From the journal RSC Chemical Biology Peer review history

12-Apr-2024

Dear Dr Kelly:

Manuscript ID: CB-ART-01-2024-000027
TITLE: Metabolically Activated Proteostasis Regulators that Protect Against Erastin-Induced Ferroptosis

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Reviewer 1

In their study Kline et al. improve their previously designed compound AA147 by altering the linker to a carbamate bioisostere, maintaining the capability of the compound to activate Atf6. Furthermore, the authors state that the new compound AA28 forms covalent bond to KEAP1, as did AA147, and thereby activates NRF2. Altering the B-ring to a furan moiety increased the specificity for NRF2. Consequently, the authors state, that the activation of NRF2 protects cells against ferroptosis induced by Erastin or Glutamate in HT22 cells. Intriguingly, one of the analogues AA64 seems to protect the cells from ferroptosis independently of NRF2 activation. The study is well done, however there are some minor limitations, which should be addressed.

Minor points:
1. Figure 2F is only done in single lanes, thus this experiment is unconvincingly showing the covalent binding of AA28 to KEAP1. It should be repeated with duplicates and the blot should be quantified to support their statement.
2. The authors only use Erastin and Glutamate as ferroptosis inducer. They should also test RSL3 or FIN56 and include the Ferrostatin-1 controls in their ferroptosis experiments.
3. It would be nice to include immunoblots and/or qPCR for Nqo1 or Hmox1 in figure 3, as the foldchange of Nqo1 in response to the compounds in the HT22 cells is a lot lower than in the IMR32 cell line and basing the protection from ferroptosis on this small effect is unconvincing.
4. Why did the authors use Thapsigargin as control for ER-stress, as tunicamycin would have been the more obvious choice, as the authors try to address the Atf6 arm of the UPR?
5. I suggest using black and white color layout for the C11-Bodipy, the panels are small and green/red is not readily visible.
6. In the end, not much proteostasis is tested here and the connection to Nfe2l1, which is intricately linked to NRF2, also involved in ferroptosis, and truly regulating proteostasis (PMID: 34999280) has not been made in this study.

Reviewer 2

This study investigates the structure-activity relationship of a series of small-molecule NRF2 activators, describing improved protection against neurotoxic insults in cell culture models. The manuscript builds on prior work by the same lab (ref. 11), which identified the lead compound AA147 as an activator of NRF2 in neuronal cells, providing protection against glutamate-mediated oxytosis. The first half of the paper describes a medicinal chemistry campaign to modify the linker and the B-ring of AA147. The authors identified that the amide linker, which has presumed protease sensitivity, can be replaced with a carbamate linker (compound AA28). Then, various B-ring substitutions are explored by coupling a series of hydroxymethyl-aryl and -cycloalkane groups to the carbamate. Several compounds with improved activity are identified, and the authors narrow in on the furfuryl substitution as the B ring. Nonetheless, other compounds with promising activity in the larger SAR exploration (Fig. 3B) were not further prioritized, which was not explained. Ultimately, the authors claim that they identify compound AA28 as an improved scaffold based on two measures: 1. Improved viability of cells treated with AA28 (and related compounds) compared to the parent compound AA147; and 2. Improved potencies – although this second point is poorly defined (see comments below).
Overall, the experiments are carried out thoroughly, and the results are described logically. Newly synthesized compounds are characterized adequately. Even though this paper builds on prior work by the authors, the new analogs highlight a successful example of a medicinal chemistry campaign to improve the activity of the AA147 for future exploration as a neuroprotective agent. I only have a few comments that the authors should address before publication:


• The authors should more clearly define what they mean by improved potency. Dose-response curves are not clearly presented in a head-to-head comparison of the parent AA147 and the improved AA28 and other scaffolds. Based on comparison with ref. 11, it appears that the EC50 values are roughly similar (~2.9 µM vs. 3.9µM for ARE-LUC reporter), but lower EC50 for rescue of glutamate-induced toxicity (~0.625µM).
• Is the improved potency argument based on the reduced “off-target” activation of the unfolded protein response target BiP? If so, this should be explained further. The compounds were largely tested at 10µM concentrations in the qPCR assay, even though ARE-LUC activity and toxicity rescue had much lower EC50 values. For AA64 (and other prioritized compounds), a helpful addition would be showing full dose-response curves for this compound - either using the respective luminescence reporters (ARE-Luc vs. ERSE), or by qPCR of BiP vs. Nqo1 activation.
• In Fig. 3B, why is the activation of several compounds variable across concentrations and not following a clear dose-response curve? Especially the control compound AA28 does not appear to activate the ARE-LUC reporter at 5-10µM where it was tested before.
• Considering the ARE-LUC activity at lower doses for AA58 and AA65 in Fig. 3B, it is surprising that the authors did not pursue these compounds further. These compounds also showed high Nqo1 activation in Fig. 3C. Still, they were not systematically explored in the cell viability assays in Fig. 4. Only AA65 was tested at a single dose against elastin-mediated toxicity in Fig. 4F.
• “These results indicate that the presence of the furan B-ring altered the selectivity of our compounds towards NRF2 activation, relative to ATF6 activation in neuronal cell culture models.”: I recommend revising this statement. It is misleading to attribute the more selective NRF2 activation properties to the furan ring. The carbamate linker in AA28 also contributed to more NRF2 activation, highlighting that other features can drive this activity.
• The data in Fig. S2E shows that AA28yne also covalently modifies other proteins. Are these PDIs involved in ATF6 activation, as the authors have shown previously? This would be worth describing briefly.
• Why is the ARE-LUC signal in Fig. S3F so much higher compared to Fig. S2D? Is this due to the difference in transient transfections of the reporter?
• Fig. S4C: Why is the protection with AA147 so much lower than in the other two experiments in Fig. S4A,B?

RESPONSE TO REVIEWER COMMENTS:

REVIEWER #1

Reviewer #1 General Comments. “In their study Kline et al. improve their previously designed compound AA147 by altering the linker to a carbamate bioisostere, maintaining the capability of the compound to activate Atf6. Furthermore, the authors state that the new compound AA28 forms covalent bond to KEAP1, as did AA147, and thereby activates NRF2. Altering the B-ring to a furan moiety increased the specificity for NRF2. Consequently, the authors state, that the activation of NRF2 protects cells against ferroptosis induced by Erastin or Glutamate in HT22 cells. Intriguingly, one of the analogues AA64 seems to protect the cells from ferroptosis independently of NRF2 activation. The study is well done, however there are some minor limitations, which should be addressed.”

Response to Reviewer #1 General Comments: We thank the reviewer for the positive response to our manuscript. We address the reviewer’s remaining minor concerns related to the work as described below.

Reviewer #1 Minor Comment #1. “Figure 2F is only done in single lanes, thus this experiment is unconvincingly showing the covalent binding of AA28 to KEAP1. It should be repeated with duplicates and the blot should be quantified to support their statement.”

Response to Reviewer #1 Minor Comment #1: We have repeated the experiment in Fig. 2E (Fig. 2F in the original submission) in triplicate with the new blot showing two independent biological replicates per condition. We also now provide quantification of the three replicates in Fig. 2F to support the statement that AA28 and AA147 activate NRF2 signaling in neuron-derived cells via KEAP1 covalent modification.

Reviewer #1 Minor Comment #2. “The authors only use Erastin and Glutamate as ferroptosis inducer. They should also test RSL3 or FIN56 and include the Ferrostatin-1 controls in their ferroptosis experiments.”

Response to Reviewer #1 Minor Comment #2: We have added Figs. 5, S5 showing that our prioritized analogs AA64 and AA65 provide protection against ferroptosis induced by FINO2 and RSL-3. In addition, we demonstrate that our analogs provide ferroptosis protection across multiple cell lines. We also include Ferrostatin-1 (FS-1) as a control for many of our experiments, showing that the efficacy of protection afforded by our compounds is similar to that observed for FS-1.

Reviewer #1 Minor Comment #3. “It would be nice to include immunoblots and/or qPCR for Nqo1 or Hmox1 in figure 3, as the foldchange of Nqo1 in response to the compounds in the HT22 cells is a lot lower than in the IMR32 cell line and basing the protection from ferroptosis on this small effect is unconvincing.”

Response to Reviewer #1 Minor Comment #3: Our results show that the majority of our AA147 analogs, save for AA64, protect against ferroptosis through activation of the NRF2-regulated oxidative stress response (OSR). We demonstrate that these compounds activate the OSR in both IMR32 (Fig. 1D-F, 3B-D) and HT22 (Figs. S1E,F, S3A,B,E,F) cells and that co-treatment with the NRF2 inhibitor ML385 blocks both the induction of NRF2 target genes (Fig. 2A, S2B) and the protection afforded by these compounds (Fig. 4, S4). Importantly, the benefit of NRF2 activation in this protection is unlikely to be attributed to the expression of a single gene, but instead reflects the protection against oxidative stress induced by the entire NRF2-regulated transcriptional program. Thus, even relatively modest increases in the expression of specific genes (e.g., NQO1) do not accurately reflect the type of benefit conveyed by this class of compound. For example, we previously showed that relatively modest protein level changes in IRE1/XBP1s target genes convey protection in diverse cellular and in vivo models of disease by changing the expression of multiple different genes. We make this point clearer in the revised manuscript, as below. Notably, we, and others, use IMR32 cells to initially probe OSR activation, as these cells are well known to show highly dynamic signaling through this pathway.

We add the following line to the discussion: Page 10, Line 28 “Thus, our results indicate that pharmacologic activation of this protective pathway comprising multiple anti-oxidant factors using these AA147 analogs protects neurons against ferroptotic cell death.”


Reviewer #1 Minor Comment #4. “Why did the authors use Thapsigargin as control for ER-stress, as tunicamycin would have been the more obvious choice, as the authors try to address the Atf6 arm of the UPR?”

Response to Reviewer #1 Minor Comment #4: The SERCA inhibitor thapsigargin is commonly used by us and others to globally activate all three arms of the UPR. Notably, unlike tunicamycin, thapsigargin induced UPR activation does not require new protein synthesis and leads to stronger UPR activation at the timepoints used in this experiment. Thus, we have consistently found Tg to be a better reporter for maximal UPR activation in HEK293 cells and other cell models, as compared to tunicamycin.

Reviewer #1 Minor Comment #5. “I suggest using black and white color layout for the C11-Bodipy, the panels are small and green/red is not readily visible.”

Response to Reviewer #1 Minor Comment #5: We appreciate the reviewer’s comment. After converting the images to black and white, it becomes more difficult to accurately view changes in ratiometric differences of the fluorophore caused by lipid peroxidation in these images. Thus, we have decided to keep the images as color images, which we feel are clearer. However, we did enlarge the images in the revised manuscript to better visualize the results.

Reviewer #1 Minor Comment #6 “In the end, not much proteostasis is tested here and the connection to Nfe2l1, which is intricately linked to NRF2, also involved in ferroptosis, and truly regulating proteostasis (PMID: 34999280) has not been made in this study.”

Response to Reviewer #1 Minor Comment #5: Nfe2l1 is the gene that encodes the transcription factor NRF1. NRF1 is activated in response to various insults including ER stress (via impaired degradation) and regulates expression of proteins involved in different biological pathways, most notably the proteasome. In previous work, we defined the transcriptional signature of NRF1 activation in diverse cell lines, including HEK293 cells (Ibrahim et al (2020) Antioxidants). In our RNAseq profiling of HEK293 cells treated with proteostasis regulators such as AA147, we observe no change in the expression of putative NRF1 target genes. This indicates that this class of compound does not regulate NRF1 activity. Further, we show that co-treatment with selective NRF2 inhibitors such as ML385 blocks protection afforded by many of our prioritized compounds, save for AA64. This demonstrates that AA147 and related analogs protect against ferroptosis through the activation of NRF2.


REVIEWER #2.

Reviewer #2 General Comments. “This study investigates the structure-activity relationship of a series of small-molecule NRF2 activators, describing improved protection against neurotoxic insults in cell culture models. The manuscript builds on prior work by the same lab (ref. 11), which identified the lead compound AA147 as an activator of NRF2 in neuronal cells, providing protection against glutamate-mediated oxytosis. The first half of the paper describes a medicinal chemistry campaign to modify the linker and the B-ring of AA147. The authors identified that the amide linker, which has presumed protease sensitivity, can be replaced with a carbamate linker (compound AA28). Then, various B-ring substitutions are explored by coupling a series of hydroxymethyl-aryl and -cycloalkane groups to the carbamate. Several compounds with improved activity are identified, and the authors narrow in on the furfuryl substitution as the B ring. Nonetheless, other compounds with promising activity in the larger SAR exploration (Fig. 3B) were not further prioritized, which was not explained. Ultimately, the authors claim that they identify compound AA28 as an improved scaffold based on two measures: 1. Improved viability of cells treated with AA28 (and related compounds) compared to the parent compound AA147; and 2. Improved potencies – although this second point is poorly defined (see comments below).
Overall, the experiments are carried out thoroughly, and the results are described logically. Newly synthesized compounds are characterized adequately. Even though this paper builds on prior work by the authors, the new analogs highlight a successful example of a medicinal chemistry campaign to improve the activity of the AA147 for future exploration as a neuroprotective agent. I only have a few comments that the authors should address before publication.”

Response to Reviewer #2 General Comments: We thank the reviewer for their positive response to our work. We address the remaining reviewer comments as outlined below.

Reviewer #2 Comment #1. “The authors should more clearly define what they mean by improved potency. Dose-response curves are not clearly presented in a head-to-head comparison of the parent AA147 and the improved AA28 and other scaffolds. Based on comparison with ref. 11, it appears that the EC50 values are roughly similar (~2.9 µM vs. 3.9µM for ARE-LUC reporter), but lower EC50 for rescue of glutamate-induced toxicity (~0.625µM).”

Response to Reviewer #2 Comment #1: We agree. We have now clarified that our new compounds show reduced cellular toxicity and improved protection against glutamate- and erastin-induced ferroptosis in the revised manuscript. In the revised manuscript, we include additional dose-responsive analyses of ferroptosis protection afforded by our compounds to clearly demonstrate their improved ability to protect against erastin-induced cell death (Fig. 4).

It is important to point out that the EC50 values for the ARE-FLuc reporters were performed in human IMR32 cell lines, while protection against glutamate- and erastin-induced ferroptosis were primarily measured in mouse HT22 cell lines. We use used the IMR32 cells to monitor reporter activity, as these cells are well-established in the literature to show a large dynamic range for ARE-FLuc activation. The ARE-FLuc reporter is an engineered reporter based on the target gene NQO1; alternate NRF2 transcriptional targets with greater responsiveness to compound treatment/NRF2 activation at lower concentrations may mediate the protection against ferroptosis/glutamate-induced toxicity. Note that we now include data showing that the EC50 for AA64-dependent protection against erastin-induced toxicity in IMR32 cells is ~1.25 µM (Fig. 4C), while the EC50 for ARE-FLuc activation in these same cells is ~2.5 µM (Fig. 2B, S3A) showing similar levels of efficacy when comparing treatment with the same cell across these two cell lines.

Reviewer #2 Comment #2. “Is the improved potency argument based on the reduced “off-target” activation of the unfolded protein response target BiP? If so, this should be explained further. The compounds were largely tested at 10µM concentrations in the qPCR assay, even though ARE-LUC activity and toxicity rescue had much lower EC50 values. For AA64 (and other prioritized compounds), a helpful addition would be showing full dose-response curves for this compound - either using the respective luminescence reporters (ARE-Luc vs. ERSE), or by qPCR of BiP vs. Nqo1 activation.”

Response to Reviewer #2 Comment #2: We now include a direct comparison of ARE-FLuc and ERSE-FLuc activation in HT22 cells treated with increasing doses of either AA147 or AA64 (Fig. S3A,B). This shows that the EC50 for NRF2 activation is nearly identical between these compounds, while the EC50 for ATF6 activation is less for AA64. These results further support our assertion that compounds like AA64 show improved selectivity for NRF2 relative to ATF6.

Reviewer #2 Comment #3. “In Fig. 3B, why is the activation of several compounds variable across concentrations and not following a clear dose-response curve? Especially the control compound AA28 does not appear to activate the ARE-LUC reporter at 5-10µM where it was tested before.”

Response to Reviewer #2 Comment #3: We agree. We repeated the experiments shown in Fig. 3B, where each condition was repeated in triplicate. All three replicates are now shown in the revised Fig. 3B. This new dose-response data better defines the activity of these compounds and clearly shows the appropriate dose response for AA28. Moreover, it better highlights our rationale for selecting prioritized AA28 analogs for further study.

Reviewer #2 Comment #4. “Considering the ARE-LUC activity at lower doses for AA58 and AA65 in Fig. 3B, it is surprising that the authors did not pursue these compounds further. These compounds also showed high Nqo1 activation in Fig. 3C. Still, they were not systematically explored in the cell viability assays in Fig. 4. Only AA65 was tested at a single dose against elastin-mediated toxicity in Fig. 4F.”

Response to Reviewer #2 Comment #4: We initially prioritized AA64 for further analysis due to its apparent selectivity for NRF2 signaling over ATF6 signaling. We agree that AA65 has promise in the cell viability assays based on the potency in the OSR activation screen and similar activation of NRF2 target genes by qPCR analysis in neuron-derived cells. We add new data to the revised manuscript demonstrating that AA65 has similar potency in protecting against numerous modes of ferroptosis induction (Fig 4A,S5), exhibits similar phenotypic effects in reducing lipid peroxidation (Fig 4E, S4D), and protects across several cell types (Fig S5).
As indicated in our manuscript, we were also excited about AA58 after initial screening using the ARE-FLuc reporter. However, qPCR analysis showed that AA58 did not show specificity for NRF2 activation relative to ATF6 activation (Fig. 3C,D). Thus, we chose not to pursue it further. We did perform additional experiments with another compound that showed low specificity for NRF2 relative to ATF6, AA65. We now demonstrate that AA65 shows improved protection against ferroptotic insults across multiple different cell lines (Figs. 4,5,S5).

Reviewer #2 Comment #5. “These results indicate that the presence of the furan B-ring altered the selectivity of our compounds towards NRF2 activation, relative to ATF6 activation in neuronal cell culture models.”: I recommend revising this statement. It is misleading to attribute the more selective NRF2 activation properties to the furan ring. The carbamate linker in AA28 also contributed to more NRF2 activation, highlighting that other features can drive this activity.”

Response to Reviewer #2 comment #5: We add the statement “This effect appears to be dependent on the carbamate linker, as the AA147 analog AA51, which differs from AA64 only by loss of the carbamate linker (Fig. S3), shows protection against erastin-induced toxicity through a mechanism sensitive to co-treatment with the NRF2 inhibitor ML385 (Fig. 4E, Fig. S4E,G)” on page 9 to clarify this point. We also add data showing that an analog omitting the 2-amino-p-cresol moiety but containing the furan-B-ring and carbamate linker does not protect against erastin-induced ferroptosis. This demonstrates that covalent modification in the presence of the carbamate linker and furan B-ring may drive the emergent properties we observe with this analog.


Reviewer #2 Comment #6 . “The data in Fig. S2E shows that AA28yne also covalently modifies other proteins. Are these PDIs involved in ATF6 activation, as the authors have shown previously? This would be worth describing briefly.”

Response to Reviewer #2 comment #6: We previously showed that AA147yne modifies the PDIs across several cell lines (see Paxman et al (2018) ELIFE). However, as ATF6 inhibitors, such as Ceapin-A7, do not resensitize neuron-derived cells to oxidative-stress induced cell death, we do not focus specifically on this protective aspect of metabolically activatable proteostasis regulators. While we have observed covalent PDI modification to be important for several pathobiological assays where AA147 shows benefits, for instance reduction in amyloidogenic light chain secretion, the majority of our protection is driven by NRF2-mediated activation of the oxidative stress response driven by KEAP1 modification. This is evident by the sensitivity of protection to co-treatment with the selective NRF2 inhibitor ML385 (see Figs. 4, S4) and is consistent with our previous work (see Rosarda et al (2022) ACS Chem Biol).


Reviewer #2 Comment #7. “Why is the ARE-LUC signal in Fig. S3F so much higher compared to Fig. S2D? Is this due to the difference in transient transfections of the reporter?”

Response to Reviewer #2 comment #7: The ARE-FLuc signals presented in S3F and S2D are dose response-experiments for two different compounds AA51 and AA28yne. We observe different potencies and efficacies in activating the OSR-selective ARE-FLuc reporter in IMR32 cells for multiple different analogs (Fig 3B).


Reviewer #2 Comment #8. “Fig. S4C: Why is the protection with AA147 so much lower than in the other two experiments in Fig. S4A,B?”

Response to Reviewer #2 Comment #8: AA147 activity can vary across experiments for a number of reasons including inactivation of compound from freeze-thaw cycles and varying extents of cell death induced by ferroptotic insults such as glutamate. We also found glutamate-induced cell death in HT22 cells to be quite variable. It is for this reason that we switched to primarily focus on erastin-induced ferroptotic death in Fig. 4, as it provided more consistent toxicity, as compared to glutamate. Moreover, we used fresh aliquots of AA147 for all experiments to minimize variability in protection across different experiments.

04-Jul-2024

Dear Dr Kelly:

Manuscript ID: CB-ART-01-2024-000027.R1
TITLE: Metabolically Activated Proteostasis Regulators that Protect Against Erastin-Induced Ferroptosis

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Reviewer 1

Thank you for addressing my points.