From the journal RSC Chemical Biology Peer review history

15-Jan-2023

Dear Dr Glazer:

Manuscript ID: CB-ART-12-2022-000247
TITLE: A Monoadduct Generating Ru(II) Complex Induces Ribosome Biogenesis Stress and is a Molecular Mimic of Phenanthriplatin

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary.

Please submit a revised manuscript which addresses all of the reviewers’ comments. Further peer review of your revised manuscript may be needed. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link:

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?link_removed

(This link goes straight to your account, without the need to log on to the system. For your account security you should not share this link with others.)

Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

All RSC Chemical Biology articles are published under an open access model, and the appropriate article processing charge (APC) will apply. Details of the APC and discounted rates can be found at https://www.rsc.org/journals-books-databases/about-journals/rsc-chemical-biology/#CB-charges.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy, https://credit.niso.org/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines https://www.rsc.org/journals-books-databases/author-and-reviewer-hub/authors-information/responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

************

Reviewer 1

The manuscript by Mitchell et al. reported an interesting Ru(II) compound that mimics the MoA of phenanthriplatin, a well-known monofunctional Pt(II) compound. The authors carried out a detailed study on the mechanism of this monofunctional polypyridyl Ru(II) compound, including the reactivity with GSH, cytotoxicity in 2D and 3D cell models, activity against bacterial growth and morphology, DNA damage ability, inhibition of topoisomerase and microtubules, and finally ribosome biogenesis stress. In general, this is an intriguing report on the monofunctional Ru(II) compound that displayed unique properties that are different from other types of Ru(II) compounds. However, there are still some fundamental concerns, and the authors did not prepare the manuscript carefully as indicated below. The major and minor concerns include:

1. What are the possible targets of compound 2 in cells? If DNA is considered the major target, the level of Ru in the genomic DNA of treated cells should be measured, and this level should be compared with the accumulation level in whole cells. More discussions on the target should be included.

2. What's the subcellular localization of compound 2? What’s the percentage of compound 2 that accumulates in the nucleus? The authors are suggested to isolate different parts (e.g., cyto, nucleus, mitochondria, etc.) and measure the level of Ru.

3. Why could compound 2 induce ribosome biogenesis? How did compound 2 induce nucleolar stress?

4. Page 2, right column, 2nd paragraph, please make sure the SI values cited in the text have the same decimal places as those included in Table 1.

5. Page 4, 1st paragraph, the NCI60 screening data of phenanthriplatin have been published in doi: 10.1073/pnas.1207670109 please consider discussing the difference between phenanthriplatin and complex 2.

6. Page 4, right column, 2nd paragraph, “phenanthriplatin produced the lowest nucleotide (nt) to metal center (mc) ratio, followed by cisplatin and 1, and then 2 (Fig. S20 and Table S3)”. Please check whether Fig S20 is properly cited here. Please clarify how did the nt/mc measured and the detailed conditions, such as treatment time and concentration, etc.

7. Page 7, 1st paragraph, please make sure the figures are correctly cited.

Reviewer 2

The manuscript by Glazer, Heidary and colleagues entitled: “A monoadduct generating Ru(II) complex induces ribosome biogenesis stress and is a molecular mimic of phenanthriplatin" presents a very nice work in terms of chemical synthesis and in vitro studies. There is comprehensive biochemical evaluation by a very notable number of methods in an attempt to understand the effects on the cells of the new octahedral Ru(II) complex incorporating a phenyl-modified terpyridine and a dip ligand. Experimental data are well described. This article was extremely interesting to read. Overall, I recommend publication of the manuscript in RSC Chemical Biology provided that the authors address the following minor points:
1. Regarding optical properties of the new Ru complexes: please, indicate in the Figure S29 caption the solvent used for running their UV/Vis spectra (water?). What about the emission properties of these Ru compounds (if any)?
2. Mitochondrial stress was excluded as mechanism of action (Fig. 4E). This should be discussed, at least briefly, in the text.
3. There might be some typos in the section of studies in Escherichia coli (page 4, right column: Fig. S20?), and also in the bacterial cytological profiling section (page 4, right column: Fig. 4E and 4F?).

Dear. Dr. Yang,

Thank you so much for your email and instructions for revision. We appreciate the reviewers for their comments and corrections. We have made all the requested changes, and detail them in the response to review.

All my best,

Phoebe (Edith)

Edith Glazer

This text has been copied from the Microsoft Word response to reviewers and does not include any figures, images or special characters:

Reviewer 1:

1. What are the possible targets of compound 2 in cells? If DNA is considered the major target, the level of Ru in the genomic DNA of treated cells should be measured, and this level should be compared with the accumulation level in whole cells. More discussions on the target should be included.
The reviewer asks a very challenging question regarding the specific target of 2 in cells. We took a somewhat untraditional approach, as we utilized the COMPARE analysis to suggest potential mechanisms of action, rather than targets, and then went through the process of validating or eliminating the likelihood that the studied compounds modulated the putative processes. As a result, we focused on processes, rather than target ID.
However, we are confident that DNA is NOT the major target. We have measured the level of Ru in the genomic DNA, and these results are presented in Table S3 and discussed in the paper (the text is copied below). Moreover, we have run additional assays that demonstrate that DNA damage signaling is not significantly activated by the ruthenium compounds.

Text on page 4:

“…cellular DNA was isolated and analyzed for metal content. The quantity of metal in the total genomic DNA was determined by ICP-MS, and the nucleotide to metal center ratio was calculated by dividing the number of nucleotides in the total genomic DNA present in each sample by the metal atoms. Phenanthriplatin produced the lowest nucleotide (nt) to metal center (mc) ratio, followed by cisplatin and 1, and then 2 (Table S3). These results indicate that both compounds 1 and 2 irreversibly bind DNA. However, despite the fact that compound 2 and p henanthriplatin have the same MIC value in E. coli, the Ru(II) complex formed 10-fold fewer DNA adducts. This is consistent with an interpretation that an indirect mechanism of DNA damage, such as topoisomerase inhibition, or another mechanism, such as ribosome biogenesis stress, plays a greater role in the functional activity of 2. A ratio of 1 DNA-Ru(II) adduct to 106 atoms of Ru taken up into each E. coli cell also suggests a mechanism of cell death independent of DNA damage.”

Text on page 6:

“Another commonly utilized reporter for DNA damage is phosphorylation of serine 139 on Histone 2A; phospho-H2AX (p-γH2AX) initiates DNA damage repair (DDR). Immunoblotting for p-γH2AX thus is a common approach to probe small molecules that covalently modify DNA and initiate DDR. To further investigate DNA damage, the lysates from A549 cells treated with cisplatin (5 μM), phenanthriplatin (0.5 μM), or 2 (0.5 μM) were probed for p-γH2AX at 4 and 24 h (Fig S25). The 24 h treatment with phenanthriplatin yielded a significant p-γH2AX response. Compound 2-treated samples, however, did exhibit p-γH2AX, indicating that another mechanism of action is involved.

Figure Addition:

Figure S25. Immunoblotting of p-γH2AX, H2AX, and GAPDH following treatment with cisplatin, phenanthriplatin, and 2. Approximately 106 A549 cells treated were with 5 μM cisplatin, 0.5 μM phenanthriplatin, or 0.5 μM 2 for the hours indicated. At 24 h, phosphorylation of H2AX was present for phenanthriplatin-treated cells, and to a much lower extent, cisplatin-treated cells.

2. What's the subcellular localization of compound 2? What’s the percentage of compound 2 that accumulates in the nucleus? The authors are suggested to isolate different parts (e.g., cyto, nucleus, mitochondria, etc.) and measure the level of Ru.

We understand the reviewer’s suggestion to determine the localization of compound 2. We have attempted this, but obtained unsatisfactory results, as it is very challenging to get high purity isolates. In addition, some literature has shown that cell fractionation is prone to small molecule leakage, which can result in unreliable results.1, 2 Furthermore, studies related to TLD-1433, the Ru(II) photosensitizer in Phase II clinical trials, have highlighted that subcellular localization studies yield varying results.3, 4

1. Michelsen, U.; von Hagen, J., Chapter 19 Isolation of Subcellular Organelles and Structures. In Methods in Enzymology, Burgess, R. R.; Deutscher, M. P., Eds. Academic Press: 2009; Vol. 463, pp 305-328.
2. Zheng, N.; Tsai, H. N.; Zhang, X.; Rosania, G. R., The subcellular distribution of small molecules: from pharmacokinetics to synthetic biology. Mol Pharm 2011, 8 (5), 1619-28.
3. Kaspler, P.; Lazic, S.; Forward, S.; Arenas, Y.; Mandel, A.; Lilge, L., A ruthenium(ii) based photosensitizer and transferrin complexes enhance photo-physical properties, cell uptake, and photodynamic therapy safety and efficacy. Photochem Photobiol Sci 2016, 15 (4), 481-95.
4. Monro, S.; Colón, K. L.; Yin, H.; Roque, J.; Konda, P.; Gujar, S.; Thummel, R. P.; Lilge, L.; Cameron, C. G.; McFarland, S. A., Transition Metal Complexes and Photodynamic Therapy from a Tumor-Centered Approach: Challenges, Opportunities, and Highlights from the Development of TLD1433. Chemical Reviews 2019, 119 (2), 797-828.
5. Dickerson, M.; Sun, Y.; Howerton, B.; Glazer, E. C., Modifying Charge and Hydrophilicity of Simple Ru(II) Polypyridyl Complexes Radically Alters Biological Activities: Old Complexes, Surprising New Tricks. Inorg. Chem. 2014, 53 (19), 10370-10377.
6. Gandioso, A.; Vidal, A.; Burckel, P.; Gasser, G.; Alessio, E., Ruthenium(II) Polypyridyl Complexes Containing Simple Dioxo Ligands: a Structure-Activity Relationship Study Shows the Importance of the Charge. ChemBioChem 2022, n/a (n/a).

3. Why could compound 2 induce ribosome biogenesis? How did compound 2 induce nucleolar stress?

The reviewer asks a very difficult question. The fact of the matter is, we don’t know, and that is part of what is intriguing in this report. The structural characteristics of the compound are quite different from the platinum agents that cause ribosome biogenesis stress, and in turn, those platinum compounds are not similar to the organic compounds that have this effect!
We have added some text to explain that a common feature shared between phenanthriplatin and 2 is the polyaromatic lipophilic groups that are coordinated to transition metals. The specific goal of this manuscript was to determine if we could design a molecule that induces ribosome biogenesis stress based on the available literature for Pt(II) and Pt(IV) systems. Future studies that leverage our scaffold functionalized with a chemical handle (as DeRose has done) will be utilized to determine how ribosome biogenesis stress is induced.

“These studies demonstrate individual Ru(II) compounds can exhibit the various biological activities of phenanthriplatin, and we hypothesized that this may be done in a single molecule if key structural features, such as polyaromatic lipophilic functional groups are incorporated into the system.”

4. Page 2, right column, 2nd paragraph, please make sure the SI values cited in the text have the same decimal places as those included in Table 1.

We thank the reviewer. This has been corrected.

“The SI for cisplatin was 0.9, consistent with previous reports.26 Compound 1 had an SI of 4.9, while phenanthriplatin and 2 had an SI value of 13.8 and 15.2 respectively, displaying the capacity for this scaffold to selectively target cancerous cells over healthy cells.”

5. Page 4, 1st paragraph, the NCI60 screening data of phenanthriplatin have been published in doi: 10.1073/pnas.1207670109 please consider discussing the difference between phenanthriplatin and complex 2.

The reviewer is correct, Lippard’s 2012 PNAS publication is a source for the NCI results of phenanthriplatin. However, unfortunately, the numerical values are not included on the tree diagram for the Phenanthriplatin data in Figure S3 of the PNAS paper. This prevented their inclusion in the heatmap as we cannot guess the values. However, we can, and have, included some general and qualitative remarks on the NCI data following the reviewer’s request.

“However, the data available from previous reports allowed for some comparisons. Non-small cell lung cancers and central nervous system (CNS) cancers were both sensitive to phenanthriplatin and 2.45 The response across ovarian cancers was also comparable, suggesting that phenanthriplatin and 2 exhibit similar behaviors across different cell lines of the same cancer type. The response in renal and breast cancers, in contrast, differed between the two compounds. Renal cancers were sensitive to treatment with phenanthriplatin but breast cancer response was average.45 Renal cancers were resistant to 2 and breast cancers were generally sensitive. Since phenanthriplatin has been identified as having multiple MoAs, and 2 exhibits comparable sensitivity-resistance profiles to phenanthriplatin, we decided to evaluate 2 for each of the plausible mechanisms highlighted by the NCI analysis: DNA damage, topoisomerase inhibition, microtubule dynamics, and ribosome biogenesis stress.”

6. Page 4, right column, 2nd paragraph, “phenanthriplatin produced the lowest nucleotide (nt) to metal center (mc) ratio, followed by cisplatin and 1, and then 2 (Fig. S20 and Table S3)”. Please check whether Fig S20 is properly cited here. Please clarify how did the nt/mc measured and the detailed conditions, such as treatment time and concentration, etc.

We thank the reviewer for drawing our attention to this mistake, and we have made corrections. The specific treatment conditions are indicated under DNA metalation in the methods section in the supplementary information. We have added description for nt/mc in the results section of the manuscript to aid readers in understanding how to interpret this information.

“The quantity of metal in the total genomic DNA was determined by ICP-MS, and the nucleotide to metal center ratio was calculated by dividing the number of nucleotides in the total genomic DNA present in each sample by the metal atoms.”
“Phenanthriplatin produced the lowest nucleotide (nt) to metal center (mc) ratio, followed by cisplatin and 1, and then 2 (Table S3).”

7. Page 7, 1st paragraph, please make sure the figures are correctly cited.
We greatly appreciate the reviewer alerting us to this mistake. We have corrected the following captions to accurately reflect what is present in the supplementary information.

“Notably, distinct bands for topoisomers were observed (Fig. S26D). In contrast, treatment of DNA with phenanthriplatin caused a decrease in DNA electrophoretic mobility, with significant smearing of the bands (Fig. S26A).”

Reviewer 2:
1. Regarding optical properties of the new Ru complexes: please, indicate in the Figure S29 caption the solvent used for running their UV/Vis spectra (water?). What about the emission properties of these Ru compounds (if any)?

We thank the reviewer for their request and inquiry. We have added text to the figure (now Figure S30) to clarify. The emissive properties of these compounds were not shown as the Ru(II) compounds were non-emissive.

“UV/Vis spectra plotted as extinction coefficients of compounds 1 (blue) and 2 (red) in acetonitrile. The data is representative of triplicate measurements. The compounds were not emissive.”

2. Mitochondrial stress was excluded as mechanism of action (Fig. 4E). This should be discussed, at least briefly, in the text.

We appreciate the reviewer pointing this out. We have added the following description in the text on page 7:

“Mitochondrial Dysfunction
Ru(II) molecules containing dip ligands have been shown to induce mitochondrial dysfunction,5 due to their lipophilicity and cationic charge.6 As compound 2 contains the dip ligand, its impact on mitochondrial function was assessed. The Seahorse MitoStress profile for cells pre-treated with 2 at 6x the IC50¬ value did not show any significant difference in response compared to the vehicle control (Fig. 4E). This result reveals that 2 does not interact directly with mitochondria or cause depolarization.”

3. There might be some typos in the section of studies in Escherichia coli (page 4, right column: Fig. S20?), and also in the bacterial cytological profiling section (page 4, right column: Fig. 4E and 4F?).

Thank you for identifying these mistakes. The necessary corrections have been applied.
“Phenanthriplatin produced the lowest nucleotide (nt) to metal center (mc) ratio, followed by cisplatin and 1, and then 2 (Table S3).”
“…and 2 (Fig. 2E) also exhibited filaments, with average lengths of 44 μm, 39 μm, and 94 μm, respectively (Fig. 2F).”

01-Feb-2023

Dear Dr Glazer:

Manuscript ID: CB-ART-12-2022-000247.R1
TITLE: A Monoadduct Generating Ru(II) Complex Induces Ribosome Biogenesis Stress and is a Molecular Mimic of Phenanthriplatin

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

You will shortly receive a separate email from us requesting you to submit a licence to publish for your article, so that we can proceed with the preparation and publication of your manuscript.

All RSC Chemical Biology articles are published under an open access model, and the appropriate article processing charge (APC) will apply. Details of the APC and discounted rates can be found at https://www.rsc.org/journals-books-databases/about-journals/rsc-chemical-biology/#CB-charges.

You can highlight your article and the work of your group on the back cover of RSC Chemical Biology. If you are interested in this opportunity please contact the editorial office for more information.

Promote your research, accelerate its impact – find out more about our article promotion services here: https://rsc.li/promoteyourresearch.

If you would like us to promote your article on our Twitter account @rsc_chembio please fill out this form: https://form.jotform.com/213543900424044.

We are offering all corresponding authors who are not already members of the Royal Society of Chemistry one year’s Affiliate membership as part of their APC. If you would like to find out more please email membership@rsc.org, including the promo code OA100 in your message. Learn all about our member benefits at https://www.rsc.org/membership-and-community/join/#benefit.

By publishing your article in RSC Chemical Biology, you are supporting the Royal Society of Chemistry to help the chemical science community make the world a better place.

With best wishes,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 1

The authors have satisfactorily addressed my previous concerns by including relevant discussions and correcting mistakes. This interesting piece of work is now publishable.