The Metabolic Stability of Antimicrobial Peptides IK8 in Plasma and Liver S9

Round 1

Reviewer 1 Report

In this manuscript, Lin and Yu describe the study of the metabolic stability of some potential antimicrobial peptides related to IK8. Stability is assessed in plasma and liver S9. The peptides were obtained by modifying the reference IK8 peptide by replacing one or two L-configuration amino acids with D-configuration amino acids.

The experiments appear well done and their description adequate. The authors show that the insertion of D-amino acids causes an increase in the stability of the corresponding peptides.

The increase in metabolic stability of peptides obtained by replacing L-configuration amino acids with D-configuration amino acids seems chemically obvious.

However, the authors do not demonstrate that the modified peptides retain their biological activity with biological activity data.  For this reason, I think the work is immature for publication at this stage.

Author Response

Response: Thank you for the advice. We have studied the biological activity of IK8 and its modified peptides. We have added it in discussion section. According to our published results (Biomaterials 2014, 35 (4), 1315-25), the substitution of peptide IK8-L with D-amino acids considerably strengthened antimicrobial activities. Moreover, among the three configuration peptides referred in this study, IK8-D demonstrated the most potent antimicrobial activities against five types of microorganisms(S. epidermidis, S. aureus, E. coli, P. aeruginosa and C. albicans), with the lowest geometric mean of minimum inhibitory concentrations (MIC) value of 4.3 mg/L, while the values of IK8-L and IK8-2D under identical testing conditions were 23.4 mg/L and 12.5 mg/L, respectively.

Reviewer 2 Report

Dear Authors, your manuscript is very interesting because antimicrobial peptides are highly interested in the research for new molecules to combat the spreading worldwide of antimicrobial resistance.

I suggest you to improve the introduction regarding the different antimicrobial peptides activities against bacteria (please indicate some examples of bacteria in which AMPs are effective)

Author Response

Response: Thank you for your question. We have added relevant contents to the introduction. Please refer to the section marked in red. We listed the recent examples of three antimicrobial peptides with significant antibacterial effects on some bacteria to prove their great potential and wide application value.

Reviewer 3 Report

The manuscript “The metabolic stability of antimicrobial peptides IK8 in plasma and liver S9” presented by Mao and colleagues is providing some information on the identifications of metabolites generated during the in vitro incubation of three peptides, the L and D enantiomers of amino acids and a mixture of L and D amino acids. They gave some data suggesting that according to the plasma origin (five plasma were tested) and liver S9 cells, the collection of metabolites differ. The methodology used looks appropriate even if we can regret that HPLC and mass spectrometry analyses were not conducted on line. The writing of the manuscript is not satisfactory and needs to be strongly improved. The reference list needs to be revised and the Introduction improved including more recent reviews and references that will provide to the reader an up-to-date view on the mechanisms of action (MoA) of AMPs (not limited to pore formation but with more complex MoA). The discussion proposed by the authors is really poor and limited to their peptides that are linear molecules without any structure in solution and with a richness in basic residues (R and K) that offers to proteolysis a nice structure to cleave. Changing L amino acids in D-amino acids to avoid this type of proteolysis is not novel and was already demonstrated to be useful for this class of linear cationic peptides.

Comments regarding the manuscript presentation. As already pointed the introduction is not proposing a clear overview of the AMP situation regarding their potential use as therapeutics (Pros and Cons). The Materials and Methods section is not reporting enough details to be able to reproduce the experiments with other candidates. Series of examples, to mention only a few of them. What is the plasma % use? What was the purity level of the peptides? How are cul9 cells and How they were prepared? What is the meaning of “Eventually”, it is non-sense, it is or it is not 20uL? DO the authors really performed MS analyses in TFA? Usually this counter ion is not relevant and formic acid should be used. It is unclear, lines if the authors used a linear or not a linear gradient (see end of page 2 of 11 and beginning of page 3). What is the Q-Tof equipment compared to the AB SCIEX triple TOF? If they are different why two different equipments? etc.

Comments regarding the experimental workflow. The authors performed according to their writing a duplicate. Why not a replicate and even better for statistical relevance 5 replicates (the recognised usage for such a study)? There is no control experiment without peptide (time 0 is not the best control). There is no toxicity study in parallel to see if a different toxicity is observed between the three IK8 forms. As the author have access to plasma they could easily use the red blood cells for haemolysis measurement, even if this is not the most pertinent experiment for testing toxicity this remains a reference. There is no interrogation by the authors on the stability of their peptides during the time-consuming process of vacuum concentration at 60°C. This should be addressed. Why the authors did not perform a real quantification using calibrations curves? They should also provide an HPLC chromatogram showing the chromatographic behaviour of each peptide in a single run, to visualise or not the modification of the retention time of the three peptides according to L, D or mixture of L and D amino acids.

It would be interesting to have the explanation by the authors how they can get a metabolite IRIK-COOH while the peptide was amidated at its C-terminus (IRIK-NH2)?

Finally, the result section is proposing a view of the results on the rat model. The referee is questioning the authors: Why not proposing the results using the Human model? It is quite frustrating. Looking at the different chromatograms, they are several possible additional metabolites at time 60 that are not considered (see Figure 1, page 3 of 11, one peak at a RT of approx 4 min, another one at 34 min), why? They are also peaks that are visible at Time 0 RTapprox9.5-10, 16 min. Can the authors clarify their origin (plasma or not). Are those peaks visible for all plasma models or not?

The manuscript proposed by by Mao and colleagues is not sufficiently robust to be accepted for publication.

Author Response

The manuscript “The metabolic stability of antimicrobial peptides IK8 in plasma and liver S9” presented by Mao and colleagues is providing some information on the identifications of metabolites generated during the in vitro incubation of three peptides, the L and D enantiomers of amino acids and a mixture of L and D amino acids. They gave some data suggesting that according to the plasma origin (five plasma were tested) and liver S9 cells, the collection of metabolites differ.

1. The methodology used looks appropriate even if we can regret that HPLC and mass spectrometry analyses were not conducted on line.

Response: Thank you for the advice. Because ik8 can have good chromatographic separation only when trifluoroacetic acid is contained in the mobile phase. Trifluoroacetic acid in the mobile phase, however, does great harm to the mass spectrum. At the same time, because the concentration of some metabolites is very low, the sensitivity of direct LC-MS is not enough. Therefore, the metabolites were firstly prepared by preparative liquid chromatography and then were identified by LC-MS/MS.

2. The writing of the manuscript is not satisfactory and needs to be strongly improved.

Response: Thank you for the advice. We have asked English speakers to polish the paper.

3. The reference list needs to be revised and the Introduction improved including more recent reviews and references that will provide to the reader an up-to-date view on the mechanisms of action (MoA) of AMPs (not limited to pore formation but with more complex MoA).

Response: Thank you for the advice. We have modified the introduction. See the section marked in red.

4. The discussion proposed by the authors is really poor and limited to their peptides that are linear molecules without any structure in solution and with a richness in basic residues (R and K) that offers to proteolysis a nice structure to cleave. Changing L amino acids in D-amino acids to avoid this type of proteolysis is not novel and was already demonstrated to be useful for this class of linear cationic peptides.

Response: Thank you for your question. We have modified the discussion part and added the discussion on the relationship between the metabolic stability of IK8 amino acid substitution and polypeptide activity. See the red part of discussion. As an innovation, the introduction of D-type amino acid in the last sentence at the end improves the stability. It is indeed biased. We rewrite it based on the previously obtained activity and toxicity data. The improvement of stability explained in this paper further reveals the potential of antibacterial peptides replaced by D-type amino acid.

5. Comments regarding the manuscript presentation. As already pointed the introduction is not proposing a clear overview of the AMP situation regarding their potential use as therapeutics (Pros and Cons).

Response: Thank you for your question. We have modified the introduction. See the section marked in red. After describing the mechanism of antimicrobial peptides, its advantages are explained in a separate paragraph, and several disadvantages of antimicrobial peptides briefly listed in the original are expanded.

6. The Materials and Methods section is not reporting enough details to be able to reproduce the experiments with other candidates. Series of examples, to mention only a few of them. What is the plasma % use? What was the purity level of the peptides? How are cul9 cells and How they were prepared? What is the meaning of “Eventually”, it is non-sense, it is or it is not 20uL? DO the authors really performed MS analyses in TFA? Usually this counter ion is not relevant and formic acid should be used. It is unclear, lines if the authors used a linear or not a linear gradient (see end of page 2 of 11 and beginning of page 3). What is the Q-Tof equipment compared to the AB SCIEX triple TOF? If they are different why two different equipments? etc.

Response: Thank you for your question. We have revised these in the “The Materials and Methods section” which were marked in red.

7. Comments regarding the experimental workflow. The authors performed according to their writing a duplicate. Why not a replicate and even better for statistical relevance 5 replicates (the recognised usage for such a study)? There is no control experiment without peptide (time 0 is not the best control).

Response: Thank you for your advice. We have added a chromatogram of blank plasma in Figure 1.

8. There is no toxicity study in parallel to see if a different toxicity is observed between the three IK8 forms. As the author have access to plasma they could easily use the red blood cells for haemolysis measurement, even if this is not the most pertinent experiment for testing toxicity this remains a reference.

Response: Thank you for your question. In the early stage, we had done hemolysis test and found that the addition of D configuration had little effect on its blood compatibility with the help of hc10 value (more reliable than the conventional HC50 value in the literature). Then we also conducted toxicity test on human keratinocytes with the help of MTT method and found that the cell survival rates of these three IK8 were still high even at high concentrations. We cited these two results, The result that IK8 is still relatively safe after the introduction of D configuration is supplemented.

9. There is no interrogation by the authors on the stability of their peptides during the time-consuming process of vacuum concentration at 60°C. This should be addressed.

Response: Thank you for your question. We studied the stability of the polypeptide and found they were stable at 60 °C.

10. Why the authors did not perform a real quantification using calibrations curves?

Response: Thank you for your question. Because the amount of peptide metabolites we prepared is very small and can not be weighed. So, it is impossible to carry out quantitative determination.

11. They should also provide an HPLC chromatogram showing the chromatographic behaviour of each peptide in a single run, to visualise or not the modification of the retention time of the three peptides according to L, D or mixture of L and D amino acids.

Response: Thank you for your advice. We have provide the chromatogram of blank plasma, and chromatograms of IK8-L, IK8-D and IK8-2D in blank plasma. These chromatograms were gotten in the same chromatographic condition. And, there are fully separated according to their retention time.   

12. It would be interesting to have the explanation by the authors how they can get a metabolite IRIK-COOH while the peptide was amidated at its C-terminus (IRIK-NH2)?

Response: Thank you for your advice. We also think it is interesting to claim the mechanism of IRIK-NH2 formation. It should be studied in the further.

13. Finally, the result section is proposing a view of the results on the rat model. The referee is questioning the authors: Why not proposing the results using the Human model? It is quite frustrating.

Response: Thank you for your advice. Due to the high price of human liver microsomes and human plasma, rat model is used for the preparation of metabolites. Therefore, the chromatograms listed in manuscript are from rat models.

14. Looking at the different chromatograms, they are several possible additional metabolites at time 60 that are not considered (see Figure 1, page 3 of 11, one peak at a RT of approx 4 min, another one at 34 min), why? They are also peaks that are visible at Time 0 RTapprox9.5-10, 16 min. Can the authors clarify their origin (plasma or not). Are those peaks visible for all plasma models or not?

Response: Thank you for your question. Indeed, there may be some metabolites that we failed to identify. In this study, we mainly identified some metabolites with retention time after 16 min. In future work, we will further identify possible metabolites before 16 min. The possible metabolite at 34 min were not prepared in sufficient amount for analysis because the concentration was too low.

Round 2

Reviewer 1 Report

The revised version can be published without further changes.

Reviewer 3 Report

The new version proposed by the authors has been sufficiently improved. However, some answers proposed by the authors are not convincing. At least the long discussion proposed on the use of TFA versus formic acid is known from years, this is the reason why users are performing their analysis in formic acid.

The authors made efforts to propose a more solid manuscript. It remains some minor editing adjustments to do.