In Vitro Activity of Novel Topoisomerase Inhibitors against Francisella tularensis and Burkholderia pseudomallei

Round 1

Reviewer 1 Report

Dear authors, after carefully revising the manuscript entitled "In vitro activity of topoisomerase inhibitors against Francisella tularensis and Burkholderia pseudomallei" describing many in vitro analyses (broth microdilution, time-kill and cell infection assays) needs little improvements before its full acceptance.

As I'm a researcher on antibacterial development, I deeply analyzed this manuscript and aim to help the authors improve their manuscript and I'm providing some additional, specific comments such as:

- since the authors have in vitro assays and their chemical structures, I think they could analyze them by Structure-Activity Relationship (SAR) approach. These analyses could be made by https://pharmit.csb.pitt.edu server.

- based on SAR and 2D structures the authors could also study their pharmacophoric features by https://www.bio.tools/pharmagist#! server.

- since 2D and topoisomerase structures are available (PDB code: 4KQV, 3SLG), the authors could employ some docking methods to evaluate their binding energies and poses that could help to understand better their in vitro results.

- after docking studies, they could employ Molecular Dynamics (MD) to evaluate their bacterial topoisomerase/ cyclohexyl-oxazolidinone interactions.

Hopefully, these comments will help the authors to revise my comments, and then I think it would have a much better chance of surviving the peer review process.

Author Response

Dear authors, after carefully revising the manuscript entitled "In vitro activity of topoisomerase inhibitors against Francisella tularensis and Burkholderia pseudomallei" describing many in vitro analyses (broth microdilution, time-kill and cell infection assays) needs little improvements before its full acceptance.

As I'm a researcher on antibacterial development, I deeply analyzed this manuscript and aim to help the authors improve their manuscript and I'm providing some additional, specific comments such as:

1. since the authors have in vitro assays and their chemical structures, I think they could analyze them by Structure-Activity Relationship (SAR) approach. These analyses could be made by https://pharmit.csb.pitt.edu server.
2. based on SAR and 2D structures the authors could also study their pharmacophoric features by https://www.bio.tools/pharmagist#! server.
3. since 2D and topoisomerase structures are available (PDB code: 4KQV, 3SLG), the authors could employ some docking methods to evaluate their binding energies and poses that could help to understand better their in vitro results.
4. after docking studies, they could employ Molecular Dynamics (MD) to evaluate their bacterial topoisomerase/ cyclohexyl-oxazolidinone interactions.

 

Hopefully, these comments will help the authors to revise my comments, and then I think it would have a much better chance of surviving the peer review process.

 

Author Response: Thank you for your suggestions. Whilst we appreciate that there may be merit in performing additional structural analysis of these compounds, this was not the focus of the manuscript. This manuscript is intended as a concise report to demonstrate the exploitation, and therapeutic potential, of these novel compounds against two biothreat pathogens. Structure-activity relationship and molecular modelling was reported in the previous referenced publication (Lyons et al, reference 18). The Lyons et al paper also provided the enzyme/mechanism of action data for these compounds. Consequently, we do not consider further structural analysis appropriate at this time, at least in the context of this manuscript.

Reviewer 2 Report

The manuscript entitled “In vitro activity of topoisomerase inhibitors against Francisella tularensis and Burkholderia pseudomallei” revealed the following research studies:

·       Investigation of the in vitro activity of a series of five cyclohexyl-oxazolidinone bacterial topoisomerase inhibitors (INFEX993, INFEX2017, INFEX2018, INFEX2019 and INFEX2020) against the Gram-negative bacterial strains Francisella tularensis and Burkholderia pseudomallei applying MIC Assay, Time Kill Assays, Intracellular assays and Statistical Analysis.

·       Broth microdilution, time-kill and cell infection assays were performed to determine activity against these biothreat pathogens.

·       The two cyclohexyl-oxazolidinone candidates, INFEX2017 and INFEX2019 demonstrated in vitro activity in multiple assays which, in some instances, was equivalent to ciprofloxacin and doxycycline as the authors described.

 

Overall, the work is well done and organized and can be accepted for publication in Antibiotics. However, there are some remarks are recommended to be taken in consideration before publication as follows:

1.     The original source of the tested cyclohexyl-oxazolidinone compounds under study was not mentioned, and clearly mentioned if they were synthesized by “Infex Therapeutics” or and if these compounds were synthesized before and by which research group were reported previoulsy?

2.     The authors did not mention the mode of action of these compounds clearly or studied it, and they did not refer if this class of compounds has alternative bioactivities reported in literatures before!

 

Author Response

Overall, the work is well done and organized and can be accepted for publication in Antibiotics. However, there are some remarks are recommended to be taken in consideration before publication as follows:

 

Dear reviewer, thank you for your comments. These have been addressed as described below. Please also see track changes to edits to the original manuscript.

 

1. The original source of the tested cyclohexyl-oxazolidinone compounds under study was not mentioned, and clearly mentioned if they were synthesized by “Infex Therapeutics” or and if these compounds were synthesized before and by which research group were reported previously?

Author Response: The NTBI compounds evaluated were originally synthesised and supplied by Redx Anti-Infectives Ltd prior to subsequent transfer of intellectual properties and technical knowledge to Infex Therapeutics Ltd. This has been clarified in the Materials and Methods section. The development of these compounds is described in reference 13 which is co-authored by individuals from both Redx Anti-Infectives and Infex Therapeutics.

2. The authors did not mention the mode of action of these compounds clearly or studied it, and they did not refer if this class of compounds has alternative bioactivities reported in literatures before!

Author Response: The mode of action of these NTBIs is via targeting of the DNA gyrase and topoisomerase IV, through different binding sites to fluoroquinolones, thereby reducing potential for cross-resistance to FQs. This is stated, and referenced, in the Introduction and the Discussion. Structure-activity relationship and molecular modelling of these compounds was reported in the previous referenced publication (Lyons et al, reference 18). The Lyons et al paper also provided the enzyme/mechanism of action data for these compounds. We did not formally confirm that this was the mechanism of action for the F. tularensis or B. pseudomallei strains evaluated in this paper which has now been acknowledged in the Discussion section. We agree that formal confirmation of this, or alternative mechanisms of action, would be warranted in any future developmental work. However, the purpose of this manuscript was intended as a concise report to demonstrate the exploitation, and therapeutic potential, of these novel compounds against two biothreat pathogens.

Reviewer 3 Report

 1.       It is concerning that no evidence was produced for the topoisomerase activity of the inhibitors tested against Francisella tularensis and Burkholderia pseudomallei. Without this evidence, it is difficult to determine the mechanism of action of these inhibitors and whether they are truly effective against these pathogens.

2.       It is essential to include the minimum inhibitory concentrations (MICs) for B. pseudomallei and F. tularensis (Table 1 ) in the results section of the research paper.

3.       The description of the intracellular activity comparison between INFEX2017, INFEX2019, and ciprofloxacin in J774.2 murine macrophages appears to be insufficiently detailed, making it difficult to assess the validity of the results.

4.       Future studies should include appropriate topoisomerase inhibitory assays to provide evidence for the mechanism of action of these NBTIs. This information would help to establish the potential clinical utility of these inhibitors and inform the design of more effective inhibitors.

 

5.       A lack of proper alignment between the introduction, methods, objective, results, and discussion sections can lead to confusion and make it difficult for readers to comprehend the research.

The manuscript's English language proficiency appears to be good

Author Response

Reviewer 3

Dear reviewer, thank you for your comments. These have been addressed as described below. Please also see track changes to edits to the original manuscript.

 

1. It is concerning that no evidence was produced for the topoisomerase activity of the inhibitors tested against Francisella tularensis and Burkholderia pseudomallei. Without this evidence, it is difficult to determine the mechanism of action of these inhibitors and whether they are truly effective against these pathogens.

 

Author Response: The mode of action of these NTBIs is through targeting of DNA gyrase and topoisomerase IV and has been characterised for other Gram negative bacteria. Whilst we consider it likely that they exhibit a comparable mode of action in F. tularensis and B. pseudomallei, we acknowledge that we did not formally demonstrate it in this study. This has been acknowledged in the Discussion section. It should be noted that structure-activity relationship and molecular modelling of these compounds was reported in the previous referenced publication (Lyons et al, reference 18). The Lyons et al paper also provided the enzyme/mechanism of action data for these compounds. We agree that formal confirmation of this, or alternative mechanisms of action, would be warranted in any future developmental work. However, the purpose of this manuscript was intended as a concise report to demonstrate the exploitation, and therapeutic potential, of these novel compounds against two biothreat pathogens. Despite the lack of confirmation of the mode of action for these pathogen, this does not invalidate the demonstration of antibacterial effect reported for these compounds against F. tularensis and B. pseudomallei strains. The methodologies used to assess antibacterial effect in this study are well characterised and commonly used in vitro methods. We are confident in the validity of the data reported.

2. It is essential to include the minimum inhibitory concentrations (MICs) for B. pseudomallei and F. tularensis (Table 1 ) in the results section of the research paper.

Author Response: The reporting of the MIC data in Table 1 has been moved to the Results section.

 

3. The description of the intracellular activity comparison between INFEX2017, INFEX2019, and ciprofloxacin in J774.2 murine macrophages appears to be insufficiently detailed, making it difficult to assess the validity of the results.

Author Response: The intracellular activity comparison compared intracellular bacterial burden of the test compound with the untreated (media only) control wells using a Mixed Effect Analysis with Dunnett’s multiple comparison test. The description of this analysis has been clarified in the figure legend associated with Figure 2.

4. Future studies should include appropriate topoisomerase inhibitory assays to provide evidence for the mechanism of action of these NBTIs. This information would help to establish the potential clinical utility of these inhibitors and inform the design of more effective inhibitors.

Author Response: We would agree that confirmation of the mechanism of action in the context of these biothreat pathogens would be warranted in any future developmental work. This has been acknowledged in the Discussion section.

5. A lack of proper alignment between the introduction, methods, objective, results, and discussion sections can lead to confusion and make it difficult for readers to comprehend the research.

Author Response: I am unclear on how the sections of the manuscripts are not aligned. The structure and narrative of the paper follows the commonly accepted Introduction, Material and Methods, Results and Discussion formatting style. Our expectation is that the information provided in each section provides a logical narrative to inform the reader on the background, results and significance of the work, presented in the context of relevant literature. 

Reviewer 4 Report

Manuscript Review:

In vitro activity of topoisomerase inhibitors against Francisella tularensis and Burkholderia pseudomallei

Adam O Whelan, Ian Cooper, Nicola Ooi , Victoria Lee , David Orr , Kevin Blades , James Kirkham , Amanda Lyons , Kay B Barnes , Mark I. Richards , Anne-Marie Salisbury , Mark Craighead , Sarah V. Harding

Manuscript Summary:

In the manuscript by Whelan et al., the authors describe the antibacterial activity of two novel type II topoisomerase inhibitors for the biothreat bacteria, Burkholderia pseudomallei and Francisella tularensis.  The authors focus on two inhibitors, INFEX2017 and INFEX2019, that have demonstrated better drug metabolism and pharmacokinetic properties in vitro.  Both inhibitors produced time-kill and intracellular inhibition properties similar to that of ciprofloxacin.  This manuscript will be of interest to those in antibacterial drug discovery, especially those interested in topoisomerase inhibitors and intracellular bacteria.

Comments for Authors:

Italicize bacterial names: LL 55, 59, 61, 62, 160, 162, 163 (check document for others)

L4: There is a comma missing in the author list after “David Orr”

L60: Define DMPK

LL69-70: Be consistent with the use of ml vs mL.  Check document for other inconsistencies.

Figure 1:  Should follow line 73 (place after the Reagents heading).

Table 1: 

There is no in-text reference for Table 1, and it is misplaced.  Suggest putting it after paragraph 1 of the Results section (LL141-151).

The in-text reference should be on L142 after the first sentence: “All compounds demonstrated…in MIC assays (Table 1).

LL94-95:  There is a font change for LL94-95.   Also, please clarify the statement “All assays were performed in triplicate.”   Are you referencing the later statement “Each experiment was performed in triplicate…. (L136), or are you stating that each experiment used triplicate replicates?  For example, in the MIC assays, were there 3 wells for each dilution of inhibitor?  It is confusing as to whether all experiments used triplicate samples or whether these (and the time-kill assays) used single wells (or tubes).  It is clearly stated for the intracellular assays that triplicate wells were used (L117).

L152 and L170: “x” should be capitalized as for the other uses of 4X.

LL159-168:  Move Fig 2 legend under Fig 2.

L160 and L165:  There is a hyphen in “as-sessed” and “ex-periments”

LL177-188:  Move Fig 3 legend under Fig 3.

L179 and L181:  Instead of J7742s (too colloquial), suggest J7742 cells (not that much longer!).

L201:  Be consistent with italicization of “in vitro.”  Elsewhere, it is italicized (L147 and L208).  Check document for others. 

L216: Use a lowercase “s” with MICs.

LL215-217:  Please clarify that the activity is against B. pseudomallei.  For example: “A fatty acid synthesis inhibitor… had MICs for B. pseudomallei of 8 μg/mL (strain Bp400) and 1 μg/mL (strain 1026b), respectively (20,21).

L217:  There is a “closed parenthesis” and a comma missing (see above comment).

L217:  There is a font change for “respectively (20,21).”

L219: “…again using B. pseudomallei strain 1026b.”

References: 

There are inconsistencies in the author listing: for example, reference 1 is last name followed by initials, while reference 2 has the initial and then the last name.

There are also inconsistencies throughout with author initials.  See reference 1 which uses periods and reference 11 which does not.

There are inconsistencies with reference titles.  For example, reference 4 uses capitalization for only the first word.  Reference 5 uses title case for all words.  Please be consistent.

References 11-22 have a change in indentation.

References 14 and 16 inconsistently reference CLSI documents.  Please provide the full reference for number 16.

Minor editing required.

Author Response

In the manuscript by Whelan et al., the authors describe the antibacterial activity of two novel type II topoisomerase inhibitors for the biothreat bacteria, Burkholderia pseudomallei and Francisella tularensis.  The authors focus on two inhibitors, INFEX2017 and INFEX2019, that have demonstrated better drug metabolism and pharmacokinetic properties in vitro.  Both inhibitors produced time-kill and intracellular inhibition properties similar to that of ciprofloxacin.  This manuscript will be of interest to those in antibacterial drug discovery, especially those interested in topoisomerase inhibitors and intracellular bacteria.

Comments for Authors:

1. Italicize bacterial names: LL 55, 59, 61, 62, 160, 162, 163 (check document for others)

Author response: Editing corrections made.

2. L4: There is a comma missing in the author list after “David Orr”

Author response: Formatting of references corrected.

3. L60: Define DMPK

Author response: Definition provided on first use.

4. LL69-70: Be consistent with the use of ml vs mL.  Check document for other inconsistencies.

Author response: Corrections made to ensure mL is used consistently throughout document.

5. Figure 1:  Should follow line 73 (place after the Reagentsheading).

Author response: Figure 1 is now place directly after Reagents section 2.1. Final positioning will be dependant to copy editors.

6. Table 1: 

Author response: Table 1 has been moved to the Results section in compliance with comments from reviewer 3.

7. There is no in-text reference for Table 1, and it is misplaced.  Suggest putting it after paragraph 1 of the Results section (LL141-151).

Author response: Table 1 moved to results section and is now referenced in the first paragraph of this section.

8. The in-text reference should be on L142 after the first sentence: “All compounds demonstrated…in MIC assays (Table 1).

Author response: Correction made, see above.

9. LL94-95:  There is a font change for LL94-95.   Also, please clarify the statement “All assays were performed in triplicate.”   Are you referencing the later statement “Each experiment was performed in triplicate…. (L136), or are you stating that each experiment used triplicate replicates?  For example, in the MIC assays, were there 3 wells for each dilution of inhibitor?  It is confusing as to whether all experiments used triplicate samples or whether these (and the time-kill assays) used single wells (or tubes).  It is clearly stated for the intracellular assays that triplicate wells were used (L117).

Author response: Font corrected. All assays used triplicate assay wells (as a minimum) and data is reported from 3 independent experiments. Section 2.2 has been edited to state this.

10. L152 and L170: “x” should be capitalized as for the other uses of 4X.

Author response: Corrected.

11. LL159-168:  Move Fig 2 legend under Fig 2.

Author response: Figure legends for all figures have been moved so that they are below the corresponding figure.

L160 and L165:  There is a hyphen in “as-sessed” and “ex-periments”

Author response: Corrected.

12. LL177-188:  Move Fig 3 legend under Fig 3.

Author response: Author response: Figure legends for all figures have been moved so that they are below the corresponding figure.

L179 and L181:  Instead of J7742s (too colloquial), suggest J7742 cells (not that much longer!).

Author response: Edited to reference either J774.2 cells or murine macrophage-like cells.

13. L201:  Be consistent with italicization of “in vitro.”  Elsewhere, it is italicized (L147 and L208).  Check document for others. 

Author response: Formatting corrections made.

14. L216: Use a lowercase “s” with MICs.

Author response: Correction made.

15. LL215-217:  Please clarify that the activity is against  pseudomallei.  For example: “A fatty acid synthesis inhibitor… had MICs for B. pseudomalleiof 8 μg/mL (strain Bp400) and 1 μg/mL (strain 1026b), respectively (20,21).

Author response: Editing clarification made as suggested.

16. L217:  There is a “closed parenthesis” and a comma missing (see above comment).

Author response: Editing corrected as suggested.

17. L217:  There is a font change for “respectively (20,21).”

Author response: Font corrected.

18. L219: “…again using  pseudomallei strain 1026b.”

Author response: Edited as suggested.

19. References: There are inconsistencies in the author listing: for example, reference 1 is last name followed by initials, while reference 2 has the initial and then the last name.

Author response: Formatting of references has been corrected and made consistent in accordance with journal guidelines.

20. There are also inconsistencies throughout with author initials.  See reference 1 which uses periods and reference 11 which does not.

Author response: Formatting of references has been corrected and made consistent in accordance with journal guidelines.

21. There are inconsistencies with reference titles.  For example, reference 4 uses capitalization for only the first word.  Reference 5 uses title case for all words.  Please be consistent.

Author response: Formatting of references has been corrected and made consistent in accordance with journal guidelines.

22. References 11-22 have a change in indentation.

Author response: Formatting of references has been corrected and made consistent in accordance with journal guidelines.

23. References 14 and 16 inconsistently reference CLSI documents.  Please provide the full reference for number 16.

Author Response: Reference 16 is cited for the CLSI definition of bactericidal/bacteriostatic definition in the time kill assay (section 2.4). This was accidentally deleted. The manuscript has been edited to reintroduce the reference. CLSI references citations have been edited to be in accordance with CLSI guidance.

Reviewer 5 Report

This communication addresses a subject of interest: the research for effective new antibiotics. The topic is relevant for the field as the impact of antimicrobial resistance has been a theme of intensive research for several groups during recent years.

The paper deserves to be published in Antibiotics, however, there are several points in which improvements should be made to strengthen the manuscript.

·       I suggest the reformulation of the title, to better reflect the explorative activities described in the paper for some representatives of the class.

·       Please ensure that all the names of the species are written in italics.

·       The format of the manuscript is outdated. The affiliation of the authors, the way references are mentioned in the text, and the order of the sections are some of the items that should be updated. Although the journal accepts free format submissions, the style of the references is not consistent for all entries.

·       Lines 11-12: Why did you consider introducing a running title and another set of key words although the Instruction for authors states “Please do not include abbreviated or short forms of the title, such as a running title or head”?

·       Lines 26-36: Please add references for the first paragraph of the Introduction.

·       Lines 30 – 32: Please mention the classes of antibiotics used to treat melioidosis, as you did for tularemia.

·       Line 60: Please explain here the abbreviation DMPK.

·       Line 82: Please mention the sources of the bacterial strains.

·       Figure 1: Please use higher resolution.

·       Reference 16 is not mentioned in the text.

·       Lines 196 – 197: Please keep the same format (Roman numerals or Arabic numerals) when referring to clinical trials phases.

·       Please add a discussion of the reasons why you have chosen ciprofloxacin and doxycycline to the detriment of other antibiotics when performing MIC tests.

·       Please add some information regarding novel candidates against F. tularensis as you did for B. pseudomallei.

·       A paragraph regarding the limitations of the study would be beneficial to the reader.

·       The perspectives of the study should be better outlined.

·       A separate Conclusions paragraph ought to be considered.

Author Response

This communication addresses a subject of interest: the research for effective new antibiotics. The topic is relevant for the field as the impact of antimicrobial resistance has been a theme of intensive research for several groups during recent years. The paper deserves to be published in Antibiotics, however, there are several points in which improvements should be made to strengthen the manuscript.

1. I suggest the reformulation of the title, to better reflect the explorative activities described in the paper for some representatives of the class.

Author Response: Whilst being mindful of the brevity required for the title, it has been to indicate activity assessment of ‘novel’ topoisomerase inhibitors. This provides a distinction from topoisomerase inhibitors in their broader sense (eg fluoroquinolones).

2. Please ensure that all the names of the species are written in italics.

Author Response: Manuscript edited accordingly.

3. The format of the manuscript is outdated. The affiliation of the authors, the way references are mentioned in the text, and the order of the sections are some of the items that should be updated. Although the journal accepts free format submissions, the style of the references is not consistent for all entries.

Author Response: Formatting of references has been corrected and made consistent in accordance with journal guidelines.

4. Lines 11-12: Why did you consider introducing a running title and another set of key words although the Instruction for authors states “Please do not include abbreviated or short forms of the title, such as a running title or head”?

Author Response: The running title has been removed. The duplicated keywords have also been removed.

5. Lines 26-36: Please add references for the first paragraph of the Introduction.

Author Response: Relevant references added.

6. Lines 30 – 32: Please mention the classes of antibiotics used to treat melioidosis, as you did for tularemia.

Author Response: There are very few antibiotics recommended for the treatment of melioidosis. The specific antibiotics commonly recommended have been identified in the edited manuscript. In addition, a reference has been added to the first paragraph with provides information on melioidosis treatment regimens.

7. Line 60: Please explain here the abbreviation DMPK.

Author Response: Drug metabolism and pharmacokinetics (DMPK). Manuscript edited accordingly.

8. Line 82: Please mention the sources of the bacterial strains.

Author Response: These strains are held at Dstl. References have been provided for the previous genome sequencing of these strains at Dstl. Further background on these strains are provided in these references.

9. Figure 1: Please use higher resolution.

Author Response: Higher resolution TIFF file provided.

10. Reference 16 is not mentioned in the text.

Author Response: Reference 16 is cited for the CLSI definition of bactericidal/bacteriostatic definition in the time kill assay (section 2.4). This was accidentally deleted. The manuscript has been edited to reintroduce the reference.

11. Lines 196 – 197: Please keep the same format (Roman numerals or Arabic numerals) when referring to clinical trials phases.

Author Response: Edited to use Roman numerals for both.

12. Please add a discussion of the reasons why you have chosen ciprofloxacin and doxycycline to the detriment of other antibiotics when performing MIC tests.

Author Response: Ciprofloxacin and doxycycline were chosen as comparator antibiotics because their in vitro activity against B. pseudomallei and F. turlarensis has previously been established by ourselves and others. This has now been stated in the Results section. The manuscript was not intended to be a comprehensive assessment of large panels of antibiotics against these bacterial pathogens, as this has been the subject of previous publications.

13. Please add some information regarding novel candidates against  tularensis as you did for B. pseudomallei.

Author Response: Where alternative non-traditional antibiotics could be identified, these were primarily against B. pseudomallei, hence the focus of examples for this pathogen. This has been stated in Discussion section.

14. A paragraph regarding the limitations of the study would be beneficial to the reader.

Author Response: Limitations, and future development requirements, are now more clearly stated in the final paragraph of the manuscript.

15. The perspectives of the study should be better outlined.

Author Response: In addressing the comments from all reviewers, significant editing of the document has been made. We would hope that this improves the contextual framing and interpretation of the manuscript.

16. A separate Conclusions paragraph ought to be considered.

Author Response: This manuscript is intended to be concise report to demonstrate the exploitation, and therapeutic potential, of these novel compounds against two biothreat pathogens. In the interest of brevity, conclusions (and limitations) have been more clearly stated in the final paragraph of the manuscript.

Round 2

Reviewer 3 Report

The authors' responses to the comments of the reviewer are presented in an organized and clear manner.

Reviewer 5 Report

Dear authors, I appreciate the answers received to my comments.