Neuroprotective Activities of New Monoterpenoid Indole Alkaloid from Nauclea officinalis

Round 1

Reviewer 1 Report

Comments:

1. In the structure elucidation section, the authors used rings A, B, C … - unclear, where ring names are defined?

2. Which kind of AChE and BuChE enzyme was used to measure the inhibitory activity of compound?

3. As mixed-type inhibitor on AChE and BuChE, nauclediol could bind in free enzymes with a K_i value and could also inhibit substrate-bound enzymes with a K_is value, which were determined by secondary plots. Please discuss in your revised manuscript.

4. The x- and y- axis in Fig. 4 and 5 is difficult to read. Please revise.

5.Lines 263 and 272: please specify the loop (numbers).

6. Abbreviation for TcAChE and hBChE is missed in the main text.

7. English should be reviewed by a specialist.

8. Typing error.

Line 67: “of the plant”

Line 110: “were conducted”

Line 206: “two diol groups”

Line 274: “In addition”

Author Response

1. In the structure elucidation section, the authors used rings A, B, C … - unclear, where ring names are defined?

Thank you for notifying the missing information. The labels for ring A, B and C have been added in Figure 1.

2. Which kind of AChE and BuChE enzyme was used to measure the inhibitory activity of compound?

Thank you for the comment. The AChE is TcAChE from Torpedo californica. While BuChE from homo sapiens. It has been included in the text. 

3. As mixed-type inhibitor on AChE and BuChE, nauclediol could bind in free enzymes with a Ki value and could also inhibit substrate-bound enzymes with a Kis value, which were determined by secondary plots. Please discuss in your revised manuscript.

Thank you for the suggestion and it is an excellent point to be included. The first and secondary plots of the Ki value of nauclediol have been added in the supplementary. The discussion for the kinetic part has been added. 

4. The x- and y- axis in Fig. 4 and 5 is difficult to read. Please revise.

The Fig 4 and 5 have been amended.

5.Lines 263 and 272: please specify the loop (numbers).

The elaboration of details about binding interactions between nauclediol and TcAChE in line 263 (now become line 282) has been done in lines 283-290.

 The elaboration of details about binding interactions between nauclediol and hBChE in line 272 (now become line 291) has been done in lines 292-294.

6. Abbreviation for TcAChE and hBChE is missed in the main text.

The abbreviation for TcAChE and hBChE have been added.

7. English should be reviewed by a specialist

Thank you for the suggestion. The grammatical mistakes, typos, and hanging sentences have been amended. 

8.Typing error.

Line 67: “of the plant”

Line 110: “were conducted”

Line 206: “two diol groups”

Line 274: “In addition”

All the stated typing errors have been corrected as highlighted in yellow.

Reviewer 2 Report

The manuscript entitled “Neuroprotective activities of new monoterpenoid indole alkaloid from Nauclea officinalis” is devoted to isolation, structure elucidation and bioactivity studying of a new compound, nauclediol. The experiments are well designed and discussed, but the text contains some inaccuracies in the results discussion. The comments are provided in the attached file of the manuscript. 

Also, there are some questions and suggestions:

1. The COSY correlations of the protons of the aromatic ring A are not provided in the Figure 2 and not mentioned in the text. Were they observed? If not, how do authors can explain their absence?

2. Nauclediol expose promissing cholinesterase inhibitory activity,  it would be interesting to explore the series of these biogenetically related compounds (indole alkaloids) isolated from Nauclea officinalis earlier for their bioactivity to analyze structure-activity relationships. 

Generally, the manuscript can be published after minor revision of result discussion and English editing.

Comments for author File: Comments.pdf

Author Response

1. The COSY correlations of the protons of the aromatic ring A are not provided in the Figure 2 and not mentioned in the text. Were they observed? If not, how do authors can explain their absence?

Thank you for notifying the missing information.

A sentence describing the COSY correlations of the aromatic protons in ring A has been added in sub-section 3.1. Structural Elucidation of Nauclediol as highlighted in yellow. The COSY correlations also have been added in Figure 2

2. Nauclediol expose promissing cholinesterase inhibitory activity,  it would be interesting to explore the series of these biogenetically related compounds (indole alkaloids) isolated from Nauclea officinalis earlier for their bioactivity to analyze structure-activity relationships. 

Generally, the manuscript can be published after minor revision of result discussion and English editing

Thank you for the suggestion.

 A brief structure-activity relationships studies on the indole alkaloids in inhibiting cholinesterase has been discussed in sub-section 3.2. Cholinesterase Inhibition of Nauclediol.

Reviewer 3 Report

In this article, a new monoterpenoid indole alkaloid, nauclediol was isolated from the bark of Nauclea officinalis. Then the compound was identified through extensive spectroscopic analysis. This compound displayed cholinesterase inhibitory activities towards AChE and BChE. Besides, neuroprotective potential of the new found compound was studied in a dose-dependent manner.

 1.   In part “2.2. Plant Materials”. The plant material must be identified by a relevant experts.

2.   Line 82-84. This paragraph seems more like the result than the method.

3.   Note the formatting. Table 1 has a large font.

4.   In the molecular docking of nauclediol, the scores should be provided when binding with the two enzymes.

Author Response

1.   In part “2.2. Plant Materials”. The plant material must be identified by a relevant experts.

The sentence “The botanical identification was conducted by the botanist, Mr. Teo Leong Eng.” has been added in sub-section 2.2. Plant Materials as highlighted in yellow.

 

2.   Line 82-84. This paragraph seems more like the result than the method.

The paragraph has been removed from sub-section 2.3. Extraction, Isolation, and Purification of Compound since all the data have been written in sub-section 3.1. Structural Elucidation of Nauclediol.

3.   Note the formatting. Table 1 has a large font.

The font size of Table 1 has been corrected.

4.   In the molecular docking of nauclediol, the scores should be provided when binding with the two enzymes.

The number of conformations in the cluster for the molecular docking of nauclediol on both enzymes has been added in Table 2.

Reviewer 4 Report

We found the manuscript relevant to the field, containing interesting information about indole alkaloid molecules targeting AChE and BChE inhibition.

The introduction part covers both old and new references concisely and has a perfect integration of the main aspects of the theme.

The cited core references are recent and appropriate to the discussion.

This article is well written, with a good organization of the contents and a clear and pertinent methodology, particularly the statistical analysis verifying the toxicity on SH-SY5Y cells and neuroinflammation in BV2 cells, though there are some concerns regarding enzyme kinetic methodology which are discussed below.

Methodologic concerns were raised on the appropriate statistical tools needed to support the conclusion about the inhibition mechanism of the new molecule toward cholinesterases. The experimental design used (linearization of the MM equation hyperbole) lacks adequate depth and precision to test the hypothesis about the enzyme kinetic mechanism (it´s very risky to make conclusions based on 3 data points on Dixon plots!). The reciprocal rearrangement of the MM equation, to give a linear dependence of 1/v upon 1/[S] (and the other linearizations), has commonly been used. It is useful for displaying the differences between the kinetic behavior of the different inhibitor types, but it is an extremely inaccurate way of determining kinetic parameters for which a direct, nonlinear regression fit to the Michaelis–Menten equation is to be preferred to any of the linear rearrangements.

The in vitro methodology is the core issue to test the inhibition mechanism, along with molecular docking studies to support it. In our opinion, an inappropriate study of enzyme inhibition, at the early stages of drug development, can make it more expensive and delay greatly the process of the "new lead" process, and it seems that this new molecule is worthy of it.

The main factor of novelty is the presentation of a new monoterpenoid indole alkaloid, nauclediol, from Nauclea officinalis which is a dual cholinesterase inhibitor with two-fold more selective against butyrylcholinesterase enzyme.

Specific comments:

#1_Figure 4 (L249) The information presented in graph bars of AChE and BChE seems to be redundant. We suggest maintaining the graph with data points with error bars and the adjusted line. It's more visually appealing.

#2_ L251-255 +Figure 5. Please perform a non-linear regression in the estimation of Ki, if you want to conclude about the inhibition mechanism for nauclediol. It´s not difficult to find scripts in R for the 4 main linear inhibitions or even to perform it in Excell or any other statistical package of choice. Please provide also the precision of estimates (SE)and the residual graph (residuals vs predicted values) for the predictive model.

#3_The quality of the images of the binding interaction (Figures 6 ,7) is quite good, unlike the graphs of Figures 8 and 9. Please increase the quality of these graphs.

#4_ Reference 9 (L378-379) Please refer to the Patent Number/Code.

Author Response

#1_Figure 4 (L249) The information presented in graph bars of AChE and BChE seems to be redundant. We suggest maintaining the graph with data points with error bars and the adjusted line. It's more visually appealing.

Thank you for the suggestion. Figure 4 has been revised and amended. 

2_ L251-255 +Figure 5. Please perform a non-linear regression in the estimation of Ki, if you want to conclude about the inhibition mechanism for nauclediol. It´s not difficult to find scripts in R for the 4 main linear inhibitions or even to perform it in Excell or any other statistical package of choice. Please provide also the precision of estimates (SE)and the residual graph (residuals vs predicted values) for the predictive model.

 

Thank you for the effort in reviewing the calculation for the mode of inhibition. We have revised the mode of inhibition graph using Excel (appendix) and performed the non-linear regression (attach in the reviewer file). We choose to include the data from Excel as it is more make sense than using the statistical tool.

A number of manuscripts calculated the Ki and Kis using excel including 

https://www.sciencedirect.com/science/article/abs/pii/S0023643813001175

https://pubmed.ncbi.nlm.nih.gov/26712615/

It is also as per suggested by another reviewer to include the Kis value and discussion for the particular section. We thank you for your kind consideration and understanding.

3_The quality of the images of the binding interaction (Figures 6 ,7) is quite good, unlike the graphs of Figures 8 and 9. Please increase the quality of these graphs.

The quality of Figures 8 and 9 has been improved. 

#4_ Reference 9 (L378-379) Please refer to the Patent Number/Code.

Patent number (5290777) has been added in reference number 9.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

We appreciate the author’s effort in their responses. However, in our opinion, remains a major gap in the methodology and results, to support the conclusions.

The Processes Journal has in its scope the modeling of biochemical/enzymology processes; therefore, the level of exigency forces us to be more rigorous regarding modeling. In vitro methodology, in enzyme kinetics, is the core matter to test the inhibition mechanism. The concerns raised in the previous version, unfortunately, persist. The experimental design used lacks adequate depth and precision to test the hypothesis about the enzyme kinetic mechanism. We must emphasize here again that it´s very risky to make conclusions based on 3 data points on Dixon plots! Detailed reasoning of the stated is given below.

In the previous version, the authors were asked: “…. Please perform a non-linear regression in the estimation of Ki, if you want to conclude about the inhibition mechanism for nauclediol. It´s not difficult to find scripts in R for the 4 main linear inhibitions or even to perform it in Excell or any other statistical package of choice. Please provide also the precision of estimates (SE)and the residual graph (residuals vs predicted values) for the predictive model…”. After carefully reading the output of the supplement, it was verified that only the competitive model is presented, and inconsistencies between the MM graph and the results of the output were noticed. Also, physical unities are lacking.  It seems that the analysis was performed only on 4 experimental data. How it is possible? Furthermore, from our reading of the output, the competitive inhibition model was not suitable for giving acceptable constant estimates. Where is the data adjustment for mixed model, claimed by the authors?

The reciprocal rearrangement of the MM equation it´s is useful for displaying the differences between the kinetic behavior of the different inhibitor types, but it is an extremely inaccurate way of determining kinetic parameters!!! The weight of experimental data for low concentrations of the substrate is overemphasized and these are the values associated normally with the largest experimental error. The linearizations are suitable in the first phase of the design of the kinetic testing, to check the unexpected behavior of the data, while the experiment is still in progress. It can be used also as initial estimates in non-linear regression estimation. Per se, linearizations are not suitable to conclude about the complexity of mechanisms that a given enzyme has towards some inhibitor, especially if the inhibitor is a non-linear one (and most of the potent ones are!).

These linearizations have been heavily criticized by enzymologists for decades, to name a few:

I)“Improper use of the double-reciprocal plot” in Kinetic Mechanisms of Enzyme Inhibition and Activation: Tutorials, examples, mechanism validation, and advice for publishing by Professor Antonio Baici. (taken from his page  https://www.enzyme-modifier.ch/constructive-criticism-new/. )

The most popular method to analyze steady-state data enzyme-catalyzed reactions, graph results, and calculate kinetic constants is still the double-reciprocal plot. The statistical problems of this method have been discussed by the authors themselves (1), even before describing its applications in more detail (2).

Lineweaver, Burk and Deming recommended:

The estimated limits of experimental error … , based on a weighting according to the reciprocals of the squares of the assigned deviations, … [(1), p. 227].

Other authors analyzed in detail the problems of the double-reciprocal plot, starting with Dowd and Riggs, who came to the conclusion:

…  the Lineweaver-Burk transformation tends to give a deceptively “good” fit, even with unreliable points. The marked inferiority of the Lineweaver-Burk plot strongly suggests that it should be abandoned as a method for estimating Km and V from unweighted points …  [(3), p. 869]

II) "...KM estimates made using double-reciprocal plots and the MM equation were consistently inferior to estimates made with nonlinear least-square fitting methods" [Schnell, S. and Maini, P.K. (2003) A Century of enzyme Kinetics: Reliability of the KM and Vmax Estimates. Comments on Theoretical Biology, 8, 169-187. http://dx.doi.org/10.1080/08948550302453].

II) "Today, there is no reason for fitting data using either the linear transformation of the Michaelis–Menten equation in analyzing the concentration dependence of the initial velocity" in [ Johnson 2013 A century of enzyme kinetic analysis, 1913 to 2013, FEBS Lett. 2013 Sep 2;587(17):2753-66. doi: 10.1016/j.febslet.2013.07.012.]

For that reason, the conclusion of the inhibition mechanism is considered to be unreliable. From the results presented, there is an enormous possibility of imprecision and statistical inaccuracy, whether in the case of linear inhibitions or not (if the inhibitor is non-linear this is even more serious!). If a compound seems to be a promising inhibitor, a precise mechanism identification is urgent!!

So, in our opinion, we suggest a resubmission, after a throughout reedition of this manuscript, where the conclusions about the real mechanism are absent. Given the presented data, the methods/results are not sufficient to provide conclusions, in a statistically satisfactory way, about the real mechanism of any of the enzymes. 

Author Response

Dear reviewer, 

Thank you very much for the comments and useful articles to conduct a kinetic study for nauclediol. 

We have rerun the experiment and re-analysis the data using statistical tool. 

We hope the latest version of manuscript is appropriately revealed the mechanism of action of nauclediol. 

Thanks 

Round 3

Reviewer 4 Report

We congratulate the authors on their effort!

The manuscript was greatly improved regarding the kinetic modeling methodology. 

The inclusion of the Bayesian Information Criterion in the discrimination of the models, although incomplete, was nicely attempted. Furthermore, it was verified a high correlation between experimental data vs estimated values.  

We believe that the conclusion of the mechanism was supported by the statistical analysis. Thank you! Very nice job!

Specific comments:

#_L283-287_Figure 5_ The graphs are nicely done, however, the physical units are lacking. Please add them accordingly.

Author Response

Dear reviewer, 

Thank you for your kind suggestion and patience in reviewing our manuscript. We very much appreciate the idea not only for this paper to be published but also because it will be beneficial to our future research. 

We have added the physical units to Figure 5.

Thank you