore features
  • The influence of substituent position and electronic properties
  • Any steric effects observed
  • Correlations between structural modifications and antibacterial potency
  • The concluding section should be better aligned with the title and overall objective of the study. The authors should explicitly state whether the original hypothesis—namely, that screening industrial photoactive compounds could yield novel antimicrobial scaffolds—was supported by the findings.

Answer.- We thank the reviewer for this valuable suggestion. The SAR analysis has been substantially expanded in the revised manuscript. Specifically, the Results section now includes a more detailed discussion of the proposed pharmacophore features and the influence of substituent position. Regarding correlations between structural modifications and antibacterial potency, we have added a brief discussion (line 437); however, we note that the relatively small number of synthesized derivatives limits the robustness of quantitative SAR conclusions. This limitation has now been explicitly acknowledged in the manuscript to avoid overinterpretation of the available data.

In addition, the concluding section of the Discussion has been revised to better align with the overall objective of the study (line 496). We now explicitly state that our findings support the original hypothesis that screening industrial photoactive compounds can yield novel antimicrobial scaffolds, while acknowledging that further optimization and investigation are required to assess therapeutic potential.

Comment 7: Supplementary file - Title of this file should match with that of the manuscript.

Answer.- We have modified the title of the file to match that of the manuscript.

Comment 8: Figure - Figures 2 and 4 are blur.

Answer.- Figures 2 and 4 have been replaced with higher-resolution versions. Additionally, we have submitted the original figures in .tiff format separately.

Comment 9: Abbreviations - All abbreviations should be defined at first mention and used consistently throughout the manuscript. The authors should revise this throughout

Answer.- All abbreviations have been defined at first mention and used consistently throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Comments are attached as PDF

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

Recommendation: Accept with minor revision

The authors report a well-structured screening study of industrial photoactive compounds that uncovers 4H‑pyran‑4‑ylidenes as a new bactericidal scaffold active against Gram‑positive bacteria, including MRSA, with low mammalian cytotoxicity at effective concentrations. They also identify resistance‑associated mutations in the rny (RNase Y) locus linked to Compound 18. The manuscript is clearly written, the experimental hypothesis is well motivated, and the data are presented in a way that is accessible to the journal’s readership. The following are minor suggestions to improve clarity and increase the appeal to readers.

We sincerely thank the reviewer for the positive evaluation of our manuscript and for recommending acceptance with minor revision. We are grateful for the constructive suggestions provided to improve clarity and increase the appeal to readers. We have carefully considered all comments and revised the manuscript accordingly.

Minor comment

Comment 1: The image quality of Figure 2 and Figure 4 should be improved; at the current resolution, data points and values (axes, labels, legends) are not very clear. Please provide higher‑resolution versions and ensure all elements are legible at the final print size.

Answer.- Figures 2 and 4 have been replaced with higher-resolution versions. Additionally, we have submitted the original figures in .tiff format separately.

Comment 2: Please add 1–2 sentences positioning the 4H‑pyran‑4‑ylidenes relative to common organic dye families (e.g. xanthene, phenothiazinium, triarylmethane, cyanine). If feasible with existing data, briefly compare basic descriptors (e.g. cLogP, TPSA, predicted charge at pH 7.4, HBD/HBA, aromatic ring count) to illustrate how “dye-like” vs “drug-like” this scaffold is.

Answer.- We thank the reviewer for this helpful suggestion. To better position the 4H-pyran-4-ylidene scaffold within the broader context of organic dye families, a brief comparison with common dye frameworks has been added to the Introduction

Page 3, Introduction: the following sentence has been added:

" Organic dyes used in photochemical and optoelectronic applications encompass several well-known structural families, including xanthenes, phenothiazinums, triarylmethanes, and cyanines. In comparison with these classical dye scaffolds, 4H-pyran-4-ylidene derivatives also belong to the broader class of π-conjugated organic dyes but display a distinct structural framework and substitution pattern. The 4H-pyranylidene unit is a proaromatic electron donor that becomes aromatic upon charge transfer [16]. This enhanced aromatic stabilization strengthens its electron-donating capability, and its chemical structure can be readily modified through organic synthesis. Notably, representative compounds within this family comply with Lipinski’s Rule of Five, suggesting that this scaffold occupies a chemical region closer to drugs than purely dye-like compounds.”

Comment 3: In the Discussion, please clarify, briefly, how Gram‑negative activity was assessed (including the efflux‑mutant experiments) and note that the present work is primarily focused on Gram‑positive pathogens.

Answer.- We thank the reviewer for this suggestion. The Discussion has been revised to clarify that Gram-negative activity was assessed using standard broth microdilution assays against wild-type E. coli and isogenic strains lacking major efflux systems. As no activity was observed against them, subsequent investigations were focused on just Gram-positive strains. This clarification has been incorporated into the revised manuscript.

Comment 4: In the Discussion, briefly compare practical pros/cons versus existing antibacterials (e.g. cytotoxicity window, physicochemical liabilities such as the carboxylate, tert‑butyl, and thiophene motifs) and note that these issues could, in principle, be addressed by standard medicinal chemistry optimization.

Answer.- We thank the reviewer for this important suggestion. The Discussion has been revised to better align with the study’s overall objective and title (lines 454). We now explicitly state that our findings support the original hypothesis that screening industrial photoactive compounds can yield structurally novel antimicrobial scaffolds.

In addition, we have expanded the SAR section to position the 4H-pyran-4-ylidene scaffold relative to currently used antibacterial agents. Specifically, we now highlight its structural divergence from established anti-staphylococcal drug classes (e.g., β-lactams, glycopeptides, lipopeptides, and oxazolidinones), emphasizing its chemical novelty as a potential advantage in the context of antimicrobial resistance. At the same time, we discuss key medicinal chemistry liabilities of the scaffold (e.g., carboxylate functionality, tert-butyl oxidation susceptibility, and thiophene metabolic concerns) to provide a balanced assessment of its strengths and limitations compared to clinically optimized agents.

These additions aim to present a more comprehensive and realistic evaluation of the scaffold’s potential while avoiding overinterpretation of the current dataset.

Conclusion remarks/suggestions

Overall, this is a well‑executed and clearly written study that identifies a genuinely novel Gram‑positive–active scaffold from an unconventional chemical space. The microbiology, SAR, and genetic data around rny/RNase Y are solid and appropriately interpreted. My comments are minor and focus on positioning the work, clarifying spectrum and mechanism language, and adding small pieces of chemical/contextual information. They do not require additional experimental work. I recommend acceptance after minor revision.

Reviewer 4 Report

Comments and Suggestions for Authors

1. In Line 121: MIC of compound 18 against MSSA mentioned as 25 μM, but in the table 1 it is reported as 12.5 μM. Please clarify.
2. The authors may briefly describe how the frequency of mutation of aureus MSSA to Compound 18 was calculated.
3. The authors must specify the final percentage of DMSO used in each experiment and consider including DMSO controls.
4. Did authors use any internal control antibiotics while determining the MIC and MBC for assay validation?
5. The authors may provide insights about the potential target of compound 18 while discussing the mechanistic perspective.

Author Response

Comments and Suggestions for Authors

Comment 1: In Line 121: MIC of compound 18 against MSSA mentioned as 25 μM, but in the table 1 it is reported as 12.5 μM. Please clarify.

Answer.- We thank the reviewer for noting this inconsistency. The most frequently observed MIC value for Compound 18 against MSSA was 25 µM. The value of 12.5 µM reported in Table 1 was an inadvertent error introduced during manuscript preparation. This has now been corrected in the revised manuscript.

Comment 2: The authors may briefly describe how the frequency of mutation of aureus MSSA to Compound 18 was calculated.

Answer.- The frequency of mutation was calculated as the ratio of resistant colonies to the total number of viable CFU plated. This sentence has been included in the Materials and Methods section

Comment 3: The authors must specify the final percentage of DMSO used in each experiment and consider including DMSO controls.

Answer.- In all experiments, DMSO was added to the untreated control conditions at the highest final concentration present within each assay to control for potential solvent effects. Specifically, the final DMSO concentration was 2.5% for single-shot and cytotoxicity assays, and up to 1% for time-kill kinetics assays depending on the highest concentration used in the assay. The Methods section has been updated to clearly state the solvent concentration used in each experimental setting.

Comment 4: Did authors use any internal control antibiotics while determining the MIC and MBC for assay validation?

Answer.- We did not include internal control antibiotics when determining the MIC and MBC of 4H-pyran-4-ylidenes.

All antimicrobial susceptibility assays were performed using standardized broth microdilution methodology under established laboratory conditions.  Appropriate internal controls were included in each experiment, including growth controls treated with DMSO, sterility controls, and independent biological replicates, which allowed clear validation of assay performance. Notably, the S. aureus strains (ATCC 29213 – MSSA and ATCC 43300 – MRSA) used in this study are routinely handled in our laboratory, and MIC determinations for reference antibiotics oxacillin (0.12 µg/mL and 4 µg/mL, respectively; the latter consistent with the breakpoints defined for resistance) and rifampicin (0.006 µg/mL) consistently match the values reported in CLSI M100.

We have explicitly included this information in the manuscript:

“For S. aureus MSSA and MRSA dose-response assays, performance was validated by MIC determinations for reference antibiotics oxacillin (0.12 µg/mL and 4 µg/mL, respectively; the latter consistent with the breakpoints defined for resistance) and rifampicin (0.006 µg/mL), which matched the values reported in CLSI M100” [line 562].

When the activity of 4H-pyran-4-ylidenes was characterized across multiple strains, at least one of them had been previously characterized for susceptibility to these compounds using standardized methodology, thereby providing an internal reference to validate assay performance. Given that the objective of these experiments was comparative evaluation of the tested compounds under consistent conditions, we believe that the implemented controls were sufficient to ensure data reliability. However, we acknowledge that the inclusion of reference antibiotics may provide additional benchmarking, and we will consider incorporating such controls in future studies.

Comment 5: The authors may provide insights about the potential target of compound 18 while discussing the mechanistic perspective.

Answer.- Whole genome sequencing of resistant MSSA mutants revealed point mutations in the rny locus, encoding RNase Y, a key regulator of RNA homeostasis in S. aureus. These findings suggest that RNA metabolism could play a role in the mechanism of resistance to Compound 18. However, it remains to be determined whether RNase Y represents the direct molecular target of the compound or whether the mutations in RNase Y affect the expression levels of the actual target. The Discussion has been expanded to clarify this point and to outline future directions.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

acceptable

Author Response

Acceptable

We thank the reviewer for their positive assessment of the manuscript. 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been extensively modified now based upon the comment. However, minor revision is recommended.

1. Abstract – The abstract contains first-person expressions (e.g., "we found," "we demonstrate"). The journal style typically favors an objective scientific tone; therefore, the authors are encouraged to use passive voice consistently throughout the abstract and other sections of the manuscript.

“This needs to be checked and modified throughout the manuscript”.

2. References – This entire section needs to be checked thoroughly, for e.g., DOI are added, etc.
3. Results – NMR can be a representative of purity determination, but HPLC provide exact information of the purity and therefore HPLC chromatograms for all synthesized compounds should be included. This is important.
4. Supplementary file – Font size should be consistent throughout.

Author Response

The manuscript has been extensively modified now based upon the comment. However, minor revision is recommended.

Abstract – The abstract contains first-person expressions (e.g., "we found," "we demonstrate"). The journal style typically favors an objective scientific tone; therefore, the authors are encouraged to use passive voice consistently throughout the abstract and other sections of the manuscript.

“This needs to be checked and modified throughout the manuscript”.

We thank the reviewer for this observation. We have carefully revised the rest of the manuscript, replacing first-person expressions with passive voice constructions, in line with the journal’s preferred scientific tone. The manuscript has been updated accordingly.

References – This entire section needs to be checked thoroughly, for e.g., DOI are added, etc.

We have thoroughly reviewed the entire reference list and incorporated the missing DOIs and ISBNs.

Results – NMR can be a representative of purity determination, but HPLC provide exact information of the purity and therefore HPLC chromatograms for all synthesized compounds should be included. This is important.

We thank the reviewer for this comment and agree that HPLC analysis can provide additional information regarding compound purity. In the present work, however, the compounds used in the biological assays were purified by chromatographic methods and subsequently characterized by NMR spectroscopy and high-resolution mass spectrometry (HRMS). These orthogonal analytical techniques are widely accepted for establishing the identity and purity of small organic molecules.

The compound set investigated in this study includes both newly synthesized derivatives and compounds originating from a previously established molecular library. All compounds were purified prior to biological evaluation, and their identity and purity were assessed based on NMR and HRMS data. On this basis, the purity of the compounds was estimated to be higher than 95%, which is generally considered sufficient for biological evaluation.

Importantly, obtaining HPLC chromatograms at this stage from archived samples would not necessarily reflect the exact material batches originally used in the biological experiments. For this reason, we believe that the analytical characterization already provided represents the most reliable documentation of compound identity and purity for the purposes of this study.

To clarify this point for readers, we have added a sentence in the Supporting Information explicitly stating the analytical criteria used to assess compound purity.

Supplementary file – Font size should be consistent throughout.

We thank the reviewer for this remark. We have carefully revised the supplementary file ensuring formatting uniformity across the document.