Identification of Bioactive Compounds from Marine Natural Products and Exploration of Structure-Activity Relationships (SAR)

Corrections recommended

Response

In the cytotoxic study, why no non-transformed cells were used? The authors should identify if these compounds have selectivity or not.

In this study we used

-Human alveolar epithelial cells (A549)

-Madin-Darby canine kidney epithelial cells (MDCK)

-Human leukaemia cells (THP-1)

-Human hepatocellular carcinoma cells (Hep-G2)

-Human embryonic kidney cells 293 (HEK293)

 All these cell lines grow as a monolayer when attached to culture flask. They collected from carcinoma and normal cell. These cell lines chosen because they are widely used in different fields of medical research, especially basic cancer research and drug discovery. Due to low amounts of compound isolated, we couldn’t run selectivity test. But selectivity, cyto-toxic against the cell lines and primary cells, as well as pharmacodynamic functions of proposed structures confirmed through analysis of the synthetic derivatives. This part is published doi: 10.3389/fmicb.2020.551189

The chemical structures of the compounds with strong potencies should be displayed.

We elucidated chemical structures of strong biological activities compound 1-8. They displayed in Figure 2. Line 178-180.

Any known compound with similar structure and biological activities? If yes, their structure should be shown. Also, they should be included into the cyto-toxic study.

The structure–activity relationships (SAR) has shown the modification of existing molecules can be used to produce new antimicrobial molecules and improve the biological activity by increasing antibacterial activity and decreasing toxicity. In the bellow tables antimicrobial activity and cyto-toxicity of similar structure presented.

Table 3. Screening of commercially available amines with structures similar to 3 and 13. Lines 250.

Table 4. Screening of commercially available amines with structures related to 1a, 1b and 2a. Lines 256.

Table 5. Screening of commercially available compounds with structures inspired by ascididemin. Line 315.

Table 6. Screening of compounds with structures inspired by ascididemin which include three aromatic rings. Line 353.

Table 7. Screening of compounds with structures inspired by ascididemin which include three aromatic rings plus further modifications. Line 420.

Table 8 present summary of anti-bacterial activity and toxicity of compounds inspired by the natural products characterised in this project. Line 439.

Corrections recommended

Response

The author could provide a structure of methicillin, vancomycin, ascididemin, quinoline, phenanthroline, and ω-conotoxin MVIIA

In this work we present the structures that we discovered and re discovered from marine natural products as well as, commercially available compounds with structures similar to synthesised compound which inspired by natural product structures. Structures of ascididemin (line 170-175), quinoline (lines 284,320) and phenanthroline (lines 284,324-335) are provided in this work.  The structures of methicillin, vancomycin and ω-conotoxin MVIIA did not present because structurally, they are not close to natural product structures or synthesised compound.  Also, the activities and cyto-toxicity of methicillin and ω-conotoxin MVIIA didn’t test.  We cannot compare they activities with isolated natural product. The vancomycin only used as a positive control.

The author could provide the structure of the most potent reported marine natural products having antibacterial activities.

Change done. Lines 69-81

The author could provide the structures of 7, 8, and 9 along with 1-5 in figure 1. If possible, for all the compounds discussed in this manuscript (eq. isoquinoline, quinoline, 4,7- 158 phenanthroline, 1,7-phenanthroline, 1,10-phenanthroline, and bipyridine).

Change done.

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The author could change “(j + k) = 14; m =5, n = 9; (q +r) = 20; (t + 5u + v) = 19” to simplified equation; like j = 7…..

Change done.

lines 178

Any reason why cyclic amines are not active compare to cyclic amine?

All the cyclic amines reduced the activities, and they are not toxic. We only observed two different result for two Gram negative bacteria. This phenomenon can explain via their patterns of susceptibility and growth rate.

Any activity profile for Benzyl amine having electron-withdrawing/or donating groups.

We didn’t consider other positions of disubstituted benzene ring to test the effect of an electron donating groups on molecules antimicrobial activities.  The main aim of this study was to modify structure of molecules by adding or removing parts/atoms (C, O, N..).  The chemically modified electrodes were partially covered.

The author could move “Purification of Natural Products from Extracts and Structure Elucidation” and “Identification and Structure Elucidation” to the supporting information and move “STRUCTURE−ACTIVITY RELATIONSHIP” from supporting information to the manuscript.

Change done.

Structure−Activity Relationship results moved to the manuscript.

Table 3. Screening of commercially available amines with structures similar to 3 and 13. Lines 250.

Table 4. Screening of commercially available amines with structures related to 1a, 1b and 2a. Lines 256.

Table 5. Screening of commercially available compounds with structures inspired by ascididemin. Line 315.

Table 6. Screening of compounds with structures inspired by ascididemin which include three aromatic rings. Line 353.

Table 7. Screening of compounds with structures inspired by ascididemin which include three aromatic rings plus further modifications. Line 420.

Table 8 present summary of anti-bacterial activity and toxicity of compounds inspired by the natural products characterised in this project. Line 439.

Purification of Natural Products from Extracts and Structure Elucidation” and “Identification and Structure Elucidation” to the supporting information are the main parts of methodology, if this is possible, we keep them in the manuscript, please.

The author could show structures while discussing the activity of series of compounds and then show the activity profile (MIC) in the table.

Change done.  

Table 8 present summary of anti-bacterial activity and toxicity of compounds inspired by the natural products characterised in this project. Compounds structures added to Table. Line 439.

Any comparison of the activity of different series of compounds.

In this study we work with Gram positive, Gram-negative and mycobacteria pathogens. Modification of molecule shown different effect on test pathogens. In summary, we reported by varying the number of carbons in the linear chains, the bioactivity against the Gram positive and Gram-negative pathogens tested increased. By extending the carbon linker to the aromatic rings then modifying and increase the aromatic rings activity of the molecules against tested pathogens reduced. This suggests that the mechanism of action may be based on targeting complex cell wall structures. Many factors influence the activity of a molecule, including side chain length, symmetry, length of the alkyl chain, the presence of charged pyridinium moiety, presence and position of the carboxylate in the structure, position of the methyl group, distance between the two pyridinium moieties and chain linker between the two pyridinium rings. Thus, the mechanism of action of these molecules may be based on the space between the rings and distance between charged moieties, which possibly manifests through interaction with membrane sites. In the next step of my work, I synthesised antimicrobial compound with fluorescent activity similar to natural product structure to track the molecule in the live cell and find the  compound  mechanism of action. I will publish that part separately.

In line 315: “¹H NMR (500 MHz, DMSO-d6)” it should not be bold.

Change done.

line 530

In Line 320: Use the abbreviation of “rdbe”.

rdbe is abbreviation of ring or double bond equivalent.

This definition presented in line 137.

In Line 247: Change “37°C” to “37 °C”.

Change done. line 462

For the references use the same format: need space and year should be in bold. Change “2014,20(4), 3-18” to 2014, 20(4), 3- 18” and make changes for the other references as well. In Line 517: change “Mar Drugs 2014,12 (1), 462-76.” To “Mar Drugs 2014,12 (1), 462-476”. And make changes for the other references as well.

Change done.

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In Table S5, the author should draw the COSY relationship in structure 6 with arrows for better understanding as like compound 8 (Fig S25).

The arrows in the Fig S25 are based on the HMBC NMR spectrum analysis.  Due to low amounts of compound isolated we didn’t run HMBC for compound 6 and 7.

All NMR should have pick peaking, integration with the structure embedded.

Change done.  Fig S27. Also, since, mass spectrometry (MS) used in this study provides an exhaustive strategy to elucidate chemical structures from insufficient resources such as natural products. I put compound 6, 7 and 8 mass patterns with the structure embedded. Fig S26

Table S8-12 should move to the manuscript instead of supporting documents.

Change done.

Table 3. Screening of commercially available amines with structures similar to 3 and 13. Lines 250.

Table 4. Screening of commercially available amines with structures related to 1a, 1b and 2a. Lines 256.

Table 5. Screening of commercially available compounds with structures inspired by ascididemin. Line 315.

Table 6. Screening of compounds with structures inspired by ascididemin which include three aromatic rings. Line 353.

Table 7. Screening of compounds with structures inspired by ascididemin which include three aromatic rings plus further modifications. Line 420.