Chalcones Isolated from Arrabidaea brachypoda Flowers as Inhibitors of NorA and MepA Multidrug Efflux Pumps of Staphylococcus aureus

Round 1

Reviewer 1 Report

NMR signal assignments and Rf values of the isolated chalcones 1, 3, 4, and 5 were should be showed in Materials and Methods Section.

Author Response

1. NMR signal assignments and Rf values of the isolated chalcones 1, 3, 4, and 5 were should be showed in Materials and Methods Section.

R: The compounds were isolated in low quantities and it was not possible to perform NMR analyzes of 1H and 13C. The chalcones have fragmentations that are particular and several works report the identification of these compounds only by mass spectrometry. We added the high-resolution mass spectrum, which allows the exact determination of molecular mass. In the Results section, we have added the description of the characterization by mass spectrometry. In addition we compare it with spectral data from our laboratory standards

Reviewer 2 Report

This manuscript written by Luís Mário Rezende-Júnior and co-workers described the Norfloxacin action inhibition of chalcones isolated from Arrabidaea brachypoda flowers. In my point of view, the natural product identification part should be improved.

 The comments are list below:

1. The authors reported the identification of four compounds. However, the numbers of those compounds are 1, 3-5. What is compound 2? If the authors cannot identify the structure. They should name those four compounds 1-4.
2. Figure 1, these structures are drawn in a strange style. Please use ChemDraw with ACS1996 style to redraw your structures.
3. The authors should provide the ¹H and ¹³C NMR data of four chalcones or put the data in supporting material.
4. Only one sentence in lines 83-81 described the identification of four Please describe more to let the readers know how these four compounds were identified.
5. Refs 44 and 45 reported the methoxylation degree of chalcones increased the NorA inhibition, which dramatically decreases the novelty of this research. The authors should provide more data (additional experiments) to improve the novelty of your research.

Author Response

This manuscript written by Luís Mário Rezende-Júnior and co-workers described the Norfloxacin action inhibition of chalcones isolated from Arrabidaea brachypoda flowers. In my point of view, the natural product identification part should be improved.

1. The authors reported the identification of four compounds. However, the numbers of those compounds are 1, 3-5. What is compound 2? If the authors cannot identify the structure. They should name those four compounds 1-4.

R: We isolated other classes of compounds from the flowers extract of Arrabidaea brachypoda, and the numbering was done in the order they were isolated.

2. Figure 1, these structures are drawn in a strange style. Please use ChemDraw with ACS1996 style to redraw your structures.

R: done

3. The authors should provide the ¹H and ¹³C NMR data of four chalcones or put the data in supporting material.

R: The compounds were isolated in low quantities and it was not possible to perform NMR analyzes of 1H and 13C. The chalcones have fragmentations that are particular and several works report the identification of these compounds only by mass spectrometry. We added the high-resolution mass spectrum, which allows the exact determination of molecular mass. In the Results section, we have added the description of the characterization by mass spectrometry. In addition we compare it with spectral data from our laboratory standards

4. Only one sentence in lines 83-81 described the identification of four Please describe more to let the readers know how these four compounds were identified.

R: In the Results section, we have added the description of the characterization by mass spectrometry.

5. Refs 44 and 45 reported the methoxylation degree of chalcones increased the NorA inhibition, which dramatically decreases the novelty of this research. The authors should provide more data (additional experiments) to improve the novelty of your research.

R: Dear Reviewer, in the cited references the authors did not work with chalcones, but they work with flavonoids and Phyllantin. In the present study we verified that an increasing in the methoxylation degree also enhanced the ability of chalcones to inhibit NorA. We think that this is a novelty.

Reviewer 3 Report

In this manuscript, the authors studied the effect to various chalcones isolated from A. brachypoda flowers as potential efflux pump inhibitors for S. aureus. The compounds were isolated and characterized via Mass-Spec. Molecular docking studies were also carried out. All the experiments have been conducted logically and the experimental design is well-suited. Overall, the manuscript is written well. A few corrections are required

Please expand the introduction section. I would like to see more details on global issue of antibiotic resistance, different kinds of efflux pumps present in S. aureus strains. Also include the current treatment regimen for various important diseases caused MRSA and other drug-resistant S. aureus strains.

Page 2 line 82 - Please explain the results for 2.1 in detail and discuss it appropriately

Page 3 line 99 - I cannot find the results for the sentence 'isolated chalcones were inactive against all S. aureus strains at the concentration'. Are the authors missing any table with relevant data?

Page 3 fig. 2 - Please label the graphs properly. Also, how is the data represented? In the figure legend, its written for geometric mean. Describe the statistical analysis performed in this figure properly in the figure legend

Page 8 line section 3.1 - why did the authors replace MH-II broth by BHI? You need to follow CLSI guidelines in order to perform micro-dilution assay. I would also like to see MBC results since it is important to know if the chalcones are bacteriostatic or bactericidal in nature

I would highly recommend that the authors perform RT-PCR experiments to determine the effect to chalcone exposure on different efflux pump genes that are described in table 3.

Author Response

In this manuscript, the authors studied the effect to various chalcones isolated from A. brachypoda flowers as potential efflux pump inhibitors for S. aureus. The compounds were isolated and characterized via Mass-Spec. Molecular docking studies were also carried out. All the experiments have been conducted logically and the experimental design is well-suited. Overall, the manuscript is written well. A few corrections are required

Please expand the introduction section. I would like to see more details on global issue of antibiotic resistance, different kinds of efflux pumps present in S. aureus strains. Also include the current treatment regimen for various important diseases caused MRSA and other drug-resistant S. aureus strains.

R: done.

Page 2 line 82 - Please explain the results for 2.1 in detail and discuss it appropriately

R: done

Page 3 line 99 - I cannot find the results for the sentence 'isolated chalcones were inactive against all S. aureus strains at the concentration'. Are the authors missing any table with relevant data?

R: Dear Reviewer, we include the Table 4 showing MIC values found for chalcones against S. aureus strains. We also added the following sentence: “Natural products from plants have been considered as clinically irrelevant when they show MIC values above 1000 µg/mL. MIC values found for chalcones tested are presented in the Table 3. These results indicate that chalcones isolated from A. brachypoda were inactive against all S. aureus strains.

Page 3 fig. 2 - Please label the graphs properly. Also, how is the data represented? In the figure legend, its written for geometric mean. Describe the statistical analysis performed in this figure properly in the figure legend

R: done.

Page 8 line section 3.1 - why did the authors replace MH-II broth by BHI? You need to follow CLSI guidelines in order to perform micro-dilution assay. I would also like to see MBC results since it is important to know if the chalcones are bacteriostatic or bactericidal in nature

R: Dear Reviewer, take in account that chalcones showed MIC values above 1000 µg/mL, which are considered clinically irrelevant (Houghton et al., 2007), in this case we consider that it is not necessary determine MBC value. CLSI guidelines must be followed strictly when it is intended to diagnose the resistance profile of a pathogen in order to guide the prescription of antibiotics. This is not the purpose of the present study. We have used BHI broth with excellent results, since it favors the growth of the tested strains. In our experiments, we always do a growth control to compare with the wells where there was growth inhibition.

I would highly recommend that the authors perform RT-PCR experiments to determine the effect to chalcone exposure on different efflux pump genes that are described in table 3.

R: In fact, inhibition of the expression of genes that encode efflux pump is one of the possible mechanisms of action of efflux pump inhibitors (EPIs). Other possible mechanisms proposed are the interaction of the EPI with the membrane leading to the dissipation of the proton-motive force, or causing conformational changes in the efflux pump. Another mechanism could be the blocking of the efflux pump after connection of the EPI in the efflux pump, and competition for the connection at the same binding site as the antibiotic. Quantifying efflux pump mRNA using the quantitative PCR technique would enrich the work, however it would not exhaust all possibilities. For example, the reduction in gene expression may be the result of inhibition not of the gene encoding the pump, but it could also be the inhibition of the expression of a regulator gene. It could also be, the inhibition of a gene that encodes a signaling protein involved in signal transduction that would lead to activation of the expression of this regulatory gene. And so on. In the present study, we found that compound 4 had a good modulating effect and the tests with EtBr and the results of molecular docking support the hypothesis that this chalcone inhibits the NorA efflux pump probably due to competition with the antibiotic. However, we cannot rule out the other possibilities.

Round 2

Reviewer 2 Report

The authors have already revised their manuscript according to my suggestion. However, some flaws were still found in this manuscript.

In figure 1, the structure is even worse. Please redraw.

Table 1, the exact mass data are all XXX.11. I think it's wrong. The authors should use the values in table 2.

Author Response

The authors have already revised their manuscript according to my suggestion. However, some flaws were still found in this manuscript.

In figure 1, the structure is even worse. Please redraw.

R: done

Table 1, the exact mass data are all XXX.11. I think it's wrong. The authors should use the values in table 2.

R: done

Reviewer 3 Report

I am satisfied with the revisions. Please make sure that fig. 1 chemical structures are clear. Structures of compounds 3 and 5 are not clear.

Author Response

I am satisfied with the revisions. Please make sure that fig. 1 chemical structures are clear. Structures of compounds 3 and 5 are not clear.

R: done