From the journal RSC Chemical Biology Peer review history

03-Mar-2023

Dear Dr Zhao:

Manuscript ID: CB-ART-11-2022-000230
TITLE: Identification of multidentate tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors that simultaneously access the DNA, protein and catalytic-binding sites by oxime diversification

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, which indicate that major revisions are necessary. It is important to be addressed the reviewer’s comment about the cellular activity and selectivity of novel inhibitors supported by new results and followed by expended discussion for TDP1 inhibitors as potential cancer therapeutics.

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

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Reviewer 1

The authors report on the synthesis of a series of TDP1 inhibitors using a microarray. A number of oxime, triazole and ethers derivatives were made and tested against the enzyme. Moderate activity was found with 380 nM for 6-D1 being the best. Furthermore, an X-ray structure of 9c in TDP1 is reported. Useful insights are gleaned into the binding mechanism of inhibitors to TDP1.
In terms of drug discovery, the ligands produced are not particularly drug-like, large and the most active one containing an oxime. In addition, using both peptide and DNA binding regions for better binding is a poor strategy since the inhibitors will be quite large and therefore with poor pharmacokinetic profiles. Nevertheless, the information provided enhances the understanding of the binding mechanism, which is useful for further development of therapeutically relevant compounds.
The abstract is quite long and has some small mistakes in it. It needs to be focused and cleaned up.

I am happy to recommend this manuscript for publication.

Reviewer 2

TDP1 is a promising target for inhibitors that wi enhance the activity of topoisomerase I inhibitors that have been used to treat cancer for many years. While a number of TDP1 inhibitors have been reported, there are concerns about their activity, specificity and clinical utility. In this study the authors have combined a rational, structured-based approach combined with synthesis and screening of targets chemical libraries to identify novel high affinity TDP1 inhibitors. Using a previously identified inhibitor that binds to the TDP1 catalytic core, the authors developed a synthetic strategy to build larger versions of the inhibitor that had the potential to extend binding into other regions of TDP1 involved in engaging with its substrate. While this is a nice result that validates their approach, there are no cell-based studies to address the activity and selectivity of the novel compounds and no discussion of their potential utility as leads for drug development.

Minor comment; The first part of the results section (Pages 4 and 5) was confusing and repetitive

Response to Reviewers' Comments

Reviewer 1
Comment: The authors report on the synthesis of a series of TDP1 inhibitors using a microarray. A number of oxime, triazole and ethers derivatives were made and tested against the enzyme. Moderate activity was found with 380 nM for 6-D1 being the best. Furthermore, an X-ray structure of 9c in TDP1 is reported. Useful insights are gleaned into the binding mechanism of inhibitors to TDP1. In terms of drug discovery, the ligands produced are not particularly drug-like, large and the most active one containing an oxime. In addition, using both peptide and DNA binding regions for better binding is a poor strategy since the inhibitors will be quite large and therefore with poor pharmacokinetic profiles. Nevertheless, the information provided enhances the understanding of the binding mechanism, which is useful for further development of therapeutically relevant compounds.

Response: We appreciate the many positive comments given by the Reviewer. TDP1 is an extremely challenging molecular target and part of this difficulty arises from the wide and open nature of the substrate binding pocket. To address this, we are exploring multidentate inhibitors that can simultaneously interact with the small catalytic pocket and the DNA and peptide substrate binding channels. Multidentate binding can afford significant thermodynamic advantages, which can theoretically approach the product of the affinities of individual components. For example, combining three millimolar-affinity fragments (ie, Kd = 10-3 M) can potentially result in an inhibitor with nanomolar affinity (ie, Kd = 10-9 M). A tridentate approach necessarily results in larger overall ligand size. However, this doesn't necessarily mean that the constructs are not "drug-like." Concepts of what constitutes a viable drug are evolving and expanding. This is exemplified by the high cellular efficacy of "proteolysis targeting chimeras" (PROTACs), which are heterobifunctional molecules composed of two active domains and a linker. Several recent publications are challenging the traditional tenets of the "Lipinski Rule of 5," which constrain parameters of what it means to be drug-like. We now make this clear by stating; “In spite of the fact that increases in molecular size and complexity required to realize these multidentate interactions may place such compounds outside parameters defined by the "Lipinski rule of 5” [Adv. Drug Deliv. Rev., 2001, 46, 3-26], this does not necessarily limit their suitability as drug candidates. [Nat. Rev. Chem., 2023, 7, 3-4]” (second paragraph, left column of page 2).

Comment: The abstract is quite long and has some small mistakes in it. It needs to be focused and cleaned up. I am happy to recommend this manuscript for publication.

Response: We have simplified and refocused the Abstract and reduced the word count.


Reviewer 2

Comment: TDP1 is a promising target for inhibitors that will enhance the activity of topoisomerase I inhibitors that have been used to treat cancer for many years. While a number of TDP1 inhibitors have been reported, there are concerns about their activity, specificity and clinical utility. In this study the authors have combined a rational, structured-based approach combined with synthesis and screening of targets chemical libraries to identify novel high affinity TDP1 inhibitors. Using a previously identified inhibitor that binds to the TDP1 catalytic core, the authors developed a synthetic strategy to build larger versions of the inhibitor that had the potential to extend binding into other regions of TDP1 involved in engaging with its substrate. While this is a nice result that validates their approach, there are no cell-based studies to address the activity and selectivity of the novel compounds and no discussion of their potential utility as leads for drug development.

Response: We appreciate the Reviewer's highly positive comments. In response to the concerns raised, we have added data from cell-based studies as well as data that measures selectivity toward TDP1 versus TDP2. We have also added cytotoxicity data in human colorectal carcinoma cell line HCT116, and for the lead compounds, including oxime (E)-6-D1, triazole 7d and ether 8b, we have shown synergistic effects with the TOP1 inhibitor camptothecin (CPT) in HCT116 cell line (see Table S5 and Figures S5-S7 in the revised ESI). We have also included a discussion of this new data; “Lead compounds, including oxime (E)-6-D1, triazole 7d and ether 8b, were selected to evaluate their TDP1 selectivity over TDP2 using in vitro gel-based assays (Table S5 and Fig. S5, ESI). Oxime (E)-6-D1 shows 74-fold greater inhibitory potency against TDP1 than against TDP2. Ether 8b is also 36-fold more potent against TDP1 than against TDP2. Potencies of triazole 7d are similar, however an 8-fold enhancement was observed against TDP1 relative to TDP2. In the HCT116 human colon cancer cell line, oxime (E)-6-D1, triazole 7d and ether 8b showed cytotoxicity CC50 values of 4.8 µM, 136 µM, and 4.3 µM, respectively (Fig. S6, ESI). The CC50 values are higher than the TDP1 in vitro IC50 values. At concentrations below their CC50 values, compounds (E)-6-D1, 7d and 8b were shown to act synergistically with the TOP1 inhibitor CPT using HCT116 cells (Fig. S6 and Fig. S7, ESI). These data are consistent with the compounds selectively targeting TDP1” (last paragraph from bottom, left column of page 5).

Comment: The first part of the results section (Pages 4 and 5) was confusing and repetitive

Response: We have condensed the first part of the Results Section to refocus and clarify its presentation (left column in page 4).

26-Mar-2023

Dear Dr Zhao:

Manuscript ID: CB-ART-11-2022-000230.R1
TITLE: Identification of multidentate tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors that simultaneously access the DNA, protein and catalytic-binding sites by oxime diversification

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

Reviewer 1

Very good(: