AtomNet-Aided OTUD7B Inhibitor Discovery and Validation

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

We deeply appreciate the constructive suggestions provided by the editor and reviewers during the initial review of our manuscript. The comments and suggests have been very helpful in guiding us to further improve our study. Over the past 9 days, we have obtained additional experimental evidence to further support our conclusions and extensively revised our manuscript. We hope that the editor and the reviewers agree that we have fully addressed all the reviewers’ concerns and substantially strengthened our paper. Therefore, we believe that the revised manuscript is now suitable for publication in Cancers.

 

Reviewer #1

This manuscript reports virtual screening to discovery OTUD7B inhibitors and investigate the compounds in NSCLC. Overall, the identified virtual screening hits are not rigorously characterized as OTUD7B inhibitors and biological results are inconsistent throughout the manuscript. Thus, it is not possible to confidently attribute a specific phenotype to OTUD7B inhibition.

Response: We thank the reviewer for raising great questions to help us further improve our manuscript. As detailed below, we have obtained new experimental results to support 7Bi as a possible OTUD7B inhibitor that suppresses OTUD7B activity but not other DUBs tested in our in vitro DUB activity assays. Furthermore, 7Bi exerts suppressive effects towards NSCLC and leukemia cell proliferation.

 

Compound related concerns

No direct read-out of the compound binding OTUD7B (no target engagement).

Response: We fully agree with the reviewer that target engagement tests are important. We originally planned determine the binding affinity of 7Bi to recombinant OTUD7B-OTU using ITC. However, we had limited amounts in hand and our vendor failed to synthesize 7Bi, so we were unable to obtain a quantity large enough to attempt ITC in a reproduceable and thoroughly tested manner. In leu of quantitative binding, we performed more in-depth structural simulations for 7Bi binding to OTUD7B. We found in the ubiquitin-bound crystal structure that served as a template for our screen, the C-terminal VLRLRG sequence (residues 70-75) of the ubiquitin ligand makes several lipophilic and charge-charge interactions with the binding site (revised Fig. S2J; light blue, thin sticks). In several of the poses for 7Bi, lipophilic cyclopentyl or methyl groups make similar lipophilic interactions with the surface of the binding site. Likewise, the amide bonds connecting the three central pyrazole rings may mimic the peptide backbone of the ubiquitin. While 7Bi lacks the charged interaction of the endogenous ligand, it can engage in more direct contact with the small sub-pocket near F391. We hope the reviewer agrees with us that further investigation to determine the direct binding between 7Bi and OTUD7B by crystallization or NMR would be needed to guide SAR studies. Compounds with improved potency, specificity and solubility would be more suitable for biotin-tagging mediated pulldown coupled mass spectrometry analysis to determine the in-cell specificity of improved 7Bi in binding and suppressing OTUD7B, which warrants a separate follow-up study.

 

No selectivity data against other OTUs, not even the close homolog OTUD7A.

Response: We fully agree with reviewer and following reviewer’s suggestion, we have purified a truncated form of OTUD7A (OTUD7A-UBA+OTU) from bacteria and included it in the in vitro deubiquitination assays using ubiquitin-AMC as substrate. As shown in the revised Fig. 3E-3F, although 7Bi suppressed OTUD7B activity it failed to suppress OTUD7A-mediated hydrolysis of AMC from ubiquitin. In addition, as shown in revised Fig. 3G-3H, 7Bi also failed to block cleavage of ubiquitin-AMC catalyzed by either USP21 or USP9X. These data further confirm the relative specificity of 7Bi in suppressing OTUD7B activity in this in vitro deubiquitination assay.

 

Limited selectivity data against other DUBs (only USP21 and USP9X, USP).

Response: We thank the reviewer for raising this question and fully agree with the reviewer. We intended to perform pan-DUB analyses with LifeSensors but was informed that this single assay would take at least 6 months. As the reviewer suggested, the most likely off-target candidate for 7Bi is its close family member OTUD7A, which has the highest sequence similarity (~70%). As shown in the revised Fig. 3E-3F, although 7Bi suppressed OTUD7B activity it failed to suppress OTUD7A-mediated hydrolysis of AMC from ubiquitin. We fully agree with the reviewer that the specificity for 7Bi is an important factor to consider and improving 7Bi specificity is the major goal for the following SAR studies in a separate project. We hope the reviewer agrees with us that 7Bi can serve as a starting chemical backbone that further medicinal chemistry can rely on to carry out rounds of SAR studies to improve its specificity and potency.

 

Intro states OTUD7B is very linkage specific (for K-63 Ubiquitin) but the in vitro assay used K-11 linkages.

Response: We thank the reviewer for raising this question and apologize for confusion raised in the introduction. In the revised manuscript, we have modified the text to clearly indicate that OTUD7B catalyzes deubiquitination of multiple ubiquitin linkages including both K63 and K11. As we stated in the revised introduction, OTUD7B has been shown to either remove K63-linked poly-ubiquitin chains from p62 [1] and GbL [2], or antagonize APC/C-mediated substrate ubiquitination by K11-linkages in cells [3]. Thus, both K63 and K11 linked ubiquitin chains are physiological substrates for OTUD7B. To avoid further confusion, we have emphasized the linkage specificity of OTUD7B in the revised manuscript.

            In addition, to further address if 7Bi-mediated OTUD7B inhibition affects non-K11 linked ubiquitin, we used ubiquitin-AMC as an additional substrate without any linkage preference and found that 7Bi largely suppressed only OTUD7B (revised Fig. 3E), but not OTUD7A (revised Fig. 3F), USP21 (revised Fig. 3G) or USP9X (revised Fig. 3H). These newly obtained experimental evidence suggests 7Bi suppresses OTUD7B activity overall and is not limited to K11-linked di-ubiquitin.

 

A prior art search needs to be included for the inhibitor 7Bi. Based on its structure, it likely has many other activities.

Response: We thank the reviewer for raising this important question and fully agree with the reviewer that this is necessary. We have searched Scifinder for literature or patents on 7Bi, and there were none. We also found no reported bioactivities for closely related compounds. This result is not unexpected with commercially available screening compounds. We have included this information in the revised manuscript.

 

A virtual screen was conducted to identify inhibitors yet a docked structure is not shown and discussed.

Response: We thank the reviewer for raising this great question. We didn’t include this information because due to the size and side-chain flexibility within the targeted protein-protein interaction site, it is challenging to select a definitive binding pose for 7Bi from the ones suggested by the AtomNet screen. We have performed more intense structural simulation during this revision and presented a docked model in the revised Fig. S2J, as well as included discussion on this model in the revised manuscript. We agree with the reviewer that inclusion of this information will be helpful for readers to better understand the study.

 

No mention of how compounds were QC’d.

Response: We thank the reviewer for raising this question. All 10 mM DMSO-compound stocks were analyzed by Mcule for purity via LC-MS. Only compounds that passed ≥85% purity were shipped and tested.

In addition, we further purified 7Bi by HPLC to 99% purity (revised Fig. S2D). Compared with 7Bi received from vendors with a 90% purity, HPLC purified 7Bi retained its activity in suppressing OTUD7B-mediated Akt-pS473 regulations in cells (revised Fig. 3J) and inhibiting OTUD7B-catalyzed K11-diubiquitin cleavage in vitro (revised Fig. 3I).

 

Western blots inconsistent and do not agree with their discussion

For their argument: OTUD7B deubiquitinates GβL, which activates Akt-pS473 signaling. so the KD or inhibition of OTUD7B should increase the degradation of GβL and decrease Akt- pS473. Except in Figure 1B: shRNA K.D. of OTUD7B hardly decreased Akt-pS473, and has no effect on GβL protein amount.

Response: We thank the reviewer for raising this question. Our previous work reports that OTUD7B removes K63-linked ubiquitination of GbL to promote GbL incorporation into mTORC2 complexes, thus activating Akt to induce Akt-pS473 signals [2]. Given it is a K63-linked ubiquitination, it does not regulate GbL protein stability but rather controls GbL binding with mTOR complex components. Thus, with either OTUD7B depletion or inhibition, we don’t observe GbL protein stability changes. This is consistent with the previous report [2]. The readout of OTUD7B deletion or inhibition by 7Bi is reduced Akt-pS473 signals, as shown in all figures. Due to the high quality of the Akt-pS473 antibody and abundance of this phosphorylation event in cells tested, the relatively smaller changes on Akt-pS473 signals are likely due to longer exposures for western blotting. We have replaced Akt-pS473 blots with shorter exposures to better reflect these changes in the revised Fig 1G and 4J. We have included more information for the regulation of OTUD7B on GbL in the revised introduction to avoid future confusion.

 

Quantification of western blots & margins for how they selected their “hits” questionable (#19 only at 0.83 relative to DMSO control, but WB does not look less intense than DMSO).

Response: We thank the reviewer for raising this question. We have re-run and re-blotted samples in Fig. 1G following the alphabetic order as the reviewer suggested below. In the newly obtained data, compound #19 suppressed ~22% Akt-pS473 signals.

 

The “top 10” compounds identified in Figure 1I are not the same as the “top ten” compounds that the group tested in different NSCLC lines (Figure 2A-D), and no explanation to why they changed the compounds. The hit #19 which they continue with is not even included here.

Response: We apologize for the confusion. The order of the compounds is not the same in Fig 2A-2D as in Fig 1I; however, Fig 2A-2D contain 8 out of 10 top hits from Fig 1I. The missing compound #37 was included in Fig S1A-S1C. Given the compound #19 is the major hit, we separated it from all others and included it Fig 2E-2F. The order of the 10 hits in Fig 1I is listed by efficiency while the order of the compounds in Fig 2A-2D is listed based on the number of compounds. This might be the reason for this confusion and hope our changes have made this more clear.  

 

Figure 2 A – D, no consistent effects from compounds across the different cell lines, contrary to what they state in discussion.

Response: We agree with the reviewer that top 10 hits behaved differently among all 4 NSCLC cell lines we tested and that is why we only focused on hits that decreased Akt-pS473 signals in at least 3 NSCLC cells as stated in discussion. We also clearly state that data in Fig 2A-2D excluded most top 10 hits from being further examined leaving only compounds #51 and #19 as candidates. Further in vitro deubiquitination assays excluded #51 (revised Fig. S2A) as a direct OTUD7B inhibitor, leaving only #19 for further analysis. To avoid further confusion, we have revised our discussion by including these compound numbers.

 

Intentionally moved data in Figure 4 to hide the THP1 (Fig 4J) because it doesn’t match the trends.

Response: We agree with the reviewer that Akt-pS473 signal changes are not dramatic upon OTUD7B inhibition in the original Fig 4J, which is more likely due to long exposure for western blotting. We have re-run and reblotted the same samples but with a shorter exposure time to better illustrate the changes.

 

Story is scatered and nonlogical

Started with virtual screen then immediately went into NSCLC cells with extremely weak compounds at 10 uM, much lower than expected for weak compounds and for a single- concentration evaluation.

Response: We agree with the reviewer that the IC50 for compounds are not high, we started a single 10 mM dose of each compound as a starting point considering the amount of work involved. Afterwards, candidates from this single dose, single time-point test were followed by time- and dose-dependent tests focusing on promising hits in Fig 2 and 3 (such as the hit #19). We fully agree with the reviewer that multiple complementary assays (such as a combination of in vitro catalytic assay coupled with in-cell assay) would be optimal. In this study, we emphasize on cellular effects given our previous experiences revealed that compounds screened form in vitro assays might not exert predicted cellular effects, which is the terminal goal for developing inhibitors. Thus, in this study we started from in-cell based screens and relied on in vitro catalytic assays for validation.

 

None of the compound lists/data are presented numerically to distract from the inconsistencies across different Figures.

Response: We thank the reviewer for raising this question. Following the reviewer’s suggestion, we have re-run and reblotted samples from Fig 1G following the numerical order. All the compounds listed in Fig 2 and Fig S1 originally follow the numerical order.

 

Somehow their hit compound #19 had an effect at 10 uM in cells, but the in vitro IC50 = 40 uM.

Response: We thank the reviewer for raising this question. Given the in vitro assays are using over-physiological doses of diubiquitin and OTUD7B, the IC50 obtained in vitro can be varied by the amount of OTUD7B or diubiquitin substrate used in the assay. Thus, it is hard to make a direct comparison between in vitro and in-cell IC50 values. Nonetheless, compound #19 inhibits OTUD7B, a known regulator of GbL, in enzyme assays.

 

Other error

Multiple inconsistencies in the text (i.e. stating 15% in Line 206, but 10% Line 219 for the same data).

Response: We thank the reviewer for pointing out this deficiency. We have corrected the figure legend to reflect the same 10% changes in the revised manuscript.

 

Misspelling of multiple works (“MINDY” Line 43, “fluorescently” Line 154, “approaches” Line 200).

Response: We thank the reviewer for pointing out this deficiency. We have corrected these typos and also carefully read through the manuscript to eliminate further grammar mistakes.

 

References:

1. Xie W, Tian S, Yang J, Cai S, Jin S, Zhou T, Wu Y, Chen Z, Ji Y, and Cui J, OTUD7B deubiquitinates SQSTM1/p62 and promotes IRF3 degradation to regulate antiviral immunity. Autophagy, 18(10): p. 2288-2302.2022.
2. Wang B, Jie Z, Joo D, Ordureau A, Liu P, Gan W, Guo J, Zhang J, North BJ, Dai X, Cheng X, Bian X, Zhang L, Harper JW, Sun SC, and Wei W, TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling. Nature, 545(7654): p. 365-369.2017.
3. Bonacci T, Suzuki A, Grant GD, Stanley N, Cook JG, Brown NG, and Emanuele MJ, Cezanne/OTUD7B is a cell cycle-regulated deubiquitinase that antagonizes the degradation of APC/C substrates. EMBO J, 37(16).2018.

Reviewer 2 Report

The paper "Artificial Intelligence-aided OTUD7B inhibitor discovery and validation" presents the usage of artificial intelligence for screening 4 million compounds for OTUD7B inhibitors. The results show that both in vivo and in vitro tests were successful. The treatment can be applied for patients who suffer from breast and lung cancer.

 

- Originality/Novelty: The paper is novel as it uses artificial intelligence to determine the activity of OTUD7B inhibitors.

 

- Significance: The results of the research are interpreted properly, the fine tuning of model parameters being performed.

 

- Quality of Presentation: The article is written appropriately, respecting the logical succession of sections. Data and analyses are presented graphically and inside tables. The results were outlined using high standards, the advantages of the machine learning method being very clear.

 

On Page 3, line 103, please arrange the line separators, such that the shRNA sequences will fit better.

 

The title of Figure 1 should appear under the figure. The other descriptions of every figure can appear inside a paragraph and not following the figure name.

 

The conclusions section has to include details about future work.

 

- Scientific Soundness: The authors copied a lot from their previous work without changing the content, figures and tables. The paper offers enough details to allow the reproduction of the results, but it needs to have a higher scientific value.

 

The paragraph "Colonies were washed 121 with distilled water, fixed with methanol for 30 min and stained with 0.5% crystal violet 122 overnight. Colonies were then washed by distilled water and air-dried. Colony numbers 123 were manually counted. Three independent experiments were performed." was taken from the paper Zhichuan Zhu, Xin Zhou, Hongwei Du, Erica W. Cloer et al. "STING Suppresses Mitochondrial VDAC2 to Govern RCC Growth Independent of Innate Immunity", Advanced Science, 2022 which is not cited.

 

Same for the paragraph "Statistical analyses were performed using the SPSS 11.5 Statistical Software. p ≤ 0.05 180 was considered statistically significant. The results are shown as means ± SD from at least 181 two or three independent experiments as indicated in figure legends. Differences between 182 control and experimental conditions were evaluated by One-way ANOVA." which was taken from Siyuan Su, Jianfeng Chen, Yao Jiang, Ying Wang et al. "Control Protein Stability to Govern Ewing Sarcoma Growth ", Advanced Science, 2021 and which was not cited.

 

- Interest to the Readers: The content of the article would surely interest the readers of the Cancers journal, and not only them.  

 

- Overall Merit:  The findings and their implications should be discussed in the broadest context possible and limitations of the work should be also further highlighted. Please present the novelty compared to the previous published papers.

 

- English Level: The level of English language is advanced. Through the entire paper, the language was appropriate and understandable, being easy to follow the flow since the beginning.

Author Response

We deeply appreciate the constructive suggestions provided by the editor and reviewers during the initial review of our manuscript. The comments and suggests have been very helpful in guiding us to further improve our study. Over the past 9 days, we have obtained additional experimental evidence to further support our conclusions and extensively revised our manuscript. We hope that the editor and the reviewers agree that we have fully addressed all the reviewers’ concerns and substantially strengthened our paper. Therefore, we believe that the revised manuscript is now suitable for publication in Cancers.

Reviewer #2

The paper "Artificial Intelligence-aided OTUD7B inhibitor discovery and validation" presents the usage of artificial intelligence for screening 4 million compounds for OTUD7B inhibitors. The results show that both in vivo and in vitro tests were successful. The treatment can be applied for patients who suffer from breast and lung cancer.

 

- Originality/Novelty: The paper is novel as it uses artificial intelligence to determine the activity of OTUD7B inhibitors.

- Significance: The results of the research are interpreted properly, the fine tuning of model parameters being performed.

- Quality of Presentation: The article is written appropriately, respecting the logical succession of sections. Data and analyses are presented graphically and inside tables. The results were outlined using high standards, the advantages of the machine learning method being very clear.

Response: We thank the reviewer for recognizing the novelty of our study. We also appreciate the constructive comments from the reviewer to help us further improve our manuscript.

 

1. On Page 3, line 103, please arrange the line separators, such that the shRNA sequences will fit better.

Response: We thank the reviewer for raising this great suggestion. In the revised manuscript, we have only included the target sequence for shRNAs and separated them in each line for better visualization.

 

2. The title of Figure 1 should appear under the figure. The other descriptions of every figure can appear inside a paragraph and not following the figure name.

Response: We thank the reviewer for raising this suggestion and we have made corresponding changes in the revised manuscript.

 

3. The conclusions section has to include details about future work.

Response: We thank the reviewer for raising this great suggestion. We have included future work in the revised conclusion.

 

- Scientific Soundness: The authors copied a lot from their previous work without changing the content, figures and tables. The paper offers enough details to allow the reproduction of the results, but it needs to have a higher scientific value.

Response: We thank the reviewer for raising this question. Following the reviewer’s suggestion, we have thoroughly revised the method section with more details, and reworked the wording of experimental procedures.

 

The paragraph "Colonies were washed 121 with distilled water, fixed with methanol for 30 min and stained with 0.5% crystal violet 122 overnight. Colonies were then washed by distilled water and air-dried. Colony numbers 123 were manually counted. Three independent experiments were performed." was taken from the paper Zhichuan Zhu, Xin Zhou, Hongwei Du, Erica W. Cloer et al. "STING Suppresses Mitochondrial VDAC2 to Govern RCC Growth Independent of Innate Immunity", Advanced Science, 2022 which is not cited.

Response: We sincerely thank the reviewer for pointing this out. Following the reviewer’s suggestion, we have cited this publication, as well as revised the method description to distinguish the statements.

 

Same for the paragraph "Statistical analyses were performed using the SPSS 11.5 Statistical Software. p ≤ 0.05 180 was considered statistically significant. The results are shown as means ± SD from at least 181 two or three independent experiments as indicated in figure legends. Differences between 182 control and experimental conditions were evaluated by One-way ANOVA." which was taken from Siyuan Su, Jianfeng Chen, Yao Jiang, Ying Wang et al. "Control Protein Stability to Govern Ewing Sarcoma Growth ", Advanced Science, 2021 and which was not cited.

Response: We sincerely thank the reviewer for pointing this out. Following the reviewer’s suggestion, we have cited this publication, as well as revised the method description to distinguish the statements.

 

- Interest to the Readers: The content of the article would surely interest the readers of the Cancers journal, and not only them.  

Response: We thank the reviewer for recognizing the novelty of our study.

 

- Overall Merit:  The findings and their implications should be discussed in the broadest context possible and limitations of the work should be also further highlighted. Please present the novelty compared to the previous published papers.

Response: We thank the reviewer for raising this great suggestion. We have searched Scifinder for literature or patents on 7Bi, and there were none. We also found no reported bioactivities for closely related compounds. For the novelty of the scientific approach, distinct from our previously published 7Ai studies (Su et al. Advanced Science 2021, 8(14):e2004846), we utilized ELISA assays that are more suitable for larger scale screens/validations, and coupled with in vitro catalytic assays using both K11-linked di-ub or ub-AMC with substrates for further selection. To the best of our knowledge, this is the first non-covalent small molecule inhibitor of OTUD7B. In addition, we included more limitations in the revised discussion section.

 

- English Level: The level of English language is advanced. Through the entire paper, the language was appropriate and understandable, being easy to follow the flow since the beginning.

Response: We thank the reviewer for this great comment.

Reviewer 3 Report

The authors reported the chemical structure of an inhibitor of OTUD7B, which is a deubiquitinase implicated in lung cancer through upregulation of the AKT signaling pathway. The authors used the AtomNet platform for virtual screening, followed by the validation of the identified inhibitors through assays measuring the AKT signaling in cell lines or the cleavage of K11-linked ubiquitin chains in vitro. The authors demonstrated that pharmacological inhibition of OTUD7B led to inhibition of AKT signaling in NSCLC and HEK293 cells, as well as in several leukemia cell lines. The identified inhibitor can be further developed into therapeutics for treating cancers, in which overexpression of OTUD7B is implicated.

Overall, the manuscript is clearly written and the methods used are all well-established and reported in previous publications. Given the importance of deubiquitinases in normal physiology and tumorigenesis, the identification of an inhibitor of OTUD7B provides not only a useful chemical probe for further functional studies of OTUD7B but also can lead to development of more potent compounds for cancer therapeutics.

I only have a few minor suggestions

1.    The introduction should include a more detailed description of the Akt-pS473 pathway and the role of OTUD7B/GbL in this pathway because it is the most relevant pathway for this study.

2.    Although the authors showed that one of the inhibitors inhibits the cleavage of K11-linked diubiquitin substrate in a biochemical assay, this experiment together with other validations in the manuscript is not sufficient to prove that the identified compound inhibits the cancer cell proliferation through on-target effects. The authors should make this point clear in the discussion.

3.    OTUD7B removes K63-linked ubiquitination on GbL to promote mTORC2 activation and this is the pathway targeted by the identified inhibitor. However, in the in vitro biochemical assays, the authors used K11-linked diubiquitin as the substrate. The authors should explain the rationale for using this substrate, which has a different linkage from the physiological substrate of relevance to NSCLC.

Author Response

We deeply appreciate the constructive suggestions provided by the editor and reviewers during the initial review of our manuscript. The comments and suggests have been very helpful in guiding us to further improve our study. Over the past 9 days, we have obtained additional experimental evidence to further support our conclusions and extensively revised our manuscript. We hope that the editor and the reviewers agree that we have fully addressed all the reviewers’ concerns and substantially strengthened our paper. Therefore, we believe that the revised manuscript is now suitable for publication in Cancers.

Reviewer #3

The authors reported the chemical structure of an inhibitor of OTUD7B, which is a deubiquitinase implicated in lung cancer through upregulation of the AKT signaling pathway. The authors used the AtomNet platform for virtual screening, followed by the validation of the identified inhibitors through assays measuring the AKT signaling in cell lines or the cleavage of K11-linked ubiquitin chains in vitro. The authors demonstrated that pharmacological inhibition of OTUD7B led to inhibition of AKT signaling in NSCLC and HEK293 cells, as well as in several leukemia cell lines. The identified inhibitor can be further developed into therapeutics for treating cancers, in which overexpression of OTUD7B is implicated.

Overall, the manuscript is clearly written and the methods used are all well-established and reported in previous publications. Given the importance of deubiquitinases in normal physiology and tumorigenesis, the identification of an inhibitor of OTUD7B provides not only a useful chemical probe for further functional studies of OTUD7B but also can lead to development of more potent compounds for cancer therapeutics.

Response: We thank the reviewer for recognizing the novelty of our study. We also appreciate the constructive comments from the reviewer to help us further improve our manuscript.

 

I only have a few minor suggestions.

1. The introduction should include a more detailed description of the Akt-pS473 pathway and the role of OTUD7B/GbL in this pathway because it is the most relevant pathway for this study.

Response: We thank the reviewer for raising this great suggestion. We have included more detailed descriptions about this signaling pathway in the revised introduction.

 

2. Although the authors showed that one of the inhibitors inhibits the cleavage of K11-linked diubiquitin substrate in a biochemical assay, this experiment together with other validations in the manuscript is not sufficient to prove that the identified compound inhibits the cancer cell proliferation through on-target effects. The authors should make this point clear in the discussion.

Response: We thank the reviewer for raising this great suggestion and fully agree with the reviewer that with current data presented, we could not draw a solid conclusion to claim 7Bi as a specific inhibitor. We have included this caveat in the revised discussion as suggested by reviewer in the revised manuscript.

 

3. OTUD7B removes K63-linked ubiquitination on GbL to promote mTORC2 activation and this is the pathway targeted by the identified inhibitor. However, in the in vitro biochemical assays, the authors used K11-linked diubiquitin as the substrate. The authors should explain the rationale for using this substrate, which has a different linkage from the physiological substrate of relevance to NSCLC.

Response: We thank the reviewer for raising this great question and apologize for the confusion. As stated in response to reviewer 1, OTUD7B has been shown to either remove K63-linked poly-ubiquitin chains from p62 [1] and GbL [2], or antagonize APC/C-mediated substrate ubiquitination by K11-linkages in cells [3]. Thus, both K63 and K11 linked ubiquitin chains are physiological substrates for OTUD7B. To avoid further confusion, we have emphasized the linkage specificity of OTUD7B in the revised manuscript.

In addition, to further address if 7Bi-mediated OTUD7B inhibition affects non-K11 linked ubiquitin, we used ubiquitin-AMC as an additional substrate without any linkage preference and found that 7Bi largely suppressed only OTUD7B (revised Fig. 3E), but not OTUD7A (revised Fig. 3F), USP21 (revised Fig. 3G) or USP9X (revised Fig. 3H). These newly obtained experimental evidence further support effects of 7Bi on suppressing OTUD7B activity is not only limited to K11-linked diubiquitin.

 

References:

1. Xie W, Tian S, Yang J, Cai S, Jin S, Zhou T, Wu Y, Chen Z, Ji Y, and Cui J, OTUD7B deubiquitinates SQSTM1/p62 and promotes IRF3 degradation to regulate antiviral immunity. Autophagy, 18(10): p. 2288-2302.2022.
2. Wang B, Jie Z, Joo D, Ordureau A, Liu P, Gan W, Guo J, Zhang J, North BJ, Dai X, Cheng X, Bian X, Zhang L, Harper JW, Sun SC, and Wei W, TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling. Nature, 545(7654): p. 365-369.2017.
3. Bonacci T, Suzuki A, Grant GD, Stanley N, Cook JG, Brown NG, and Emanuele MJ, Cezanne/OTUD7B is a cell cycle-regulated deubiquitinase that antagonizes the degradation of APC/C substrates. EMBO J, 37(16).2018.