Synthesis, Molecular Docking, and Dynamic Simulation Targeting Main Protease (Mpro) of New, Thiazole Clubbed Pyridine Scaffolds as Potential COVID-19 Inhibitors
, Amr S. Abouzied 2,3, Abdulwahab Alamri 4, Sirajudheen Anwar 4, Mukhtar Ansari 5, Ibrahim Khadra 6, Yasser H. Zaki 7 and Sobhi M. Gomha 8,9,*Round 1
Reviewer 1 Report
In this manuscript, a series of N-aminothiazole-hydrazineethyl-pyridines were synthesized and docking and dynamic simulation were performed to predict 8a as a potent SARS-CoV-2 main protease inhibitor.
1. There are many minor writing mistakes in this manuscript. In Figure 1, what is Kt? Should it be Ki? There should be a space between the numbers and the units. Cov should be CoV as in the text. What is “urboni”? In the text, the first mentioned term should be given with its abbreviation. The abbreviation of Coronavirus is CoV, and please also add the full names of SARS-CoV, MERS-CoV etc. The abbreviations should be consistent through the whole text. I can see “COVID-19” and “CoV-19”. Please use COVID-9 as defined by WHO. Its full name is coronavirus disease 2019. The first mentioned main protease should be abbreviated as (Mpro). In Figure 1, some inhibitors have IC50 at mM range. Please check again because mM IC50s are not potent at all and do not deserve to be listed.
2. Are the compounds synthesized new compounds? If yes, the assignments of the NMR (both 1H and 13C) spectra into the chemical structures should be given as supplementary information. I can see the chemical shifts data, but not the spectra. If not, please also compare the chemical shifts to confirm the identity and specify the references which prepared the compounds.
3. I only can see the molecular docking of the synthesized compounds into Mpro, but the data (IC50 or Ki) should be measured and reported here, using the commercially available assay kit or by collaboration with others. With the experimentally determined data, docking can be used to explain the structure-activity relationship of the compounds. Otherwise, docking is just a prediction of inhibition and whether 8a really can inhibit Mpro is not sure.
Author Response
Response to Reviewer 1 Comments
Dear reviewer, many thanks for your time spending and efforts to review the manuscript.
Point 1: There are many minor writing mistakes in this manuscript. In Figure 1, what is Kt? Should it be Ki? There should be a space between the numbers and the units. Cov should be CoV as in the text.
What is “urboni”?
In the text, the first mentioned term should be given with its abbreviation. The abbreviation of Coronavirus is CoV, and please also add the full names of SARS-CoV, MERS-CoV etc. The abbreviations should be consistent through the whole text. I can see “COVID-19” and “CoV-19”. Please use COVID-9 as defined by WHO. Its full name is coronavirus disease 2019. The first mentioned main protease should be abbreviated as (Mpro).
In Figure 1, some inhibitors have IC50 at mM range. Please check again because mM IC50s are not potent at all and do not deserve to be listed.
Response 1: Kt modified to Ki; a space between the numbers and the units was added; Cov modified to CoV; SARS CoV urboni inhibitors modified to SARS CoV Urbani inhibitors;
Abbreviations were added as: Corona viruses (CoV); Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2); Middle East Respiratory Syndrome CoronaVirus (MERS-CoV); The World Health Organization (WHO); Coronavirus disease 2019 (CoV-19); SARS-CoV-2 main protease (Mpro).
All the mentioned IC50 at mM range were checked and they are correct as reported
Point 2: Are the compounds synthesized new compounds? If yes, the assignments of the NMR (both 1H and 13C) spectra into the chemical structures should be given as supplementary information. I can see the chemical shifts data, but not the spectra. If not, please also compare the chemical shifts to confirm the identity and specify the references which prepared the compounds.
Response 2: 1H and 13C NMR spectra were added into supporting information file (see Supporting Information)
Point 3: I only can see the molecular docking of the synthesized compounds into Mpro, but the data (IC50 or Ki) should be measured and reported here, using the commercially available assay kit or by collaboration with others. With the experimentally determined data, docking can be used to explain the structure-activity relationship of the compounds. Otherwise, docking is just a prediction of inhibition and whether 8a really can inhibit Mpro is not sure.
Response 3:
In this study, we designed, synthesized, and characterized a novel series of N-aminothiazole-hydrazineethyl-pyridines. FTIR, MS, NMR, and elemental studies were used to identify new products, With the aim to investigate the inhibition mechanism on SARS-CoV-2 main protease which suggests pathway for studies of potential compounds development, molecular docking studies have been carried out to predict bound conformations and free energies of binding for synthesized compounds (ligands) and targets (6LU7) to find out the most promising candidate against SARS-CoV-2. Docking is widely used for the study of biomolecular interactions and mechanisms, and it is applied to structure-based drug design. The methods are fast enough to allow virtual screening of ligand libraries containing tens of thousands of compounds and yield information that is not easy to obtain in laboratory analysis, and, furthermore, is typically (much) less costly than by experiment. The physicochemical parameters, toxicity assessment and molecular docking approaches showed that compound (8a) was not toxic, did not violate Lipinski’s rule of 5, can fit well with the binding site of target protease through interactions with essential amino acids, hence inhibiting the replication function of the virus. Furthermore, compound (8a) complex with protein showed stability during the 100 ns trajectory of molecular dynamic simulation analysis. The predicted findings could be a helpful suggestion for us and other scientists to conduct further confirmation via in vitro experimental studies. Nevertheless, the preliminary results of the present study may pave the way to develop more potent agents against SARS-CoV-2 in the near future.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The present study is devoted to the synthesis of new potential inhibitors of the SARS-CoV-2 main protease. Verification of their antiviral activity was carried out by theoretical methods such as molecular docking and molecular dynamics.
The study appears to be quite logical and consistent. However, there are a few things to note:
1. Schemes 1-3 don't indicate reaction conditions.
2. Full assignment of 1H and 13C NMR peaks should be given in Supplementary Materials. Also, to convincingly prove the structure of the newly synthesized compounds, it is necessary to present the data of 2D NMR correlations for them.
3. Since crystals have been obtained for some of the synthesized compounds (13a, 14a, 14b, 14c), it is necessary to present their crystal structures.
4. Why was an obsolete modification of the main protease (6LU7) dating back to 2020 chosen as a target?
5. Figures 4(14c) and 6 are stretched horizontally.
6. Figures 3 (bottom), 4, 6 (bottom), 9 are very vague, need to be redrawn.
7. “COVID-19 main protease”, “COVID-19 receptor” in the last paragraph of page 9, the first paragraph of page 10 and the title of Table 2 is a typo. It will be correct: “SARS CoV-2 main protease”.
Author Response
Response to Reviewer 2 Comments
Dear reviewer, many thanks for your time spending and efforts to review the manuscript.
Point 1: Schemes 1-3 don't indicate reaction conditions.
Response 1: Reaction conditions were added into schemes1-3 (see revised manuscript)
Point 2: Full assignment of 1H and 13C NMR peaks should be given in Supplementary Materials. Also, to convincingly prove the structure of the newly synthesized compounds, it is necessary to present the data of 2D NMR correlations for them.
Response 2: 1H and 13C NMR spectra were added into supporting information file (see Supporting Information). 2D NMR correlations data are not available. Also these compounds are similar to several previously reported compounds
Med Chem. 2022;18(10):1100-1108. doi: 10.2174/1573406418666220413095854.
Molecules. 2022 Jul; 27(14): 4639. doi: 10.3390/molecules27144639
Point 3: Since crystals have been obtained for some of the synthesized compounds (13a, 14a, 14b, 14c), it is necessary to present their crystal structures.
Response 3: We tried several times to obtain single crystal for the synthesized compounds but we couldn’t
Point 4: Why was an obsolete modification of the main protease (6LU7) dating back to 2020 chosen as a target?
Response 4:
We thank the reviewer for raising this question. The selection of (6LU7) because it is still recently one (2020) and it has a specific binding site occupied by (N3) as inhibitor which is similar to the newly synthesized ligands based on target similarity predication (https://pubchem.ncbi.nlm.nih.gov/ ). Moreover, (6LU7) has a high resolution (2.16 Å) comparing with most recently ones as shown below,
ID Method Resolution Released
7P35 X-ray 2.25 Å 2021
7UJ9 X-ray 2.25 Å 2022
7Z0P X-ray 2.52 Å 2022
Reference “Chemoinformatics Approaches to Virtual Screening”, Editors: Alexandre Varnek, Alex Tropsha; https://doi.org/10.1039/9781847558879
Point 5: Figures 4 (14c) and 6 are stretched horizontally.
Response 5: Changed with high resolution ones (see revised manuscript)
Point 6: Figures 3 (bottom), 4, 6 (bottom), 9 are very vague, need to be redrawn.
Response 6: Changed with clearer and high-resolution ones (see revised manuscript)
Point 7: “COVID-19 main protease”, “COVID-19 receptor” in the last paragraph of page 9, the first paragraph of page 10 and the title of Table 2 is a typo. It will be correct: “SARS CoV-2 main protease”.
Response 7: Done (see revised manuscript)
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Authors did not address the assay part of their inhibitors to improve the impact of this article, which is very simple to do. I still do not know whether the synthesized compounds are really inhibitors of the main protease.
Author Response
Response: we added the following paragraph in the conclusion (see revised manuscript)
The physicochemical parameters, toxicity assessment and molecular docking approaches showed that compound (8a) was not toxic, did not violate Lipinski’s rule of 5, can fit well with the binding site of SARS-CoV-2 target protease through interactions with essential amino acids, hence inhibiting the replication function of the virus. Furthermore, compound (8a) complex with SARS-CoV-2 target protease showed stability during the 100 ns trajectory of molecular dynamic simulation analysis. The predicted findings could be a helpful suggestion for us and other scientists to conduct further confirmation via in vitro experimental studies. Nevertheless, the preliminary results of the present study may pave the way to develop more potent agents against SARS-CoV-2 in the near future.
Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
Authors did not add the assay data to greatly increase the impact of their paper, but added sentences in conclusion that could justify their contribution and future work.
