Potent Alkaline Phosphatase Inhibitors, Pyrazolo-Oxothiazolidines: Synthesis, Biological Evaluation, Molecular Docking, and Kinetic Studies

Round 1

Reviewer 1 Report

In this article, Nasab et al. have proposed a study involving synthesis, in vitro essays and molecular modeling techniques to propose new inhibitors targeting Alkaline phosphatases. 

In my opinion, this study is very interesting and combines three different and transversal approaches to propose those new thiazolones compounds. I think it deserves to be published in IJMS but first some points needs to be clarified and more explained:

- My first remarks goes to the comparison between the experimental results and the docking studies. The IC50 assays have been made on the CAIP (Calf Intestin Alkaline Phosphatase), but the docking studies on the placental isoform of the protein! Consequently, does the results could be comparable?  We do not have in the paper any precision of the degree of similarity between the four isoforms of the protein, so no clue about a relevant comparison between the two results. Authors have to detailed this information and add a justification of this choice.

I saw that there is currently no structures of the intestinal isoform, but i) there is several structure of the placental form of the protein, so why the choice of PDB ID 1EW2 against the others? and ii) why the authors have not tried to propose a homology model of the intestinal isoform, especially because the Alphafold model is available and seems qualitative?

All those points must be clarified, especially because the heart of the introduction focus on the need of designing inhibitors that specifically target one of the four isoforms, but this study compared EC50 made on CAIP and the molecular point of view made on ALP.

- Regarding the docking calculation and the methodology, I admit that the calculations were made with a rigid receptor, with no flexibility on the side-chains positions because of the lack of this information. The energy difference obtained by docking experiment between the entire set of compounds is less than 1 kcal/mol for almost all of them, meaning that the difference is not really significant. I suggest the authors to made the docking calculations again with taking into account the receptor flexibility (directly by autodock vina, or by generating alternative conformations) in order to see if it can refine the interaction energy and better separate the compounds.

Other minor points:

- What Is the difference between Calf intestin Alkaline phosphatase (CAIP), on which the experiment has been made, and Intestinal alkaline phosphatase (IALP) cited in the introduction?

-I think that the structural detailed in the results part are clearly lacking during the lecture of the manuscript, making some hard to understand without the information. In my opinion, the authors must add those details in table 1, by adding the chemical structure of each compound, or two colons with the definition of R and R'.

Author Response

 

To

The Worthy Editor in Chief,

International Journal of Molecular Sciences

We are pleased to have an opportunity to revise our manuscript entitled “Potent Alkaline Phosphatase Inhibitors, Pyrazolo-Oxothiazolidines: Synthesis, Biological Evaluation, Molecular Docking, and Kinetic Studies”. We agree that the revisions proposed by the worthy reviewers significantly enhance our manuscript in terms of scientific presentation as well as made the idea more lucid. In the revised manuscript, we have prudently considered reviewers’ suggestions and acted accordingly. As instructed, we have attempted to succinctly explain changes made in reaction to all comments. We reply to each comment in a point-by-point fashion. We have turned track changes on to allow concerned staff to see changes made in the revised version of our manuscript. The responses to the concerns raised by reviewers are shown as text and our responses are shown as text.

 

The reviewers’ comments were very helpful overall, and we appreciate such constructive feedback on our manuscript. After addressing the issues raised, we feel the quality of the paper is ameliorated.

 

 

Thank you for your consideration of this manuscript.

                                                                                                                                     Sincerely,

                                                                                                                                 Prof. Dr Song Ja Kim

              Corresponding Author

Professor, Department of Biological Sciences, College of Natural Sciences, Kongju National University, Gongju 32588, Republic of Korea.

Reviewer No. 1

In this article, Nasab et al. have proposed a study involving synthesis, in vitro essays and molecular modeling techniques to propose new inhibitors targeting Alkaline phosphatases. In my opinion, this study is very interesting and combines three different and transversal approaches to propose those new thiazolones compounds. I think it deserves to be published in IJMS but first some points needs to be clarified and more explained:

1. My first remarks goes to the comparison between the experimental results and the docking studies. The IC50 assays have been made on the CAIP (Calf Intestin Alkaline Phosphatase), but the docking studies on the placental isoform of the protein! Consequently, does the results could be comparable? We do not have in the paper any precision of the degree of similarity between the four isoforms of the protein, so no clue about a relevant comparison between the two results. Authors have to detailed this information and add a justification of this choice.

Answer: We would like to extend our deepest gratitude to the reviewers who took their precious time to evaluate our manuscript. Thank you for your worthy comments. Yes, it’s a concern in comparison with in-vitro and in-silico studies. Meanwhile, we followed the already published protocols for both in vitro and in silico studies (Mol Divers. 25(3):1701-1715; Bioorg Chem. 12; 90:103108; doi: 10.1002/ddr.21542; Arch Pharm; 353, 3 e1800278).

2. I saw that there is currently no structures of the intestinal isoform, but i) there is several structure of the placental form of the protein, so why the choice of PDB ID 1EW2 against the others? and ii) why the authors have not tried to propose a homology model of the intestinal isoform, especially because the Alphafold model is available and seems qualitative?

Answer: Thank you for bringing our attention to this issue. We agree with you, so we have chosen this structure by following the protocol of already published articles (Mol Divers. 25(3):1701-1715; Bioorg Chem. 12; 90:103108; doi: 10.1002/ddr.21542; Arch Pharm; 353, 3 e1800278). Therefore, we did not use any other computational tool to predict the protein model.

3. All those points must be clarified, especially because the heart of the introduction focus on the need of designing inhibitors that specifically target one of the four isoforms, but this study compared EC50 made on CAIP, and the molecular point of view made on ALP.

Answer: We appreciate the reviewer’s comment, yes, we have targeted our compounds on one of the isoforms of ALP, and we did experiments on that type, however, the CAIP is the source for ALP, which we have used in our in vitro experiments due to this source of protein availability.  

4. Regarding the docking calculation and the methodology, I admit that the calculations were made with a rigid receptor, with no flexibility on the side-chains positions because of the lack of this information. The energy difference obtained by docking experiment between the entire set of compounds is less than 1 kcal/mol for almost all of them, meaning that the difference is not really significant. I suggest the authors to made the docking calculations again with taking into account the receptor flexibility (directly by autodock vina, or by generating alternative conformations) in order to see if it can refine the interaction energy and better separate the compounds.

Answer: We really appreciate the reviewer for keenly reviewing our manuscript and raising salient points to improve the quality of our manuscript. Yes, the binding energy difference is quite low among all synthetic compounds. It’s just because the derivatives possessed the same basic chemical skeleton and only one position in the whole molecule is changed by incorporating different moieties. Therefore, the docking energy difference is low in our docking experiment.

5. What Is the difference between Calf intestin Alkaline phosphatase (CAIP), on which the experiment has been made, and Intestinal alkaline phosphatase (IALP) cited in the introduction?

Answer: Thank you for this comment, there is no difference between these two words, both of them CIAP and IALP are the same.

6. I think that the structural detailed in the results part are clearly lacking during the lecture of the manuscript, making some hard to understand without the information. In my opinion, the authors must add those details in table 1, by adding the chemical structure of each compound, or two colons with the definition of R and R'.

Answer: We would like to appreciate the reviewer’s worthy comment on our research. We completely agree with the reviewer’s recommendations.  Table 1 was modified as you suggested.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript by Nasab et al. reports the charaterization of inhibitors of alkaline phosphatases (AP) using pyrazolo-oxothiazolidines derivatives. The authors discovered inhibitors against AP by performing enzyme kinetics, cell-based assay, and in silico study as well as the synthesis of derivatives. Among them, the 7g showed a very good effect as a drug candidate. This work is interesting and straghtforward, and the manuscript was scientifically well organized. This manuscript can be published on IJMS after minor revisions.

 

(1) The authors describe some pharmaceutical uses of pyrazolo or oxothiazolidine derivatives alone, but do not mention some examples of their use in combination. Please add references on this.

 

(2) Please indicate the significant digits correctly in Figure 2, Table 2 and line 222. 

 

(3) The abbreviation for DPPH was written first on line 220, and the full letter was written on line 572.

 

(4) Line 220 and line 279: in-vitro -> in vitro.

 

(5) Line 226 ~ 228: In Figure 4, the cell viability of some derivatives is less than 70%, but all are described as more than 70%. Please correct this.

 

(6) Line 261 ~ 262: What is the remaining 1%?

 

(7) 3.3.1. Please check the capitalization of subheading.

Author Response

COVER LETTER FOR ARTICLE REVISION

To

The Worthy Editor in Chief,

International Journal of Molecular Sciences

We are pleased to have an opportunity to revise our manuscript entitled “Potent Alkaline Phosphatase Inhibitors, Pyrazolo-Oxothiazolidines: Synthesis, Biological Evaluation, Molecular Docking, and Kinetic Studies”. We agree that the revisions proposed by the worthy reviewers significantly enhance our manuscript in terms of scientific presentation as well as made the idea more lucid. In the revised manuscript, we have prudently considered reviewers’ suggestions and acted accordingly. As instructed, we have attempted to succinctly explain changes made in reaction to all comments. We reply to each comment in a point-by-point fashion. We have turned track changes on to allow concerned staff to see changes made in the revised version of our manuscript. The responses to the concerns raised by reviewers are shown as text and our responses are shown as text.

 

The reviewers’ comments were very helpful overall, and we appreciate such constructive feedback on our manuscript. After addressing the issues raised, we feel the quality of the paper is ameliorated.

 

 

Thank you for your consideration of this manuscript.

                                                                                                                                     Sincerely,

                                                                                                                                 Prof. Dr Song Ja Kim

              Corresponding Author

Professor, Department of Biological Sciences, College of Natural Sciences, Kongju National University, Gongju 32588, Republic of Korea.

 

 

Reviewers Comment to Author:

 

Reviewer No. 2

This manuscript by Nasab et al. reports the charaterization of inhibitors of alkaline phosphatases (AP) using pyrazolo-oxothiazolidines derivatives. The authors discovered inhibitors against AP by performing enzyme kinetics, cell-based assay, and in silico study as well as the synthesis of derivatives. Among them, the 7g showed a very good effect as a drug candidate. This work is interesting and straghtforward, and the manuscript was scientifically well organized. This manuscript can be published on IJMS after minor revisions.

1. The authors describe some pharmaceutical uses of pyrazolo or oxothiazolidine derivatives alone, but do not mention some examples of their use in combination. Please add references on this.

Answer: We would like to extend our deepest gratitude to the reviewers who took their precious time to evaluate our manuscript. As per the reviewer’s suggestion, we added some applications and biological activity of the combination of pyrazolo and oxothiazolidine derivatives. Please see the changes highlighted in blue in the manuscript.

2. Please indicate the significant digits correctly in Figure 2, Table 2 and line 222.

Answer: Thank you for the comment, these values are in the concentration unit, changing the significant digit may have an effect on the values.

3. The abbreviation for DPPH was written first on line 220, and the full letter was written on line 572.

Answer: Thank you for bringing our attention to this issue. We have added the abbreviation of DPPH at the first placed text in the manuscript.

4. Line 220 and line 279: in-vitro -> in vitro.

Answer: Thank you for your valuable suggestion. In the overall manuscript text, it was modified as you suggested.

5. Line 226 ~ 228: In Figure 4, the cell viability of some derivatives is less than 70%, but all are described as more than 70%. Please correct this.

Answer: We would like to appreciate the reviewer’s worthy comment on our research. We corrected the description of our results. Please see the changes highlighted in blue in the manuscript.

6. Line 261 ~ 262: What is the remaining 1%?

Answer: Thank you for your comment; unfortunately, we could not find any 1% value to adjust it.

7. 3.1. Please check the capitalization of subheading.

Answer: Thank you for bringing our attention to this issue. The capitalization issue has been resolved.

 

Editorial Comments

The manuscript is focused on synthesis and biological/biochemical characterization of some novel inhibitors of alkaline phosphatase. Overall, the study is relevant and the results appear to be convincing. However, there are two points where some improvements would be welcomed.

1. The need for novel medicines based on alkaline phosphatase (AP) inhibitors is discussed insufficiently. Indeed, changes in the level of AP and its enzymatic activity are associated with multiple conditions and important for diagnostics. However, pharmacological inhibitors are needed only in cases where excessive AP is critical for the development or exacerbation of an adverse condition. Such specific conditions should be listed and discussed.

Answer: Thank you for your worthy comment. More explanations of inhibitors, both traditional and synthetic, as well as the relationship between our synthesis and alkaline phosphatase inhibitors, have been presented in the introduction section. Please see the changes highlighted in blue in the manuscript.

2. Compound 7g indeed demonstrates the lowest IC50. However, several other compounds, such as 7a and 7l, demonstrate IC50s very close to that of 7g. Furthermore, 7l shows lower toxicity in cell culture (~95% vs ~70% @4uM), and both 7a and 7l show a drug likeness score higher than that of 7g (especially, 7l). Hence, focusing all attention on 7g may not be justified.

Answer: Thank you for your worthy comment. To point out the other compounds in the comparison of 7g, yes, it’s true that almost all compounds are active and showed good activity in all the experiments. The reason is to point out only the most potent compound based on the in-vitro results to make it highlighted for the next research step such as animal or future human trial for the pharmaceutical companies as a drug.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors have improved the quality of their paper, it can now be accepted for publication