Discovery of New VEGFR-2 Inhibitors: Design, Synthesis, Anti-Proliferative Evaluation, Docking, and MD Simulation Studies

Round 1

Reviewer 1 Report

This manuscript described, design, synthesis, antiproliferative evaluation, docking, and MD simulation studies of 4 new VEGFR-2 inhibitors. Based on the Biological testing in the manuscript, these 4 new pounds did not show promising result, compared to sorafenib data. Based on the manuscript, I have few questions listed here.

 

1.   Compound 6, compound 10, C13 NMRs should be adjusted the phase line, to make the peaks to be positive position.

 

2.   Compound 6 C13 NMR should show 0ppm to 200ppm, H NMR should show 0ppm to 14ppm.

 

3.   Compound 7 NMR in supporting information, 2.5ppm was DMSO solvent peak, 3.3ppm was the H2O residual, they should not do the integration.

 

4.   Compound 8 NMR in supporting information, integration should be integer

 

5.   Page 2, line 76 to line 79, authors described “sorafenib still 76 possesses some pharmacokinetic problems represented in either its bad water solubility or decreased oral bioavailability. The later problems potentiated the need for the discovery of new VEGFR-2 anti-angiogenic drugs.” But in the manuscript, the design part, did not show the logic, to solve the solubility and oral bioavailability problem. Manuscript better describe it, how authors designed the 4 compounds. Especially, the title was “Design and synthesis”.

 

6.   In vitro VEGFR-2 enzyme assay inhibition showed the new 4 compounds were not better than sorafenib. The compounds design was not good. How authors to justify this manuscript, what is the purpose to publish this manuscript?

 

 

Author Response

Reviewer 1

This manuscript described, design, synthesis, antiproliferative evaluation, docking, and MD simulation studies of 4 new VEGFR-2 inhibitors. Based on the Biological testing in the manuscript, these 4 new pounds did not show promising result, compared to sorafenib data. Based on the manuscript, I have few questions listed here.

 Thank you for your efforts and your valuable comments. All comments were considered in high interest and all changes were highlighted in the revised manuscript.

1. Compound 6, compound 10, C13 NMRs should be adjusted the phase line, to make the peaks to be positive position.

Response: For compounds 6 and 10 we carried out APT experiments

The phase was corrected also. In APT, CH3 and CH in one direction while CH2 and quaternary carbons in the other direction. In addition the proton data were clarified

Compound 6 C13 NMR should show 0ppm to 200ppm, H NMR should show 0ppm to 14ppm.

Response: DONE         

2. Compound 7 NMR in supporting information, 2.5ppm was DMSO solvent peak, 3.3ppm was the H2O residual, they should not do the integration.

Response: Done

4. Compound 8 NMR in supporting information, integration should be integer.

Response: DONE

3. Page 2, line 76 to line 79, authors described “sorafenib still 76 possesses some pharmacokinetic problems represented in either its bad water solubility or decreased oral bioavailability. The later problems potentiated the need for the discovery of new VEGFR-2 anti-angiogenic drugs.” But in the manuscript, the design part, did not show the logic, to solve the solubility and oral bioavailability problem. Manuscript better describe it, how authors designed the 4 compounds. Especially, the title was “Design and synthesis”.

Response: Thank you for this notice. This point was clarified as per requested.

6. In vitro VEGFR-2 enzyme assay inhibition showed the new 4 compounds were not better than sorafenib. The compounds design was not good. How authors to justify this manuscript, what is the purpose to publish this manuscript?

Response: You are right that the synthesized compounds have less activity than sorafenib. But these compounds have some advantages as: i) enhanced water solubility, ii) good immunomodulatory effect, iii) good binding mode in the active pocket of VEGFR-2, iv) stability in the active pocket for 100 ns, and the simple procedure for the chemical synthesis with good yield. All these advantages encourage us to publish these compounds to be lead molecules for further modifications and optimization.

Reviewer 2 Report

Ibrahim H. Eissa, Mohamed Elzahaby and al describe the design, synthesis, anti-proliferative evaluation, docking, and MD simulation studies of new VEGFR-2 inhibitors. Since the final compounds were not fully characterized (HRMS or elemental analyses are missing), the validity of the results are doubtful. Journal titles are missinig in many references.

I encourage the resubmission of this paper

Author Response

Ibrahim H. Eissa, Mohamed Elzahaby and al describe the design, synthesis, anti-proliferative evaluation, docking, and MD simulation studies of new VEGFR-2 inhibitors. Since the final compounds were not fully characterized (HRMS or elemental analyses are missing), the validity of the results are doubtful. Journal titles are missing in many references.

Response: Elemental analyses were carried out for the synthesized compound. All obtained values were in the accepted range. Also, all references were checked and corrected in the revised manuscript.

Reviewer 3 Report

 

Elkaeed et al. synthesized a series of nicotinamide-based VEGFR2 inhibitors and characterized their potency, and analyzed their cell cycle, apoptotic and immunomodulatory effects in a variety of assays. The best compound in their series was compound 6, which had similar but weaker potency than sorefenib in anti-proliferative assays. Subsequently, they performed computational studies to evaluate the binding interactions for two of the most potent compounds they have synthesized. In addition, the authors evaluated the pharmacokinetic profiles of their novel inhibitors in silico and predicted them to be safe in general. These inhibitors could be potentially interesting, and their inhibitory potency could be further developed. The conclusions are supported by the presented data. However, the manuscript needs to be re-worked extensively, in particular the overall language needs to be revised and some claims need to be substantiated or clarified. Besides, there are several major issues that the authors should address before the paper can be considered for publication.

 

Introduction: Overall language sounds awkward. Please consider revising the language.

 

Line 51: Should be "especially"

Line 52: Should be “in response” not “in a response”

Line 55: Should be “produce a safe and selective”

Line 73: Typological error. Should be “hinge”, not “hing”

Line 78: Should be “latter” instead of “later”

Line 83-85: Rephrase sentence. Perhaps “of various compounds containing benzoxazole,……scaffolds”

Line 87: Should be “four new”

Line 89: The phrase should be “hinge region”.

Line 125: Should be “latter”

Line 195: Could the authors substantiate how compound 6 “is bound tightly” to VEGFR from a computational model? Can the authors comment on the degree of tight binding?

Line 201: Should it be Ile886?

Line 208: Should it be Lys866?

Line 250: Remove the brackets.

Line 285: Please elaborate on the figure legends. They are too brief.

Line 287: The word “In silico” should be in italics

Line 316: Please reduce the number of significant figures

Line 324: Please accurately phrase the table title as “Predicted toxicity parameters and properties of the synthesized compounds”, or use a similar title.

 

Table 1: Is there a reason why median was reported, instead of mean? Please provide more information on IC50 values. The authors should indicate whether the ± values refer to s.e.m. or s.d.? For the in vitro IC50 determination against VEGFR2, please indicate IC50 in Table 1, not “VEGFR-2 protein concentration” which is vague. Does the in vitro IC50 values have an ± values (s.e.m. or s.d.)?

 

Please revise the Figure legends in general and provide more detailed figure captions.

Figure 6/7/8: Amino acid residue labels were not clear. Some were obscured behind the protein cartoon structures.

Figure 7: Please make the figure legend more informative and precise.

Figure 8: Please make the figure legend more informative and precise.

Figure 9: Please label the y-axis informatively. For example, y-axis in Figure 9b) should be RMSF (nm) and Figure 9e) should be “Number of hydrogen bonds”

Figure 10: Please ensure consistent angle or positioning of the protein structures in order for readers to have a clearer idea of the changes between 1 and 100 ns

 

Format of numbers: Please be consistent in reporting the IC50 values or percentages. Keep to 3 significant figures if possible.

 

Author Response

Reviewer 3

Elkaeed et al. synthesized a series of nicotinamide-based VEGFR2 inhibitors and characterized their potency, and analyzed their cell cycle, apoptotic and immunomodulatory effects in a variety of assays. The best compound in their series was compound 6, which had similar but weaker potency than sorefenib in anti-proliferative assays. Subsequently, they performed computational studies to evaluate the binding interactions for two of the most potent compounds they have synthesized. In addition, the authors evaluated the pharmacokinetic profiles of their novel inhibitors in silico and predicted them to be safe in general. These inhibitors could be potentially interesting, and their inhibitory potency could be further developed. The conclusions are supported by the presented data. However, the manuscript needs to be re-worked extensively, in particular the overall language needs to be revised and some claims need to be substantiated or clarified. Besides, there are several major issues that the authors should address before the paper can be considered for publication.

Thank you for your praise and valuable comments. I considered these comments with high interest. The comments and my response are summarized in the following points.

1. Introduction: Overall language sounds awkward. Please consider revising the language.

Response: Done

2. Line 51: Should be "especially"

Response: Done

3. Line 52: Should be “in response” not “in a response”

Response: Done

4. Line 55: Should be “produce a safe and selective”

Response: Done

5. Line 73: Typological error. Should be “hinge”, not “hing”

Response: Done

6. Line 78: Should be “latter” instead of “later”

Response: Done

7. Line 83-85: Rephrase sentence. Perhaps “of various compounds containing benzoxazole,……scaffolds”

Response: Done

8. Line 87: Should be “four new”

Response: Done

9. Line 89: The phrase should be “hinge region”.

Response: Done

10. Line 125: Should be “latter”

Response: Done

11. Line 195: Could the authors substantiate how compound 6 “is bound tightly” to VEGFR from a computational model? Can the authors comment on the degree of tight binding?

Response: Clarified         

12. Line 201: Should it be Ile886?

Response: Yes, it was corrected.

13. Line 208: Should it be Lys866?

Response: it is Ile886 and it was corrected.

14. Line 250: Remove the brackets.

Response: Done

15. Line 285: Please elaborate on the figure legends. They are too brief.

Response: more clarifications were added into the captions of the figures

Line 287: The word “In silico” should be in italics

Response: Done

16. Line 316: Please reduce the number of significant figures

Response: Done

17. Line 324: Please accurately phrase the table title as “Predicted toxicity parameters and properties of the synthesized compounds”, or use a similar title.

Response: Done

18. Table 1: Is there a reason why median was reported, instead of mean? Please provide more information on IC50 values. The authors should indicate whether the ± values refer to s.e.m. or s.d.? For the in vitro IC50 determination against VEGFR2, please indicate IC50 in Table 1, not “VEGFR-2 protein concentration” which is vague. Does the in vitro IC50 values have an ± values (s.e.m. or s.d.)?

Response: this typo was corrected. We used the mean and not median. In addition, we measured the IC50 of VEGFR-2 inhibition and not the protein concentration. This point was adjusted in the revised manuscript. The SD for VEGFR-2 inhibition was added.

19. Please revise the Figure legends in general and provide more detailed figure captions.

Response: Done

20. Figure 6/7/8: Amino acid residue labels were not clear. Some were obscured behind the protein cartoon structures.

Response: Done  

21. Figure 7: Please make the figure legend more informative and precise.

Response: Done

22. Figure 9: Please label the y-axis informatively. For example, y-axis in Figure 9b) should be RMSF (nm) and Figure 9e) should be “Number of hydrogen bonds”

Response: Done

23. Figure 10: Please ensure consistent angle or positioning of the protein structures in order for readers to have a clearer idea of the changes between 1 and 100 ns

Response: Both figures A and B were captured from the same angle.

The difference is due to the incidence of conformational changes in the VEGFR-2 enzyme

24. Format of numbers: Please be consistent in reporting the IC50 values or percentages. Keep to 3 significant figures if possible.

Response: Done  

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

My biggest concern was about the their molecular design logic, how to justify their idea.

Now, the authors added extra text and explaining. The revised manuscript is much clearer and easier to understand, now the manuscript is ready for publication. Good job.

 

Author Response

The authors would like to thank the reviewer for his valuable comments and questions that will make our manuscript much better.

Extensive editing for English language has been done

Reviewer 2 Report

The manuscript has been revised according to my remarks.

I thank the authors.

Author Response

The authors would like to thank the reviewer for his valuable comments and questions that will make our manuscript much better.

Extensive editing for English language has been done

Reviewer 3 Report

 

Line 50: many efforts were made to generate

Line 125: turquoise not Turquoise

Line 132: incorporation
Line 133: spacing between the and synthesized words

Line 193: Flow cytometric analysis of the cell cycle in HCT-116 after treatment with compound 6

Line 206: after treatment with 9.3 μM of compound 6 for 48 h.

Line 208: No comma

Line 236: Rephrase for clarity

Line 265: binding mode similar to that of the benzimidazole-urea inhibitor

Line 272: many HIs? Please rephrase.

Line 279: include a comma

Line 324: please correct typological error. Compounds not ompounds

Line 326: MD not M D

Line 439: confirms

Line 450: bound

Line 465: spacing needed between words are and gas. Two types

Line 517: incorrect use of the word controversy

Line 669: typological error

Line 737: the MTT procedure

 

Figure 10: It would be better to show superimposed structures (color differently), so that the conformational changes can be more clearly visualized. Are the conformational changes in the current figure highlighted in green?

 

Please ensure consistency throughout manuscript. Table X. Not Table X:

 

Italicize all mentions of in vitro

 

In the Experimental section, “In-depth discussions of….have been held in Supplementary data” is awkward sounding. Please edit.

 

The manuscript still contains many typological and formatting errors. I suggest the authors edit the manuscript more thoroughly.

Author Response

The authors would like to thank the Editor for his valuable help, kind guidance and professional work.

First, the authors would like to thank the reviewer for his valuable comments and questions that will make our manuscript much better.

All comments were considered and answered positively

In detail,

1. Line 50: many efforts were made to generate

Response: Done

2. Line 125: turquoise not Turquoise

Response: Done

3. Line 132: incorporation

Response: Done

4. Line 133: spacing between the and synthesized words

Response: Done

5. Line 193: Flow cytometric analysis of the cell cycle in HCT-116 after treatment with compound 6

Response: Done

6. Line 206: after treatment with 9.3 μM of compound 6for 48 h.

Response: Done

7. Line 208: No comma

Response: Done

8. Line 236: Rephrase for clarity

Response: Done

9. Line 265: binding mode similar to that of the benzimidazole-urea inhibitor

Response: Done

10. Line 272: many HIs? Please rephrase.

Response: Done

11. Line 279: include a comma

Response: Done

12. Line 324: please correct typological error. Compounds not ompounds

Response: Done

13. Line 326: MD not M D

Response: Done

14. Line 439: confirms

Response: Done

15. Line 450: bound

Response: Done

16. Line 465: spacing needed between words are and gas. Two types

Response: Done

17. Line 517: incorrect use of the word controversy

Response: Done

18. Line 669: typological error

Response: Done

19. Line 737: the MTT procedure

Response: Done

20. Figure 10: It would be better to show superimposed structures (color differently), so that the conformational changes can be more clearly visualized. Are the conformational changes in the current figure highlighted in green?

Response: Figure 10 is a Ribbon diagrams compound 6-VEGFR-2 complex at 1st and last ns.  It’s a schematic representation to prove that compound 6 remained bonded correctly in the active pocked till the end of study and to illustrate the overall path and conformational changes of the compound 6-VEGFR-2 complex over the 100 ns in 3D.

We tried to superimpose both structures but the resulted figure was so confusing 

21. Please ensure consistency throughout manuscript. Table X. Not Table X:

 Response: Done

22. Italicize all mentions of in vitro

 Response: Done

23. In the Experimental section, “In-depth discussions of….have been held in Supplementary data” is awkward sounding. Please edit.

 Response: Done

24. The manuscript still contains many typological and formatting errors. I suggest the authors edit the manuscript more thoroughly.

Response: Done