Enzymatic Synthesis of 2-Chloropurine Arabinonucleosides with Chiral Amino Acid Amides at the C6 Position and an Evaluation of Antiproliferative Activity In Vitro
, Maria Ya. Berzina 1, Ilya V. Fateev 1, Alexei L. Kayushin 1, Elena V. Dorofeeva 1, Olga I. Lutonina 1, Ekaterina A. Zorina 1, Konstantin V. Antonov 1, Alexander S. Paramonov 1, Inessa S. Muzyka 1, Olga S. Zhukova 2, Mikhail V. Kiselevskiy 2, Anatoly I. Miroshnikov 1, Roman S. Esipov 1 and Irina D. Konstantinova 1,*Round 1
Reviewer 1 Report
queries
1. how does the functionalgroups of the anti tumour drugs play a role on the effcetiveness of healing. does hydrogen-bonding would have been a key factor? the authors can address this question to signify the nature of inetraction.
2. does the role of polar and non polar groups in amino acid pay a role? justify
3. are there any side products formed in the synthesis of compunds 1b-12b?
minor changes should be done in the change of words like showed, used etc..
Author Response
1 Reviewer
How does the functional groups of the anti tumor drugs play a role on the effectiveness of healing. Does hydrogen-bonding would have been a key factor? The authors can address this question to signify the nature of interaction.
Which of the enzymes is responsible for the antiproliferative activity of the serine nucleoside is still unclear. In this case, it is absolutely impossible to discuss the occurrence of some kind of hydrogen bond (with what?). Moreover, only the nucleoside 4b (L-serinylamido- substituent) was active, but not the 5b (D-serinylamido- substituent) nucleoside. The functional groups are the same, but the antitumor effect is quite different.
- Does the role of polar and non polar groups in amino acid pay a role? Justify.
To reveal the possible dependence ‘antitumor activity’ - ‘substituent at C6 position’ arabinosides both with polar amino acid residues and with nonpolar amino acids were synthesized. Except of the L-serinylamido derivative, all other compounds were inactive. Correlation is not possible.
- Are there any side products formed in the synthesis of compounds 1b-12b?
No, they are not formed. HPLC-profiles of the reaction mixtures are shown on Figure 4. No by-products has been observed. The enzymatic transglycosylation is highly stereo- and regiospecific for this type of substrate.
Minor changes should be done in the change of words like showed, used etc…
Corrected.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
From my point of view the discovery is not attractive enough for publication in its current version in such a reputable journal as IJMS.
The part on biological activity is too poor and weak. The authors tested the antiproliferative activity of compounds against only one tumour cell line.
a) It is necessary to conduct additional experiments on other tumoural cell lines.
b) The cytotoxicity of all the compounds against normal non-cancerous cells should be assessed to exclude their general toxicity.
c) Additional studies need to be conducted to elucidate the mechanism of anticancer activity.
References: Abbreviations of journal names should be provided.
Text formatting should be carefully checked.
The language should be modified carefully.
Author Response
2 Reviewer
The part on biological activity is too poor and weak. The authors tested the antiproliferative activity of compounds against only one tumor cell line.
- It is necessary to conduct additional experiments on other tumor cell lines.
The objective of the biological part of this investigation was to screen a number of purine nucleosides and to evaluate active compound in comparison with Nelarabine. Nelarabine is a purine analog and antineoplastic agent used in therapy of acute leukemia, particularly acute myeloid leukemia (AML) [Ju HY, Hong CR, Shin HY. Advancements in the treatment of pediatric acute leukemia and brain tumor - continuous efforts for 100% cure. Korean J Pediatr. 2014 Oct; 57(10):434-9. doi: 10.3345/kjp.2014.57.10.434]. Therefore we used human acute myeloid leukemia cell line U937.
The antiproliferative activity of the compounds synthesized was tested on other tumor cell lines: LS174T human Caucasian colon adenocarcinoma, SKOV3 ovarian cancer cells, MCF7 breast cancer cells, and A549 non-small cell lung cancer cells. All compounds tested did not affect the cell viability: survival was >80% (within concentrations up to 50.00 μM). We have added this information into the manuscript (marked by magenta).
- The cytotoxicity of all the compounds against normal non-cancerous cells should be assessed to exclude their general toxicity.
Normal cells are not included in the screening panel of antitumor drugs. Antitumor drugs have no selective activity on tumor cells in vitro.
Side effects of antitumor compounds are subjects of in vivo investigation.
Hepatotoxicity from nelarabine is likely due to direct toxicity as is typical for other purine analogues [Zimmerman HJ. Oncotherapeutic and immunosuppressive agents. In, Zimmerman HJ. Hepatotoxicity: the adverse effects of drugs and other chemicals on the liver. 2nd ed. Philadelphia: Lippincott, 1999, pp. 673-708].
Nevertheless, cytotoxic properties of compounds against Vero E6 cells were determined by incubation of a cell culture in the presence of nucleosides at the range of concentrations up to 1,000 μg/ml during 72 hr. Nucleosides 1b - 12b did not affect the Vero E6 cells survival.
- Additional studies need to be conducted to elucidate the mechanism of anticancer activity.
We are currently studying the mechanism of cell death. Of the entire series of synthesized arabinosides, only the the nucleoside 4b (L-serinylamido- substituent) exhibits antiproliferative activity. Currently, an additional amount of the substance is being synthesized to study its pharmacodynamics and pharmacokinetics. The possibility of monophosphorylation of this nucleoside by adenosine kinase will also be studied to elucidate the possible mechanism of its action and metabolism within the cells. Right now it is impossible to make any assumptions. We hope to obtain the necessary data in the future, which can form the basis of our next publication.
References: Abbreviations of journal names should be provided.
- Сorrected
Text formatting should be carefully checked.
- Checked
The language should be modified carefully.
- Modified
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
I accept the explanations of the authors.
