Colchicine: Isolation, LC–MS QTof Screening, and Anticancer Activity Study of Gloriosa superba Seeds

Round 1

Reviewer 1 Report

Docking was performed on a static protein structure ignoring any protein flexibility essential for binding. Numerous studies demonstrated the significance of protein flexibility of ligand binding. It is likely the correct binding mode of Colchicine is not identified. A nanosecond MD simulations might provide a different binding mode. Or if available taking another protein structures (with different resolution) and docking the same ligand and check whether the results are same. It is also reported that right protonation state on the ligands and the right charges on the metal are key to relatively correct docking pose. It seems the authors missed to consider it. http://autodock.scripps.edu/news/successfully-docking-to-proteins-with-metal-ions

Minor

The modeled protein structure as well as the docked structures may be submitted to the open access repository (e.g. Figshare). It will be useful to readers

Line 166 It is unclear to readers why the authors used two UniprotID. Full length protein is unavailable? Perhaps an explanation will help

why the authors used only chains A and B and why chains C and D were removed? explanation might help

How many clusters were observed in AutoDock? Out of those how many were different? Discussing about the docking clusters instead of docking energy  in the results will be useful.

Also discuss about the protein structure quality of the modeled protein

Figure 4. legends about the small molecule structures will be useful to readers. May be about the differences.

Author Response

1).The modeled protein structure as well as the docked structures may be submitted to the open access repository (e.g. Figshare). It will be useful to readers

- We will certainly deposit the molecular docking work in Figshare open access repository once the manuscript is accepted for publication.

2). Line166 it is unclear to readers why the authors used two uniprotID. Full length protein is unavailable? Perhaps an explanation will help.

- As the full length protein is unavailable and the DAMA-colchicine prefers αβ-interface [26,38], human tubulin protein sequences were obtained from UniProt, 2013. Human tubulin α-chain (TUBA1C) (UniProt ID: Q9BQE3) was chosen as reference sequence for human α-tubulin, and βI isoform (TUBB) (UniProt ID: P07437) was chosen for human β-tubulin (47,37).

3). Why the authors used only chains A and B and why chains C and D were removed? Explanation might help.

- The chains C and D were removed from the protein structure just to avoid the complexity.

4). How many clusters were observed in autodock? Out of those how many were different? Discussing about the docking clusters instead of docking energy in the results will be useful.

- The mean free energy of binding of DAMA-colchicine with 1SA0 was -7.12 kCal/mol. There were 24 clusters, and the largest cluster consisted of 12 conformations; lowest binding energy in the cluster was -7.53 kCal/mol and the lowest binding energy among all was -8.66 kCal/mol. The mean free energy of binding for DAMA-colchicine with the modelled human tubulin was comparable to that of bovine tubulin, -7.17 kCal/mol. There were 7 clusters, and the largest cluster consisted of 71 conformations; lowest binding energy in the largest cluster was -7.84 kCal/mol, and the lowest binding energy among all was -9.38 kCal/mol. The mean free energy of binding for colchicine docked with human tubulin was -7.71 kCal/mol; there were 7 clusters with the largest cluster showing 73 conformations, and the lowest binding energy in the cluster and among all was -8.49 kCal/mol.

5). Also discuss about the protein structure quality of the modeled protein.

- 93.41% of the residues had an averaged 3D-1D score ≥0.2 in VERIFY3D check of the modelled human tubulin, whereas the overall average score of G-value was -1.14 in PROCHECK.

6). Figure 4. legends about the small molecule structures will be useful to readers. May be about the differences.

- Figure 4. Structures of DAMA-colchicine (C₂₂H₂₅NO₆S, MW: 431.503g/mol, 2-sulfanyl-N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-6,7-dihydro-5H-benzo[a]heptalen-7-yl]acetamide) and Colchicine (C₂₂H₂₅NO₆, MW: 399.443g/mol, 2-Mercapto-n-[1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydro-benzo[a]heptalen-7-yl]acetamide)

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have described novel method for extraction and purification of colchicine extract from Gloriosa superba Seeds using super critical fluid extraction. According to the data provided extraction rate depends on temperature and CO2 pressure. It is also stated that 3% of water is used as co-solvent for the extraction but there are no any data how 3% water quantity has been defined. This should be clearly defined (experimental part or reference cited) before article can be considered for publication.

Author Response

Response to Reviewer 2 Comments

Comment 1. The authors have described novel method for extraction and purification of colchicine extract from Gloriosa superba Seeds using super critical fluid extraction. According to the data provided extraction rate depends on temperature and CO2 pressure. It is also stated that 3% of water is used as co-solvent for the extraction but there are no any data how 3% water quantity has been defined. This should be clearly defined (experimental part or reference cited) before article can be considered for publication.

Response 1:Water is used as co-solvent for extraction just to enhance the extraction efficiency and the water was removed from the extract, as it was moved to the separator-2 by applying low pressure. However 3% water quantity is based on our experimental trials and on the basis of our experience.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors replied all the minor comments. However, I couldn't find any reply for the major comment. 

Major comment

Docking was performed on a static protein structure ignoring any protein flexibility essential for binding. Numerous studies demonstrated the significance of protein flexibility of ligand binding. It is likely the correct binding mode of Colchicine is not identified. A nanosecond MD simulations might provide a different binding mode. Or if available taking another protein structures (with different resolution) and docking the same ligand and check whether the results are same. It is also reported that right protonation state on the ligands and the right charges on the metal are key to relatively correct docking pose. It seems the authors missed to consider it. http://autodock.scripps.edu/news/successfully-docking-to-proteins-with-metal-ions

Author Response

Dear Sir

As per the comments from academic editor dated 19 July 2019, we have made necessary changes on the manuscript.