DNA Triplex-Formation by a Covalent Conjugate of the Anticancer Drug Temozolomide

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled “DNA Triplex-Formation by a Covalent Conjugate of the Anti-2 cancer Drug Temozolomide” attempts to establish a synthesis protocol to covalently attach temozolomide to the 5’ of an oligonucleotide that forms a triplex. Solid-supported synthesis of a covalent conjugate (Temo-TTTCTTTTCTCTCTT) of triplex-forming oligonucleotide (TTTCTTTTCTCTCTT) and a temozolomide (an oral chemotherapy drug) to lessen off-target effects is discussed. Temozolomide is unstable under basic conditions. In the solid supported synthesis, protection groups were selected to preclude basic deprotecting conditions. The authors also show that the small molecule attachment does not affect the thermal stability of triplex structure. The manuscript is valuable for demonstrating a useful synthetic protocol. However, some questions below should be addressed before publication:

1. Reaction conditions and starting materials are not given in all schemes.
2. Functionality equivalent of LCAA-CPG is not given in line# 145.
3. Schematic representation of synthesis of compounds 14, 20, 21, 22 and 23.
4. Description of ‘trityl assay’ and ‘DMT assay’ were not provided in the manuscript.
5. What is the meaning of standard conditions in line # 170 and line# 232?
6. ‘Washed with’ appeared two times in line# 175.
7. What is Tpoce8 in compounds 15 and 16?

Author Response

Comments 1: Reaction conditions and starting materials are not given in all schemes.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have added all of the necessary reaction conditions and starting materials to all of the schemes.

Comments 2: Functionality equivalent of LCAA-CPG is not given in line# 145.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have clarified the functionality by adding the appropriate wording “primary amino groups”.

Comments 3: Schematic representation of synthesis of compounds 14, 20, 21, 22 and 23.

Response 3: We agree with this comment. Therefore, we have included all of the necessary schematic information that represents synthesis of all compounds including 14, 20, 21, 22 and 23.

Comments 4: Description of ‘trityl assay’ and ‘DMT assay’ were not provided in the manuscript.

Response 4: We agree. Thank you for making this point. We have changed the wording “DMT assay” to “trityl assay” as they relate to the same identical procedure. The experimental details of the trityl assay are in the published literature and therefore we added Reference 19 to the Materials and Methods section where the trityl assay procedure has been performed.

Comments 5: What is the meaning of standard conditions in line # 170 and line# 232?

Response 5: Thank you for pointing this out. We agree with these comments. Therefore, we have in lines # 170 and 232 simplified the sentence by removing the words “standard conditions” as in the Materials and methods section General, the following explanatory wording is given; “Oligodeoxynucleotides were prepared on a Beckman Oligo 1000 DNA synthesizer according to the manufacturer’s protocol using commercially available reagents (LINK Technologies).

Comments 6: ‘Washed with’ appeared two times in line# 175.

Response 6: We agree with this comment. Therefore, we have deleted the repeated words so that sentence is now correct.

Comments 7: What is Tpoce8 in compounds 15 and 16?

Response 7: Thank you for pointing this out. We agree with this comment. Compounds 15 and 16 have been redrawn to show clearly cyanoethyl protecting groups attached to the bridging phosphate groups therefore this replaces the need to use Tpoce to represent them and therefore these instances have been removed.

Reviewer 2 Report

Comments and Suggestions for Authors

This article discusses the creation and utilization of a new synthesis procedure for base-sensitive oligonucleotides on a new separable solid support that contains the di-iso-propylsilylene chemical group. The support is demonstrated to be compatible with mild deprotection conditions and with the hydrolysis-sensitive anticancer agent temozolomide, which is covalently attached to triploid-forming oligonucleotides. This attachment enables the stable triplex formation of DNA, where its binding activity is retained even when modified. The system delivers a potentially more effective directed treatment of glioma with reduced potential side effects of conventional chemotherapy.

This research is of great importance, as it unites temozolomide, which is a key drug for glioblastoma treatment, with triploid oligonucleotide technology, thus showing a novel chemical approach to targeted gene therapy. Creating a synthesis vector with activity in mild conditions paves the way for the synthesis of pharmacological hybrid molecules that are prone to basic environments, and possible applications in other bases or medications.

To my view, the article needs several major corrections.

1. Although an abstract of the article is readable, it contains many technical information (e.g., various types of protecting groups and conditions for their removal) that can be reduced. It would be preferable to concentrate on what is new and on the significance of the method's application in therapeutic development, making it more accessible to non-specialists.
2. The introduction provides a good description of the mechanism of action of temozolomide and discusses pertinent literature. Nevertheless, the connection between the therapeutic target, glioblastoma, and the reason for selecting triploid sequence technology might be more succinctly declared. A better explanation of how triploid binding enhances selectivity or diminishes resistance would also provide stronger justification for the study.
3. The procedure is explained in great detail, and this allows repetition of the experiments. The use of numerous chemical compositions and products with numbers (e.g., 10, 11, 12.) may, however, be cumbersome to the reader. It would have been easier to read if these numbers were preceded by brief descriptions either in the text or as a table or figure to allow quick reference.
4. The results obtained from the stability tests of the triploid helices are of great importance. An observation has been made that stability is not affected by the temozolomide modification, which represents an important finding. However, it would be helpful to determine whether this means that the biological activity remains the same or if further studies in cellular or in vivo systems are required.
5. The conclusions section accurately summarizes the main findings, but would be even more valuable with some suggestions for future directions. Discussion of next steps, e.g., in vitro or in vivo testing of the biological activity of the compositions, or comparison with other delivery methods of temozolomide, would have been useful.
6. Figures and diagrams, i.e. triploid structures and chemical formulas, play a significant role in the comprehension of the article. However, their placement and numbering (i.e. "Figure 3" is used twice with different data) might get confusing. Re-numbering or rearranging the graphical content might be a suggestion.

Author Response

Comments 1: Although an abstract of the article is readable, it contains many technical information (e.g., various types of protecting groups and conditions for their removal) that can be reduced. It would be preferable to concentrate on what is new and on the significance of the method's application in therapeutic development, making it more accessible to non-specialists.

Response 1: Agreed. We have reworded the relevant parts of the abstract to focus on the novelty and significance of the method. We have removed the technical information and explicit use of protecting group terminology to sharpen the focus on novelty for the benefit of non-specialists.

Comments 2: The introduction provides a good description of the mechanism of action of temozolomide and discusses pertinent literature. Nevertheless, the connection between the therapeutic target, glioblastoma, and the reason for selecting triploid sequence technology might be more succinctly declared. A better explanation of how triploid binding enhances selectivity or diminishes resistance would also provide stronger justification for the study.

Response 2: We agree with this comment. Therefore, we have explained how conjugate formation can conceivably enhance selectivity by delivering temozolomide to its target site; the middle G in runs of three (GGG) compared with use of temozolomide alone.

Comments 3: - The procedure is explained in great detail, and this allows repetition of the experiments. The use of numerous chemical compositions and products with numbers (e.g., 10, 11, 12.) may, however, be cumbersome to the reader. It would have been easier to read if these numbers were preceded by brief descriptions either in the text or as a table or figure to allow quick reference.

Response 3: – We appreciate your kind comment that the methods section is explained in detail. For all of the compounds mentioned, including 10, 11 and 12, every numbered compound is prefixed by a descriptor or name. The structures of every numbered compound referred to in the body of the text is shown in the appropriate Figure or Scheme as close to first mention as possible. Further to this we have included the names of the compounds as well as the numbers in the Table of Contents at the beginning of the Supplementary Materials document.

Comments 4: The results obtained from the stability tests of the triploid helices are of great importance. An observation has been made that stability is not affected by the temozolomide modification, which represents an important finding. However, it would be helpful to determine whether this means that the biological activity remains the same or if further studies in cellular or in vivo systems are required.

Response 4: Thank you for your support in pointing out that triploid  stability is not affected by temozolomide modification is an important finding. Now that we have established reliable methodology, we have opened up new avenues of research whereby in vivo and in vitro evaluation of activity can be assessed in the next phase of the development process and have updated the concluding paragraph to reflect this point.

Comments 5: The conclusions section accurately summarizes the main findings, but would be even more valuable with some suggestions for future directions. Discussion of next steps, e.g., in vitro or in vivo testing of the biological activity of the compositions, or comparison with other delivery methods of temozolomide, would have been useful.

Response 5: Agreed. We have, accordingly added the suggested wording to the concluding paragraph to emphasize this point.

Comments 6: Figures and diagrams, i.e. triploid structures and chemical formulas, play a significant role in the comprehension of the article. However, their placement and numbering (i.e. "Figure 3" is used twice with different data) might get confusing. Re-numbering or rearranging the graphical content might be a suggestion.

Response 6: Thank you for pointing this out. We agree. Figure 3 legend that appears a second time with different data and should have been numbered as Figure 4. Reviewer 1 makes the point in their Comment 3 that “Schematic representation of synthesis of compounds 14, 20, 21, 22 and 23 compounds” is missing. We have therefore used what would have been Figure 4 as an additional Scheme 3 to show the reactions and conditions used to address Comment 3 from reviewer 1 and to address Comment 6 from Reviewer 2.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made all the suggested corrections to the article. I recommend its publication.