Identification of small molecule inhibitors of G3BP-driven stress granule formation

Two small molecules, G3Ia and G3Ib, block a protein interaction surface of G3BP1, leading to stress granule inhibition and/or dissolution. These new tools will aid in understanding the biology of stress granules and hold promise for therapeutic targeting of stress granules.

Please note that JCB now requires authors to submit Source Data used to generate figures containing gels and Western blots with all revised manuscripts.This Source Data consists of fully uncropped and unprocessed images for each gel/blot displayed in the main and supplemental figures.Since your paper includes cropped gel and/or blot images, please be sure to provide one Source Data file for each figure that contains gels and/or blots along with your revised manuscript files.File names for Source Data figures should be alphanumeric without any spaces or special characters (i.e., SourceDataF#, where F# refers to the associated main figure number or SourceDataFS# for those associated with Supplementary figures).The lanes of the gels/blots should be labeled as they are in the associated figure, the place where cropping was applied should be marked (with a box), and molecular weight/size standards should be labeled wherever possible.Source Data files will be made available to reviewers during evaluation of revised manuscripts and, if your paper is eventually published in JCB, the files will be directly linked to specific figures in the published article.
Source Data Figures should be provided as individual PDF files (one file per figure).Authors should endeavor to retain a minimum resolution of 300 dpi or pixels per inch.Please review our instructions for export from Photoshop, Illustrator, and PowerPoint here: https://rupress.org/jcb/pages/submission-guidelines#revised The typical timeframe for revisions is three to four months.While most universities and institutes have reopened labs and allowed researchers to begin working at nearly pre-pandemic levels, we at JCB realize that the lingering effects of the COVID-19 pandemic may still be impacting some aspects of your work, including the acquisition of equipment and reagents.Therefore, if you anticipate any difficulties in meeting this aforementioned revision time limit, please contact us and we can work with you to find an appropriate time frame for resubmission.Please note that papers are generally considered through only one revision cycle, so any revised manuscript will likely be either accepted or rejected.
When submitting the revision, please include a cover letter addressing the reviewers' comments point by point.Please also highlight all changes in the text of the manuscript.
We hope that the comments below will prove constructive as your work progresses.We would be happy to discuss them further once you've had a chance to consider the points raised in this letter.
The authors have previously discovered that two proteins, G3BP1 and G3BP2, are necessary and sufficient for stress granule formation.In this new paper, they rationally design small molecule G3BP1/2 inhibitors and show that they have potent activity against stress granules formed under diverse conditions.They also show that their compounds can either prevent the formation of stress granules or dissolve pre-existing ones and can do so in neurons.The compounds (but not their inactive enatiomers) can also block stress granule formation induced by mutant neurodegenerative disease proteins VCP and FUS.Importantly, these compounds exhibit little to no cellular toxicity.This is a very exciting paper and the compounds presented here will be of immediate and broad interest to the cell biology field.The paper is very well written and the data are compelling.I recommend this paper be published in the JCB.I have some comments and suggestions for the authors to consider.1) In this U2OS cell model, inducing stress granules using arsenite has been show to also cause mislocalization of another ALS protein, TDP-43, from the nucleus to the cytoplasm.Does treatment with G3BP1/2 inhibitors reduce TDP-43 mislocalization to the cytoplasm?
2) The authors show that the stress granules indued by mutant FUS expression are dissolved by treatment with their inhibitors, but the FUS inclusions seem resistant to the compounds.This could mean, as the authors propose, that the FUS inclusions are more stable than the stress granules.It could also mean that the FUS inclusions, contrary to expectation, do not directly coalesce with stress granules.Can the authors test this by pre-treating with their compounds before expressing mutant FUS?Does this treatment prevent FUS from forming cytoplasmic puncta?
Reviewer #2 (Comments to the Authors (Required)): Freibaum et al describe the identification and characterization of two small molecule inhibitors (G3Ia and G3Ib) of G3BP-driven stress granule assembly.They show that G3Ia and G3Ib (and not their enantiomers) bind to G3BP1 and both block SG assembly and facilitate dissolution of pre-formed SGs.These small molecules affect SG formation in different cell types (U2OS and Hela) and in response to different stresses: sodium arsenite and heat shock.Freibaum et al provide evidence that G3Ia and G3Ib function by binding to G3BP1 and specifically interfering with the recruitment of caprin to the NTF2L domain of G3BP1.The authors also show that these small molecules can perturb SGs in a disease-relevant context, suggesting a potential avenue for therapeutic development.This work is exciting and well-written, and the small molecule inhibitors of SG formation are likely to be of significant value to the condensate biology community.I have very few concerns about this work -see below.
Results/discussion: • The author's claim that the effect of these compounds is long-lived would be better supported by an experiment in which cells are pre-treated with the compound over the time period used in other experiments (30 or 60 minutes), incubated for 24 hrs in the absence of inhibitor, and then assayed.This would show that the effects are long-lasting.The current data show that cells can tolerate the inhibitors for 24 hrs and maintain their effects on SG formation, but these data are insufficient to conclude that the effects are long-lasting.
• Paragraph 2 of the discussion suggests that the compounds do not affect translation.While I think it is unlikely that they do, the statement will be improved by showing this data.This would also strengthen the claim that the compounds are not toxic (Fig 2A).
• Most experiments were performed in GFP-G3BP stable cell lines.While I understand the utility of this cell line for live cell imagining, the effect of the compounds should also be shown in a WT cell line with endogenous G3BP1 to assure the validity and applicability of the conclusions.1st Revision -Authors' Response to Reviewers: November 28, 2023

Reviewer #1
Biomolecular condensation is an emerging principle that underlies many cellular processes.Stress granules are bimolecular condensates that form in the response to cellular stresses and have been connected to the pathogenesis of several diseases, including cancer and neurodegeneration.
The authors have previously discovered that two proteins, G3BP1 and G3BP2, are necessary and sufficient for stress granule formation.In this new paper, they rationally design small molecule G3BP1/2 inhibitors and show that they have potent activity against stress granules formed under diverse conditions.They also show that their compounds can either prevent the formation of stress granules or dissolve pre-existing ones and can do so in neurons.The compounds (but not their inactive enatiomers) can also block stress granule formation induced by mutant neurodegenerative disease proteins VCP and FUS.Importantly, these compounds exhibit little to no cellular toxicity.This is a very exciting paper and the compounds presented here will be of immediate and broad interest to the cell biology field.The paper is very well written and the data are compelling.I recommend this paper be published in the JCB.I have some comments and suggestions for the authors to consider.1) In this U2OS cell model, inducing stress granules using arsenite has been show to also cause mislocalization of another ALS protein, TDP-43, from the nucleus to the cytoplasm.Does treatment with G3BP1/2 inhibitors reduce TDP-43 mislocalization to the cytoplasm?
Author response: As suggested by the reviewer, we have now examined whether our G3I compounds reduce the localization of TDP-43 to cytoplasmic stress granules.First, we used immunofluorescence to assess TDP-43 localization in U2OS cells treated with vehicle, inactive enantiomer, or active compound.We found that TDP-43 accumulates in stress granules in cells treated with vehicle, G3Ia′ (inactive enantiomer), or G3Ib′ (inactive enantiomer) but not G3Ia or G3Ib (inhibitor compounds) (new Figure S2G, H).Thus, as the reviewer anticipated, the G3BP1 inhibitors also prevent mislocalization of TDP-43 protein.To confirm this result, we used a CRISPR-modified U2OS cell line in which endogenous TDP-43 and G3BP1 were labeled with GFP and mRuby3, respectively.Again, we found that treatment with G3Ib (inhibitor), but not G3Ib′ (inactive enantiomer), blocked the accumulation of TDP-43 into stress granules (new Figure S2I, Supplemental Videos 5 and 6).
2) The authors show that the stress granules indued by mutant FUS expression are dissolved by treatment with their inhibitors, but the FUS inclusions seem resistant to the compounds.This could mean, as the authors propose, that the FUS inclusions are more stable than the stress granules.It could also mean that the FUS inclusions, contrary to expectation, do not directly coalesce with stress granules.Can the authors test this by pre-treating with their compounds before expressing mutant FUS?Does this treatment prevent FUS from forming cytoplasmic puncta?
Author response: We have performed this experiment as suggested and found that pretreatment with G3Ia and G3Ib inhibits the incorporation of G3BP1 into stress granules but does not affect the accumulation of mutant FUS into cytoplasmic puncta (new Figure 5E, F), suggesting that the formation of mutant FUS inclusions is independent of stress granules.

Reviewer #2
Freibaum et al describe the identification and characterization of two small molecule inhibitors (G3Ia and G3Ib) of G3BP-driven stress granule assembly.They show that G3Ia and G3Ib (and not their enantiomers) bind to G3BP1 and both block SG assembly and facilitate dissolution of pre-formed SGs.These small molecules affect SG formation in different cell types (U2OS and Hela) and in response to different stresses: sodium arsenite and heat shock.Freibaum et al provide evidence that G3Ia and G3Ib function by binding to G3BP1 and specifically interfering with the recruitment of caprin to the NTF2L domain of G3BP1.The authors also show that these small molecules can perturb SGs in a disease-relevant context, suggesting a potential avenue for therapeutic development.This work is exciting and well-written, and the small molecule inhibitors of SG formation are likely to be of significant value to the condensate biology community.I have very few concerns about this work -see below.
Results/discussion: • The author's claim that the effect of these compounds is long-lived would be better supported by an experiment in which cells are pre-treated with the compound over the time period used in other experiments (30 or 60 minutes), incubated for 24 hrs in the absence of inhibitor, and then assayed.This would show that the effects are long-lasting.The current data show that cells can tolerate the inhibitors for 24 hrs and maintain their effects on SG formation, but these data are insufficient to conclude that the effects are long-lasting.
Minor comments: • Define acronyms/explain jargon: example, Fig 1E details, Fig 2A • Supplementary Videos 9-24 not present with submitted files • Unclear which fig number and legend corresponds to each supplemental video provided.Panels of videos are labeled with an inhibitor and concentration, but the figure legend just lists pre-incubation with one inhibitor at one concentration.Please clarify experimental procedure and figure legends associated with each video.• Fig S5 B -difficult to see images, please enlarge.• Discussion first paragraph: reference to Fig S1 should be S2; discussion paragraph 2 refers to Fig 1, but should be Fig 2 here.• Discussion paragraph 2 states that G3Ia and G3Ib leave the dimerization capability of G3BP intact, however the compounds bind to the NTF2L domain, which according to Sanders et al. 2020 and Yang et al. 2020 is dimerization domain.Please clarify.