Novel Polymyxin-Inspired Peptidomimetics Targeting the SARS-CoV-2 Spike:hACE2 Interface

Round 1

Reviewer 1 Report

In their manuscript the authors A. Contini,, K. Vermeire  and F. Zanardi et. al. report a discussion addressed to found molecules that could simultaneously bind to the sub-regions BS1 and BS2 of SARS-CoV2 and simultaneal hinder the interaction with hACE2 thus to hamper the infective process of SARS-CoV-2 into human cells. To do this, starting from the natural occurring polymyxin B(PMX) they adopt a very interesting strategy for the development of a polymyxin-Inspired peptidomimetics (a medium-sized molecules) addressed to SARS-CoV-2 Spike-hACE2 interface. Their metodologies combined molecular modelling, chemical strategies, binding affinity of peptidomimetics to Spike RBD and antiviral effect against SARS-CoV2. The overall manuscript is well-craft, hypothesis and modeling well-fit with the following design of the peptidomimetic. Even the chemical strategy carried out on a mixed solution and SPPS protocols sound very interesting, and include feature of innovation. The schematic representation of the chemical strategy result very clear, as the corresponding methodologies of synthesis and characterizations of the molecules produced.  

I my opinion the manuscript at this level is quite clear and scientifically is well-sound. I recommended it publication basically as it is.

Minor editing of English language required

Author Response

In their manuscript the authors A. Contini,, K. Vermeire  and F. Zanardi et. al. report a discussion addressed to found molecules that could simultaneously bind to the sub-regions BS1 and BS2 of SARS-CoV2 and simultaneal hinder the interaction with hACE2 thus to hamper the infective process of SARS-CoV-2 into human cells. To do this, starting from the natural occurring polymyxin B(PMX) they adopt a very interesting strategy for the development of a polymyxin-Inspired peptidomimetics (a medium-sized molecules) addressed to SARS-CoV-2 Spike-hACE2 interface. Their metodologies combined molecular modelling, chemical strategies, binding affinity of peptidomimetics to Spike RBD and antiviral effect against SARS-CoV2. The overall manuscript is well-craft, hypothesis and modeling well-fit with the following design of the peptidomimetic. Even the chemical strategy carried out on a mixed solution and SPPS protocols sound very interesting, and include feature of innovation. The schematic representation of the chemical strategy result very clear, as the corresponding methodologies of synthesis and characterizations of the molecules produced. I my opinion the manuscript at this level is quite clear and scientifically is well-sound. I recommended it publication basically as it is.

Comments on the Quality of English Language: Minor editing of English language required

Reply to Rev. 1

We thank the reviewer for appreciation of our work and the positive feedback.

As for minor editing of the English language, we already asked an English mother-tongue (chemistry-educated) professional translator to carefully proofread our manuscript before submission to the Journal (as mentioned in the acknowledgements).

Reviewer 2 Report

Dear authors,

The manuscript gives a nice and compelling intro to SARS-CoV2 and the concerns related to treatment of COVID-19. However, the rational for building novel antivirals on the scaffold of PolymyxinB is not very clear. In light of the renal toxicity issues tied to Polymyxin, the design approach might in fact look a bit stupid.

Thus, to improve the scientific soundness I would urge the authors to address this more clearly. Why Polymyxin? Which advantages is it to use this scaffold? Why is toxicity no longer an issue, this should ideally be corroborated with experimental evidences. In fact, the level of antiviral activity is hard to interpret without also knowing how the compounds affect viability of the host cells.

COVID is furthermore often linked to subsequent bacterial pneumonia. Given that Polymyxin is an approved antibacterial drug, it seems logical to also question the antibacterial activity of the Polymyxin derivatives developed within this paper.

Author Response

The manuscript gives a nice and compelling intro to SARS-CoV2 and the concerns related to treatment of COVID-19. However, the rational for building novel antivirals on the scaffold of PolymyxinB is not very clear. In light of the renal toxicity issues tied to Polymyxin, the design approach might in fact look a bit stupid. Thus, to improve the scientific soundness I would urge the authors to address this more clearly. Why Polymyxin? Which advantages is it to use this scaffold? 

We thank the reviewer for critical reviewing our manuscript and for the points of attention raised.

Concerning the reason of choosing the PMX structure as a starting point for construction of our peptidomimetics (PM).

As widely documented in the introduction and Results and Discussion sections of our manuscript, we started from the PMX structure as suggested from a previous computational modelling study (Ref. 42 in the revised version) wherein the PMX molecule resulted to be the best-ranked compound to theoretically fit in both the BS1 and BS2 regions of Spike protein interface. However, the PMX molecule was just the starting point of our investigation, and thorough structural variation of original PMX was imparted in both computational studies and in the synthesis, resulting in the creation of completely novel PMs with structural features which resemble the PMX core, but with highly marked deviations from it (i.e. they are deprived of the octanoyl chain, they incorporate diverse amino acid residues, they are dimerized, they incorporate unnatural triazolyl linker, as described in the manuscript). For a clear immediate evaluation of the structural differences see, for example, the structural description and the corresponding MWs of PMX as compared to PMs in Table 1.

For these reasons, we respectfully disagree with the reviewer that our approach is not well-founded. 

Why is toxicity no longer an issue, this should ideally be corroborated with experimental evidences. In fact, the level of antiviral activity is hard to interpret without also knowing how the compounds affect viability of the host cells.

Concerning the toxicity of the synthesized PMs.

Indeed, this issue was considered, and cellular toxicity has been evaluated for our PMs, as reported in Table A2 of the Supplementary Material, and also already commented in the Results section (page 13). No cytotoxic effect was observed for all peptidomimetics. To underline better this result, we also specified this toxicity issue in the discussion section (page 14) and in the description of the Supplementary Material (page 19). Also, we better specified the caption of Table A2 in the Supplementary Material to indicate the toxicity data.

COVID is furthermore often linked to subsequent bacterial pneumonia. Given that Polymyxin is an approved antibacterial drug, it seems logical to also question the antibacterial activity of the Polymyxin derivatives developed within this paper.

Concerning evaluation of the anti-bacterial activity of PMs.

The following note (ref. 53 in the revised version) has been added. Ref. 53: With these marked deviations from the original PMX structure, it is presumable that the PMs of this work are deprived of the antibacterial activity of PMX. Biological evaluation of antibacterial activity of PMs was not carried out here, going beyond the scope of this antiviral work.

Concerning possible improvement of background and inclusion of relevant references.

We added one recent reference (ref. 35 of the revised version), describing an interesting synthesis of stapled peptides published a few weeks ago.