Chemical Synthesis and Structure–Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript titled "Tick Anticoagulant Peptide (1-60) from the Hematophagous Parasite Ornithodoros moubata as a Highly Potent and Selective Inhibitor of the Blood Coagulation Factor Xa: Chemical Synthesis, Inhibition Mechanism, and SAR Studies with Noncoded Amino Acids"., is considered as one of interesting manuscript that i have reviewed. The idea of manuscript is not new but it is good for screening and chemical synthesis a new anticlotting agent. The manuscript is written well and the results are represented in good manner. But I have some comments which could be summarized in;
Title:
It is too long and should be summarized.
Abstract
Abstract is missing these parts;
Introduction
Materials and methods
Conclusion and convey.
Introduction
Lines 115 to 124 is kind of repetition and should be deleted
Lines 132 and 133 should be deleted.
Materials and methods
Too long should be summarized
Results
Too long should be shorten.
Discussion
Written well but needs more citation which will help the authors to finalized and interpretation their work in good manner.
Author Response
Reviewer #1
The manuscript titled "Tick Anticoagulant Peptide (1-60) from the Hematophagous Parasite Ornithodoros moubata as a Highly Potent and Selective Inhibitor of the Blood Coagulation Factor Xa: Chemical Synthesis, Inhibition Mechanism, and SAR Studies with Noncoded Amino Acids" is considered as one of interesting manuscript that i have reviewed. The idea of manuscript is not new but it is good for screening and chemical synthesis a new anticlotting agent. The manuscript is written well and the results are represented in good manner. But I have some comments which could be summarized in.
- Title
It is too long and should be summarized.
Response: We thank the Reviewer for her/his positive attitude toward our work and, accordingly, the Title text has been shortened from 35 to 21 words (-40%).
- Abstract
Abstract is missing these parts;
Introduction
Materials and methods
Conclusion and convey.
Response: Some parts of the abstract have been re-written in the Revised Manuscript to make the text more schematic and focused on the Results. The text has been reduced by 17% (from 249 to 207 words)
- Introduction
Response: The Introduction has been reduced by 18% (from 790 to 647 words).
Lines 115 to 124 is kind of repetition and should be deleted
Response: Accordingly, this part of the text has been deleted in the Revised Manuscript.
Lines 132 and 133 should be deleted.
Response: Accordingly, this part of the text has been deleted in the Revised Manuscript.
- Materials and methods
Too long should be summarized
Response: Accordingly, this part of the text has been reduced by 36% (from 2722 to 1741 words) in the Revised Manuscript.
- Results
Too long should be shorten.
Response: Accordingly, this part of the text has been reduced by 13% (from 1503 to 1314 words) in the Revised Manuscript.
- Discussion
Written well but needs more citation which will help the authors to finalized and interpretation their work in good manner.
Response: In the Discussion of the Revised manuscript four additional citations, regarding the structure and dynamics of TAP and BPTI in the free/unbound and complexed/bound form, have been added to the 21 references already present in the Original manuscript. Some parts of the Introduction, regarding the “degeneracy of the stereochemical code” in the TAP/BPTI pair have been amended and moved to the Discussion, where another example of this “degeneracy” has been reported, i.e. the Ubiquitin/Thi-S protein pair, along with the corresponding two additional references.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript titled "Tick Anticoagulant Peptide (1-60) from the Hematophagous Parasite Ornithodoros moubata as a Highly Potent and Selective Inhibitor of the Blood Coagulation Factor Xa: Chemical Synthesis, Inhibition Mechanism, and Structure-Activity Relationship Studies with Noncoded Amino Acids" evaluates the new functionalities in the molecular scaffold of TAP, which has significance in research and clinical applications.
It presents good quality of presentation, appropriate study design and adequate discussion. The reviewer raises only one comment on adding method details of HPLC including the instrument type and mobile phase in the kinetic studies.
Author Response
Reviewer #2
Comments and Suggestions for Authors
The manuscript titled "Tick Anticoagulant Peptide (1-60) from the Hematophagous Parasite Ornithodoros moubata as a Highly Potent and Selective Inhibitor of the Blood Coagulation Factor Xa: Chemical Synthesis, Inhibition Mechanism, and Structure-Activity Relationship Studies with Noncoded Amino Acids" evaluates the new functionalities in the molecular scaffold of TAP, which has significance in research and clinical applications.
It presents good quality of presentation, appropriate study design and adequate discussion. The reviewer raises only one comment on adding method details of HPLC including the instrument type and mobile phase in the kinetic studies.
Response: We thank the Reviewer for her/his positive attitude toward our work. As requested, the experimental details of RP-HPLC analyses of kinetic studies have been included in the Revised Mnauscript, as follows: “At time intervals, aliquots (20 µg) were taken, quenched with 4% aqueous TFA (40 µl), and fractionated with a Vydac (4.6 x 250 mm, 5 µm particle size) Cl8 analytical column, eluted with a linear acetonitrile-0.1% TFA gradient from 20 to 40% in 30 min at a flow rate of 0.8 ml/min and connected to a model 1500 Jasco (Tokyo, Japan) HPLC system. The absorbance of the effluent was monitored at 226 nm.”
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript from De Filippis et al reports the chemical synthesis of tick anticoagulant peptide (TAP) as a means of introducing non-native amino acids to undertake a structure activity relationship study. This is an interesting idea and has been well carried out, revealing some interesting differences in the biophysical behaviour of TAP and BPTI.
The manuscript appears to include conflicting ideas on the behaviour of the TAP N-terminus. Lines 637-639 state 'these observations indicate that the amino terminal binding site in TAP must remain in a highly constrained and specific spatial conformation in order to support high-affinity binding with FXa.' Whereas lines 982 to 984 state that 'the enhanced conformational flexibility TAP appears to have been “evolutionarily engineered” to lower the energetic cost of TAP adaptation to FXa active site.' These two statements seem to be in conflict. I note that the observations mentioned in lines 637-639 do not support 'a highly constrained and specific conformation', rather they support the presence of a specific chemical structure that supplies the atomic interactions that favour increased affinity and specificity. The dynamic nature of the TAP N-terminus has not been demonstrated in this paper because a local HDX-MS analysis was not possible. Some information on the dynamics of the N-terminus may be gleaned by examining the ATP NMR structures in the PDB and comparing the structures in the deposited ensembles. Molecular dynamics simulations may also provide useful information. Clarification of the author's position would strengthen the manuscript.
Some other minor suggestions follow:
Figure 1A should be labeled to indicate which sequence is TAP and which BPTI.
Figure 1B does not appear to have any magenta regions identifying interaction sites. Perhaps these regions could be marked with text?
The structures selected from the ensemble as the most representative should be identified, ie number 1 or number 12
Software version is normally abbreviated as "ver" rather than "vs"
Table S1 should state how the molar absorptivity numbers were obtained, ie which software or website was used.
Figure 2C might be enhanced by adding lines at the N and R retention times across the five HPLC traces to emphasise the difference in the major peaks. Alternatively, plotting the intensity of the N and R peaks against time should show a decrease in the R form and increase in the N.
Table S2. Adding numbers to the first and last residues in the identified sequences would help convince the reader that the correct disulfide pairing had been identified.
Figure 4. Why is there undeuterated BPTI present in the fully deuterated mass spectrum when there was none present in the earlier mass spectra?
Figure 7 shows just one structure of free TAP from the NMR ensemble superimposed on the bound structure. How was this structure selected? Were the other members of the NMR ensemble equally distant? Do the other members of the NMR ensemble support a rigid or flexible N-terminus?
Comments on the Quality of English LanguageThe English is quite good, needing only minor copy editing.
Author Response
Reviewer #3
The manuscript from De Filippis et al reports the chemical synthesis of tick anticoagulant peptide (TAP) as a means of introducing non-native amino acids to undertake a structure activity relationship study. This is an interesting idea and has been well carried out, revealing some interesting differences in the biophysical behaviour of TAP and BPTI.
Response: We thank the Reviewer for her/his positive attitude toward our work.
- The manuscript appears to include conflicting ideas on the behaviour of the TAP N-terminus. Lines 637-639 state 'these observations indicate that the amino terminal binding site in TAP must remain in a highly constrained and specific spatial conformation in order to support high-affinity binding with FXa.' Whereas lines 982 to 984 state that 'the enhanced conformational flexibility TAP appears to have been “evolutionarily engineered” to lower the energetic cost of TAP adaptation to FXa active site.' These two statements seem to be in conflict. I note that the observations mentioned in lines 637-639 do not support 'a highly constrained and specific conformation', rather they support the presence of a specific chemical structure that supplies the atomic interactions that favour increased affinity and specificity.
Response: We thank the Reviewer for bringing this point to our attention and essentially agree with the Reviewer’s observation. Accordingly, in the Revised manuscript lines 637-639 were replaced with the following sentence: “Altogether, these observations indicate that the chemical integrity of the N-terminal tripeptide sequence in TAP is mandatorily required to support high-affinity binding with fXa”.
- The dynamic nature of the TAP N-terminus has not been demonstrated in this paper because a local HDX-MS analysis was not possible. Some information on the dynamics of the N-terminus may be gleaned by examining the ATP NMR structures in the PDB and comparing the structures in the deposited ensembles. Molecular dynamics simulations may also provide useful information. Clarification of the author's position would strengthen the manuscript.
Response: We thank the Reviewer for bringing this point to our attention and, accordingly, in the Revised manuscript we have more deeply discussed the structure/dynamics of TAP and BPTI free in solution and bound to fXa and trypsin, respectively. The following sentence has been inserted in the Discusion “Our interpretation is strongly supported by the solution NMR structure of the inhibitor showing that, in contrast to what observed in the NMR structure of BPTI [Berndt et al., 1992], the N-terminal tripeptide of TAP has only few NOE distance constraints and that all the aromatic amino acids undergo rapid flipping motions about the Cb-Cg bonds [Antuch et al., 1994], indicative of both local and global enhanced flexibility of TAP compared to BPTI.”
Some other minor suggestions follow:
- Figure 1A should be labeled to indicate which sequence is TAP and which BPTI.
Response: Done.
- Figure 1B does not appear to have any magenta regions identifying interaction sites. Perhaps these regions could be marked with text?
Response: Done.
- The structures selected from the ensemble as the most representative should be identified, ie number 1 or number 12.
Response: Although OLDEARDO software by Kelley and Sutcliffe (1997 is still present in the list of NMR software utilities in the Expasy portal, we were not able to run the program on the server for the conformer ensembles of TAP and BPTI. Hence, in this study we have always used the first (#1) conformer of the NMR ensemble of TAP and BPTI for ASA and electrostatic calculations and for superposition of the inhibitor structure in the free and protease-bound form. Notably, conformer #1 of TAP and BPTI is superimposable to the “biological assembly” structure provided in PDB. In the Revised manuscript, the citation for OLDERADO has been removed and the selection of conformer #1 has been reported in the Materials and Methods.
- Software version is normally abbreviated as "ver" rather than "vs"
Response: We thank the Reviewer for bringing this point to our attention and, accordingly, in the Revised manuscript we have converted vs. into ver.
- Table S1 should state how the molar absorptivity numbers were obtained, ie which software or website was used.
Response: The molar absorptivity values of wild-type TAP and synthetic analogues substituted at position 3 were calculated using the absorptivity values at 280 nm of 1280 M-1·cm-1 for Tyr, 5690 M-1·cm-1 for Trp, and 120 M-1·cm-1 for Cystine, as reported by Gill, S. G., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326. Notably, p-amino-Phe, p-aminomethyl-Phe, p-guanido-Phe and piridyl-Ala do not significantly absorb at 280 nm. The molar absorptivity value of the TAP analogue Tyr1b-naphthyl-Ala was calculated using an absorptivity value for b-naphthyl-Ala at 280 nm of 5380 M-1·cm-1, according to Liang, J., Edlsbrunner, H., and Woodward, C. (1998) Protein Sci. 7, 1884-1897.
- Figure 2C might be enhanced by adding lines at the N and R retention times across the five HPLC traces to emphasise the difference in the major peaks. Alternatively, plotting the intensity of the N and R peaks against time should show a decrease in the R form and increase in the N.
Response: We thank the Reviewer for bringing this point to our attention and, accordingly, in the Revised manuscript we have added continuous and dashed lines to help the reader to better follow during time the decrease of the reduced “R” peak and the concomitant increase of the S-S oxidised native “N” peak.
- Table S2. Adding numbers to the first and last residues in the identified sequences would help convince the reader that the correct disulfide pairing had been identified.
Response: Done.
- Figure 4. Why is there undeuterated BPTI present in the fully deuterated mass spectrum when there was none present in the earlier mass spectra?
Response: We thank the Reviewer for bringing this point to our attention. The presence of trace amounts of the undeutereted species in the mass spectrum of the fully deuterated BPTI form is due to a carry over chromatographyc effect of the undeuterated samples (analysed first) on the chromatographic analysis of the fully deuterated BPTI (analysed second). In the Revised manuscript, the correct mass spectrum has been included.
- Figure 7 shows just one structure of free TAP from the NMR ensemble superimposed on the bound structure. How was this structure selected? Were the other members of the NMR ensemble equally distant? Do the other members of the NMR ensemble support a rigid or flexible N-terminus?
Response: As outlined above (major point n. 2; Reviewer #3), in the Revised manuscript, we have always used conformer #1 of TAP in the superposition with the structure of TAP complexd to fXa. Conformer #1 is identical to the “biological assembly” structure provided by PDB. The lack of significant NOE constrains in the N-terminal tripeptide is a proof of a loose/disordered structure.
Author Response File: Author Response.pdf
