In Vitro and In Silico Evaluation of Cholinesterase Inhibition by Alkaloids Obtained from Branches of Abuta panurensis Eichler
, Anton Cherednichenko 4,5, Ingrity Suelen Costa Sá 1, LÃlian Macedo Bastos 1, Felipe Moura Araújo da Silva 1, Rita de Cássia Saraiva Nunomura 1,6 and Andriy Grafov 7,*Round 1
Reviewer 1 Report
Authors have reported in vitro and in silico evaluation of cholinesterase inhibition by five different alkaloids obtained from the branches of Abuta panurensis. Out of five, three alkaloids were isolated for the first time from A. panurensis. The isolated compounds were characterized for their structural elucidation and evaluated for the in vitro and in vivo cholinesterase inhibition. This work is carried out systematically and scientifically sound. There are some issues that the authors should address to improve their manuscript before its publication. My main concerns are as follows:
Abstract: Authors are advised to include some quantitative information regarding the characterization of isolated compounds in order to enhance the readability of the article.
Abbreviations: Authors are advised to define each abbreviation in its first appearance in abstract, manuscript, Figure and Table.
Supplementary materials: please write the caption for each supplementary figure as required by the journal.
Several subsections such as Institutional review board statement, Informed consent statement, Data availability statement, and Sample availability are missing. Authors are advised to download the latest template and include all missing information.
The resolution of all figures is low. Please improve it.
Author Response
Dear Editor,
We thank the Reviewer 1 for the comments and additional suggestions, we took all of them into account in the revised version of the manuscript.
Abstract: Authors are advised to include some quantitative information regarding the characterization of isolated compounds in order to enhance the readability of the article.
Quantitative information on the isolated compounds was added to the abstract.
Abbreviations: Authors are advised to define each abbreviation in its first appearance in abstract, manuscript, Figure and Table.
The authors checked the manuscript and defined all abbreviations in abstract, manuscript, figures, and Table.
Supplementary materials: please write the caption for each supplementary figure as required by the journal.
The authors wrote captions for each supplementary material and made a table of contents there for better readability.
Several subsections such as Institutional review board statement, Informed consent statement, Data availability statement, and Sample availability are missing. Authors are advised to download the latest template and include all missing information.
We added the required information and updated the template.
The resolution of all figures is low. Please improve it.
The majority of figures were produced in vector format, they were transformed into raster ones of high quality.
Reviewer 2 Report
This is about a very interesting work on the study of potential pharmacological properties, based on the inhibition of aceticolinesterase and butyrylcholinesterase, of five alkaloids isolated from Abuta panurensis.
Table 1 must include the meaning of the acronyms MM/GBSA.
The authors mention that the cholinesterase inhibition assays were replicates of 3 measurements, however, in Table 1 authors do not mention the standard deviation. Authors must include it.
What was the reason for putting “n/a” (not applicable) in Table 1 for the IC50 measurement of N-trans-feruloyltyramine? If the compound did not present activity, authors must indicate: “without activity”.
The authors should discuss in more detail why neostigmine has the highest affinity energy value and the lowest IC50.
With respect to Table 1, why was a positive control not used for the BChE inhibition assay?
In Figure 2, the authors should indicate the position of the amino acids Phe330, Glu199 and Trp84.
The most promising compound seems to be compound 4, why do the authors conclude that compounds 1 to 4 are promising? What is your main argument for concluding that all 4 compounds are promising?
In Figures 3 and 5, authors do not indicate what the pink arrows mean (lines 291 and 384).
In line 102 “Table S1” is mentioned. However, I did not find such a table in the article or in the supplementary material. It's a mistake? Should it say Figure 1? Same for line 112 (says Table S2), line 121 (says Table 3), line 132 (says Table S4), and line 140 (says Table 5).
Author Response
Dear Editor,
We thank the Reviewer 2 for the comments and additional suggestions, we took all of them into account in the revised version of the manuscript.
Table 1 must include the meaning of the acronyms MM/GBSA.
The acronym means
MM/GBSA = molecular mechanics/generalized Born and surface area continuum solvation method.
The explanation was inserted into the Table caption.
The authors mention that the cholinesterase inhibition assays were replicates of 3 measurements, however, in Table 1 authors do not mention the standard deviation. Authors must include it.
Standard deviation was added to the Table 1.
What was the reason for putting “n/a” (not applicable) in Table 1 for the IC50 measurement of N-trans-feruloyltyramine? If the compound did not present activity, authors must indicate: “without activity”.
We agree that putting “n/a” (not applicable) is not a correct phrase. We meant that the compound showed the inhibition percentage of the enzyme below 20%, which is considered as inactive. The description was changed to “not active”.
The authors should discuss in more detail why neostigmine has the highest affinity energy value and the lowest IC50.
The biases of docking protocol for higher molecular size and lipophilicity due to the increase in van der Waals term are well known. Additionally, the purpose of molecular docking is a quick classification of ligands by their affinity to a receptor, in contrast to more sophisticated methods, such as ab initio or free energy perturbation calculations, aimed at more exact description of all bonding terms. Hence, it is desirable to compare molecules of similar size to be more accurate. The neostigmine is the smallest molecule in the range of compounds under investigation. Moreover, one has to bear in mind that both molecular docking and molecular mechanics are predictive modelling methods, which always have to be pharmacologically validated, e.g., by IC50 determination.
Therefore, the authors consider that the issue is discussed in sufficient detail in the manuscript and there is no necessity to provide more discussion that could be speculative.
With respect to Table 1, why was a positive control not used for the BChE inhibition assay?
The data was added to the Table 1.
In Figure 2, the authors should indicate the position of the amino acids Phe330, Glu199 and Trp84.
The Figure 2 was revised and positions of the three amino acids were indicated.
The most promising compound seems to be compound 4, why do the authors conclude that compounds 1 to 4 are promising? What is your main argument for concluding that all 4 compounds are promising?
The values of IC50 determined for the alkaloids under investigation together with a comparison of those with the IC50 values reported in the literature for the same groups of alkaloids (bisbenzyltetrahydroisoquinoline, proaporphine, protoberberine, etc.) were used as the assessment criterion to consider whether a compound is promising or not. Two of the alkaloids under investigation demonstrated the IC50 values close to the lower limit reported for the group as e.g., lindoldhamine 39.38 (literature range for bisbenzyltetrahydroisoquinoline alkaloids 34.6 – 78.22) or even slightly better as e.g., palmatine 35.25 (literature range for protoberberine alkaloids 36.6 – 141.8). The N-methylmaytenine (4) revealed the lowest IC50 value among the compounds isolated in this study and there are no data reported on cholinesterase enzymes inhibition by polyamine alkaloids. Therefore, the compound 4 was considered the most promising.
The corresponding sentence in the manuscript was revised as follows:
”Taking into a consideration the values of IC50 determined for the alkaloids under investigation together with a comparison of those with the IC50 values reported in the literature for the same groups of alkaloids, the compound 4 appears to be the most promising, since it showed the lowest IC50 value” (lines 372 -375)
In Figures 3 and 5, authors do not indicate what the pink arrows mean (lines 291 and 384).
Thank you for the comment it was our inaccuracy. The pink arrow means a hydrogen bond, the explanation is included into the revised legends of the figures.
In line 102 “Table S1” is mentioned. However, I did not find such a table in the article or in the supplementary material. It's a mistake? Should it say Figure 1? Same for line 112 (says Table S2), line 121 (says Table 3), line 132 (says Table S4), and line 140 (says Table 5).
Thank you for the comment it was our inaccuracy. The tables are now included in the Supplementary material, we also provided a table of contents to the SM to improve the readability.
