HuR modulation counteracts lipopolysaccharide response in murine macrophages

  1. Isabelle Bonomo
  2. Giulia Assoni
  3. Valeria La Pietra
  4. Giulia Canarutto
  5. Elisa Facen
  6. Greta Donati
  7. Chiara Zucal
  8. Silvia Genovese
  9. Mariachiara Micaelli
  10. Anna Pérez-Ràfols
  11. Sergio Robbiati
  12. Dimitris L. Kontoyannis
  13. Marilenia De Matteo
  14. Marco Fragai
  15. Pierfausto Seneci
  16. Luciana Marinelli
  17. Daniela Arosio
  18. Silvano Piazza
  19. Alessandro Provenzani

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Abstract

Lipopolysaccharide (LPS) exposure to macrophages induces an inflammatory response, which is regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA-binding protein that regulates cytokines and chemokines transcripts containing AU/U-rich elements (AREs) and mediates the LPS-induced response. Here, we show that small-molecule tanshinone mimics (TMs) inhibiting HuR–RNA interaction counteract LPS stimulus in macrophages. TMs exist in solution in keto-enolic tautomerism, and molecular dynamic calculations showed the ortho-quinone form inhibiting binding of HuR to mRNA targets. TM activity was lost in vitro by blocking the diphenolic reduced form as a diacetate, but resulted in prodrug-like activity in vivo. RNA and ribonucleoprotein immunoprecipitation sequencing revealed that LPS induces a strong coupling between differentially expressed genes and HuR-bound genes, and TMs reduced such interactions. TMs decreased the association of HuR with genes involved in chemotaxis and immune response, including Cxcl10, Il1b and Cd40, reducing their expression and protein secretion in primary murine bone marrow-derived macrophages and in an LPS-induced peritonitis model. Overall, TMs show anti-inflammatory properties in vivo and suggest HuR as a potential therapeutic target for inflammation-related diseases.

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  1. Version published to 10.1242/dmm.050120
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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    Summary:
    The Authors report on the synthesis and characterization of a class of small molecules, the tanshinone mimics (TMs), which interfere with binding of the RNA binding protein (RBP) HuR to its mRNA targets. HuR is an important regulator of mRNA stability and translation of genes involved in key homeostatic (cell cycle, stress response) and pathologic process (inflammation, carcinogenesis). In particular, the first part of the study describes the compounds' chemical synthesis and some pharmacokinetic parameters (i.e., definition of molecular binding, solubility, bioavailability, prodrug generation etc). The second part undertakes, in in vitro and ex-vivo model of LPS-induced mouse macrophage activation, the identification of HuR-bound mRNA targets, which is then evaluated within the global LPS-induced transcriptome; finally, the study evaluates the ability of TMs to inhibit HuR-mediated proinflammatory gene regulation, indicating their use and potential value as therapeutic anti-inflammatory strategy.
    Major comments:
    The manuscript contains a wealth of data generated from different experimental systems, spanning from synthetic chemistry to preclinical models of gene regulation, requiring cultural backgrounds in chemistry and biology as well. The key conclusions are well supported by the data, but it takes a great effort to get to the core results and thus critically read and evaluate their interpretation. Although the complexity and sheer size of data sets generated lends itself to a hard read, this is further complicated by data presentation, which especially in the second part needs to be significantly improved to gain clarity and focus. For ease of referral, specific comments will be addressed related to Figures whenever possible.
    1.1 • Page 15: To measure TM7nox disrupting ability of HuR:mRNA complex for the HTRF assay (Figure 2G) and for biotin pull down assay (Figure 5C), it was chosen a biotinylated probe containing the AU rich elements of the TNFα, as known HuR target. Please comment on the rationale, and whether could it be relevant reevaluate these parameters post-hoc, based on the sequences identified in HuR targets more susceptible of modulation by TM compound (listed in table 1, Figure 5 A/B) and based on the absence of regulation of TNFa (Figures 3D, 4D, 7A) found in the tested systems.

    R1.1 - We thank the reviewer for this observation. We have been using the biotinylated probe containing the AU-rich elements of TNFα as a representative probe for HuR for biochemical assays in several articles (PMID: 29313684, PMID: 26553968, PMID: 23951323). As the reviewer suggests, a posteriori, it is worth reevaluating the representative probe to be used for evaluating the disrupting ability of TMs based on the data we present here. Indeed, we will tackle this problem in our following efforts, as it is a meaningful although time-consuming task which is outside of the scope of this manuscript.

    1.2 • Page 16-18: Description of the RNAseq data shown in Figure 3 should be more centered around the main findings regarding the effect of TMnox that are further pursued in the study: that is, (Figure 3B), the 249 downregulated DEGs found modulated by TM7nox in presence of LPS, where was observed a strong enrichment of categories related to the inflammatory response: cytokines (Il1b, Cxcl10, Il10, Il19, Il33), immune cell chemotaxis (Ccl12, Ccl22, Ccl17, Ccl6) and innate immune response.

    The description of the GO for the remaining data should be shortened to main points, perhaps reporting what described in the results with each section of the Venn in a table, while referring to the whole list in the supplements as already done. This could replace Figures 3D, E which do not add substantially to what provided in the supplementary table 2 and to which they can be added as further visualization.

    R1.2 - We thank the reviewer for this suggestion, accordingly, we simplified the text keeping only the description of the genes modulated by TM7nox during LPS treatment. The other information originally there was moved to Supplementary table 2. Revised figures 3E and 3F now focus only on the 249 downregulated genes of this group.

    1.3 • Page 18-19: Description of the results of the RIP-seq shown in Figure 4 set is very confusing: onward from the line "477 HuR-bound transcripts (log2 FC > 3) were also upregulated by LPS at the transcriptional level..." the numbers do not match or reconcile with those shown in the Venn diagram (Fig. 4B) nor with those listed in the figure legend of Figure S8.

    R1.3 - We agree with the reviewer, we apologize for having reported the wrong numbers, and we clarified this point in general by deeply revising the text. A more precise explanation of the selection procedure for the genes of interest is now reported and better explained also by adding a scheme (Fig 4D in the revised manuscript).

    1.4 Moreover, as previously remarked for Figure 3 (and even more for this dataset in which initial description of Venn in 4B is unclear), panel 4E does not add as much to the info provided in Table 1/supplementary Table 1, where they can eventually be added as further data visualization; Instead, Figure S8 displays very informative data merging together the results obtained in RNAseq (Fig. 3) and RIP-Seq (Fig.4) and should be displayed in Figure 4, as in the result section they are indeed presented together.

    R1.4 - We agree with this remark, thus we have removed the old panels 3E in S8C and 4E in S9B, and we now provide the information previously contained in old S8 in the main figure 4E of the revised manuscript.

    1.5 • Page 19-20: Description of the modulation by TM7nox of HuR binding to specific consensus sequences is summarized at the end of the relative paragraph as follows: "TM7nox reshapes HuR binding to target genes in presence of LPS by disrupting the binding of HuR towards target genes containing a lower number of HuR consensus sequences than the average observed in the HuR-bound transcripts". Understanding of these data through the provided text and the Supplementary Figure 9 is very laborious and referring of an entire dataset to a supplementary figure makes it even harder. It would be best to show this as main figure, not supplemental, either adding a Venn diagram as in 3B/4B showing to which dataset each part of the analysis refers, or even more efficaciously, extrapolate a representative gene set for the main analyses showing TM7nox activity in target genes with higher vs lower consensus sequences; same approach for the analysis in Figure 9B, where the effect on a gene with sequence #1 or #10 could be compared with one bearing sequence #3 for example.

    R1.5 - We agree with the reviewer, thus we moved the information of old S9 in figure 4C of the revised manuscript. We deeply revised the information provided also by taking into account the request to compare this experiment to the one in Lal et al. NAR 2017 (please see also R2.4). We made an effort to identify a subset of genes that follow a coherent modulation, identifying 82 genes highlighted in Supplementary Table 1. All such genes show increased expression during LPS or LPS/TMnox vs DMSO conditions, and decreased association to HuR during LPS/TMnox vs LPS. As 47 of these, i.e. more that 50%, contain less AU rich sequences than the average (highlighted in Supplementary Table 1), we can consider them as a representative gene ensemble modulated in accordance with the presence of AU rich sequences.

    1.6 • Page 21: Description of the effect of three TMs (TM6, TM7nox and TM7nred) on LPS response in macrophages at the single gene level (Figure 5 and Figure 6): TM6 and TM7nox were used in exps in Fig. 5 A and E, while only TM7nred was used for CXCL10 secretion analysis (fig.5 D and F): please describe the compound choices' rationale (as done for experiments in Figure 6).

    R1.6 – Following the reviewer suggestion, we now explain our rationale in choosing the small molecules, that is the use of the ones bearing the active quinone species. We have performed additional experiments, and now we report TM6n, TM7nox, and the control DHTS activity in decreasing the secretion of Cxcl10 (figure 5E in the revised manuscript). All compounds behave similarly in this experiment. TM7nred is now used to show its equivalence to TM7nox in figure 5E and in figure 6 of the revised manuscript.

    1.7 • Page 21-22: The effect on HuR expression of siRNA silencing and, more importantly, of TMs shown in Figure 6A, first panel, should be visualized at protein level by western blot. This is an important point as for CXCL10 and iL1b there seems to be an additive effect between decreased HuR levels and pharmacological blocking.

    R1.7 - Following the reviewer suggestion, we now show the protein level as measured by intracellular Elisa; as we were not able to detect the proteins by western blot. The protein level is in general agreement with the gene expression level. We do not observe an additive effect by pharmacological inhibition during HuR silencing, but we rather observe a slight increase in the protein level during HuR silencing. We do not have an explanation for this effect, which may depend on several reasons - for example, an aspecific effect of the TMs when their molecular target HuR is absent.

    1.8 • Page 24: please rephrase the statement 'These observations suggest the utilization of TMs in autoinflammatory and autoimmune diseases' as 'These observations suggest the evaluation of TMs in specific preclinical models for autoinflammatory and autoimmune diseases'.

    R1.8 - We fully agree with the reviewer, and we changed the text in the revised manuscript accordingly.

    1.9 • In the discussion, please include a paragraph with study limitation and possible biases (for example, the choice of RNP-IP without crosslinking has pros and cons).

    R1.9 – Thank you for the good suggestion, we added a paragraph in the discussion which describes study limitations due to the utilization of RNP-IP vs crosslinking.

    The data and the methods are correctly presented for reproducibility, replicates and statistical analysis are adequate. Minor comments: 1.10 • At least in the single gene validation experiments (Fig.5), a negative control (such as recombinant HuR with mutated RRMs in trans-, or ARE-less/non-HuR targetable sequence in cis, or inactive TM) would be advisable.

    R1.10- We thank the reviewer for the suggestion. Accordingly, we used an ARE-less/non-HuR targetable gene as RPLP0 for validation.

    1.11 • Figure 6B/C: for immunofluorescence panels, zooming on a smaller number of cells will render more visible HuR and NFkB nucleocytoplasmic shuttling, given that quantification and statistics are provided by imaging software. Negative control stainings (secondary Abs only) should be included.

    R1.11 – In accordance with this suggestion, we now report a higher magnification of the immunofluorescence images. We also report the standard DHTS effect, showing a difference vs TMnox activity which may suggest its impact on NFkB shuttling.

    1.12 • Figure 7A: in the X axis LPS+8n is indicated: is it a typo for LPD+6n or was compound TM8n indeed used?

    R1.12 – Thanks for your spotting our mistake, the prodrug 8 described in figure 1 was used.

    1.13 • In the Methods section please include protocols and materials for immunofluorescence (results shown in Fig. 6B/C).

    R1.13 – As for your suggestion, protocols and materials for immunofluorescence were added to the methods.

    1.14 • There are some typos and repetition in figure legends (legend Figure S9).

    R1.14- Thank you for this, we revised all the figure legends.

    Prior studies are referenced appropriately. Review Cross-commenting I fully agree with the Reviewer's remarks. I would add that a general concern expressed is that this manuscript in its present form has a readership issue: the first part is for chemistry/pharmacology audience, the second is biology-based. Splitting the work has been suggested; or, the Authors may decide which part is more impactful and present the other in a streamlined version.

    Reviewer #1 (Significance):

    This is a large study reporting progress in the development of synthetic antagonists of HuR function, which is the Authors' well-established line of research. The TM compounds are small molecules with anti-inflammatory effects with strong potential for therapeutic use due to selected inhibition of HuR-mediated upregulation of proinflammatory molecules. The physicochemical and early biological characterization done in this study will allow further testing of their efficacy and of the overall role of HuR-mediated regulation as targetable mechanism in several preclinical human disease models. Targeting of the RNA-binding protein HuR has been tackled as therapeutic approach in cancer, less in chronic immune and inflammatory diseases despite many common mechanisms and mediators. This study could be well received by researchers involved in basic science and drug development (chemistry, biochemistry/biophysics, pharmacology, computational modeling) and biologists/physician scientists interested in testing these compounds in translational research settings where HuR-driven functions can be relevant (cancer, chronic inflammation), though the chemical part would be less accessible to the latter audience. Reviewer's background is in preclinical human models of chronic inflammation with interest in posttranscriptional gene regulation with familiarity with RNAseq and RIPseq dataset and analysis. For the part of the manuscript regarding the synthesis and physicochemical characterization of the TN compound I requested assistance to a faculty from the chemistry department with expertise in that field, who did not request any specific clarification or addendum.

    Reviewer #2 (Evidence, reproducibility and clarity):

    In the manuscript entitled "HuR modulation with tanshinone mimics impairs LPS response in murine macrophages" the authors have described the synthesis and application of small molecule mimics of the naturally occurring compound tanshinone, which is known to inhibit the binding of the RBP HuR to a class of its mRNA targets. The authors have shown that the tanshinone mimics (TMs) used by them block the binding of RRM1-2 of HuR to ARE-containing RNA in vitro, and reduce the interaction of HuR with a set of ARE-containing mRNAs in LPS-treated mouse macrophage cells. This reduction of interaction of HuR with some of these mRNAs correlates with the reduction in their level in the cells treated with the TMs, and in the secreted level of their proteins in the serum of animals with LPS-induced peritonitis. Together, the study demonstrates the role of these TMs as modulators of the LPS-induced inflammatory response by blocking the binding of HuR to a subset of LPS-induced inflammatory mRNAs and thereby downregulating their mRNA and protein levels in inflammatory cells. The manuscript describes a comprehensive study, ranging from chemical synthesis of TMs, MD simulations to demonstrate the binding site of the TMs to the cleft formed by the RRM1-linker-RRM2 domains of HuR, which has been shown in crystal structure to be the main binding site of A/U-rich RNA molecules, in vitro studies showing the ability of the TMs to hinder ARE-containing RNA binding to HuR RRM1-2, whole transcriptome analysis to show the effect of the TMs on LPS-induced differential gene expression in murine macrophages, and on HuR binding to target mRNAs, and animal studies to show the effect of the TMs on the level of some inflammatory mediators in the serum of mice with LPS-induced peritonitis. The results are quite convincing and is in line with what is generally known about the effect of HuR on the expression of a large number of genes encoding pro-inflammatory proteins, and the ability of tanshinone derivatives/mimics in inhibiting HuR binding to target mRNAs. The authors put these two information together in this study and the results are on expected lines. While the results are convincing and quite comprehensive, I would suggest the following in order to substantiate and strengthen the results: 2.1. The experiments do not have any "positive control", such that the performance of the TMs can be compared with that of a molecule with known HuR binding inhibition activity, such as DHTS. It would be good to have such a comparison, to understand whether the TMs work similar to DHTS or differently, both qualitatively in terms of the mRNA targets which they affect and the extent of their anti-inflammatory activity.

    R2.1- We added DHTS as a comparison to TMs, following the reviewer’s comment. In this model, the net effect of DHTS is partially overlapping with TMs, at least for the parameters that we checked (see Figure 5, 6 and 7), showing some differences in the modulation of NF-kB shuttling upon LPS stimulation. Therefore, we suggest that DHTS and TMs show partially different effects on mRNA targets and in terms of anti-inflammatory activities.

    2.2. It is not clear to me whether the mRNAs which show differential expression in the RNAseq analysis of cells treated with LPS and TMs are exactly the ones which show difference in binding with HuR in the RIPseq analysis in presence of the TMs. This analysis is important for a number of reasons: all the mRNA binding targets of HuR are not affected by HuR at the level of mRNA stability, many are affected at the level of translation, without change in mRNA level. These mRNAs should therefore show change in binding of HuR in the RIPseq assay in presence of TM, but not show change in expression. Secondly, there may be mRNAs which show a change in expression in presence of TMs, but do not show binding of HuR, suggesting pleiotropic roles of the TMs. Therefore, instead of an overall correlation between differential expression and change in HuR binding of mRNAs, a table comparing the RIPseq status of individual mRNAs with that of their differential expression status, in presence and absence of LPS/TMs would be useful, further designating the different groups of mRNAs based on these differential status (change in HuR binding/change in expression, change in HuR binding/no change in expression etc.).

    R2.2 – We tried to rationalize the data following the reviewer’ suggestion, however, we could not fully adopt this strategy due to the complexity of the experiment design. Indeed, we have focused our attention on the effect of TMs during LPS stimulus, which induces a strong transcriptional response, rather than in steady state conditions. This is why we reported the overall correlation of LPS vs DMSO and TM7nox/LPS vs DMSO. Then, we evaluated whether the observed difference in the correlation may be reflected on a change of HuR binding, and we checked the RIPseq status during co-treatment vs LPS. This was the case for a subset of genes that are reported in Supplementary Table 1. Nevertheless, to be fully compliant with the reviewer’s request we now report a Supplementary Table 1 containing the entire gene list, so that the reader can immediately filter out the subsets according only to the comparison TM7nox/LPS vs LPS.

    2.3. Nuclear/cytoplasmic localization of HuR and NFkb is impossible to discern at the magnification of the immunofluorescence images in Fig 6 B and C. Higher magnification images are required to understand changes in localization.

    R2.3 – In accordance with this suggestion, we now report higher magnification, please see also R1.11. We do not observe any change in nuclear/cytoplasmic localization of HuR and NFkb due to TMs treatment. We rather observe LPS-induced NFkB nuclear accumulation, ActD-induced HuR cytoplasmic shuttling and inhibition of NFkB translocation, during LPS and DHTS treatment.

    2.4. It has been shown that DHTS-I increases the binding of HuR to the mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, whereas it reduces the interaction of HuR with the mRNAs having shorter 3'UTR and with low density of U/AU-rich elements (Lal et al., NAR, 2017). It is not clear if the same is observed in case of the TMs or not, and such a comparative analysis would be useful to address this point.

    R2.4 – We re-analysed the data, checking the density of U/AU rich elements and the length of the 3’UTR of the displaced mRNA as in Lal et al. NAR 2017. Although we could not compare DHTS and TMs within the same biological system, it appears that the rules dictating their mechanism of action are similar.

    I think that the above suggested points are feasible as most of them really involve re-analysis of existing data. Only the suggestion to add DHTS or tanshinone as a positive/comparison control will require experimentation and addition of new data.

    Review Cross-commenting

    I think most of the reviewers' comments coincide in the evaluation of the manuscript. I would especially like to draw attention to the fact that all three reviewers found that the content and form of data presented in the paper is very dense and bogs down the reader and distracts from the overall focus of the manuscript.

    Reviewer #2 (Significance):

    The work described in the manuscript is comprehensive as it ranges from chemical synthesis and in vitro evaluation of the TMs to the characterization of their effects in vivo. Although the effect of tanshinone derivatives on HuR mRNA target binding is known, and the effect of HuR on inflammatory gene expression is also known, the manuscript is significant as it brings these two information together and tests the effect of these TMs on HuR-mediated regulation of inflammatory gene expression.
    However the extensiveness of the work also makes it quite dense, and I feel that the focus of the paper is often lost in the details. Also, the text of the manuscript is dense and verbose and uses many irregular grammatical and phraseological usages, for eg "their
    modulation or mis-localization lead to the insurgence of complex phenotypes and diseases". It appears to me that it would be ideal if the chemical synthesis, MD simulation studies and in vitro studies are presented in an independent manuscript. Also, that would allow a more exhaustive referencing of the known studies in literature where tanshinone derivatives, and other small molecules, have been used to modulate HuR binding to mRNA targets.
    This work would be of interest to molecular cell biologists in general and RNA biologists in particular, especially those who are studying RNA-protein interactions, and scientists who are interested in drug development using RNA-protein interactions as drug targets.
    My interest in the work lies in my expertise in studying RNA-protein interactions, especially of RNA-binding proteins such as HuR involved in regulating the translation of mRNAs encoded by inflammatory genes. I do not have expertise in chemical synthesis and am therefore not qualified to evaluate the first set of results describing the chemical synthesis of TMs.

    Reviewer #3 (Evidence, reproducibility and clarity):

    In this study, the authors investigated the modulation of HuR by tanshinone mimics and how it mitigates LPS response in murine macrophages. This represents a nice integration of synthetic chemistry, molecular simulations, and in vitro as well as in vivo experimental validations. Overall, this is an interesting study, and will add to the growing interest in HuR in inflammatory-mediated disease. The paper contains a lot of data (actually several papers in one) which may bog down the reader and distract from the overall message. it is suggested that they condense the data and simplify the figures and use more supplemental figures.
    Major Comments:
    3.1. The authors have shown the dose response and cytotoxicity effect of tanshinone mimics; The authors show that TMs affect the overall HuR mRNA but they don't show protein levels.

    R3.1 – In accordance with the reviewer’s comment, we now show also protein levels, as we performed intracellular ELISA (Figure 6 in the revised manuscript); please see also R1.7.

    3.2. It is unclear the timing of certain experiments for LPS vs TMs (whether macrophages were pre-treated with TMs before LPS)-e.g fig 5. The authors should clarify for all experiments as the long-term clinical paradigm would be treatment after inflammation has been established.

    R3.2 – In most experiments TMs are co-administered with LPS. Only in one of the two protocols used for Actinomycin D chase experiment TMs are added after LPS with Act D, as we wanted to discriminate between transcriptional and post-transcriptional effects of TMs (see also R3.3).

    3.3. They have also identified differentially expressed genes which are RNA binding ligands of HuR by RIP-Seq. However, it would be necessary to check whether TM7nox affects the stability of those targets before conclusions can be made that TMs don't inhibit the primary transcriptional response (as mentioned in the Discussion section). Transcriptional effects of HUR chemical inhibition or genetic silencing has been reported previously in other cell systems.

    R3.3 – The reviewer is entirely correct, and we accordingly amended our conclusions. Indeed, TMs have an impact on gene transcription during co-administration with LPS as now suggested by Actinomycin D chase experiments reported in Figure 6C in the revised data and discussion in the manuscript.

    3.4. HuR competes with many RBPs (e.g. TTP and KSRP) as well as microRNAs (including miR-21 and miR-122) to regulate the stability/translational efficiency of several AU-rich transcripts. Does TM binding to HuR lead to increase access of these RBPs/microRNA to the transcripts? This could be addressed by RNA IP with antibodies to TTP or KSRP.

    R3.4 – The reviewer is suggesting an important experiment that requires multiple controls and significant efforts. Indeed, we are planning to study the specificity of TMs, and we prefer to tackle and report this point in a later publication.

    3.5. Another aspect of HuR functioning is the dimerization of HuR. HuR dimerization has been linked with many pathophysiologic conditions. The authors should show the effect of TM7nox on HuR dimerization. In figure 2, for example, there is a suggestion of this in the representative EMSAs where an intermediate shifted band is seen with the addition of TMs. Also, the legend should make clear which ligand is being tested in the modeling (purple structure) versus the RNA probe in the EMSAs. It would help the reader to identify the RNA probe used-e.g. "5′-DY681-labeled ARE RNA probe.

    R3.5 – We agree with the reviewer’s suggestion, and we investigated whether TM7nox influences HuR dimerization in the absence of RNA as performed in PMID 17632515 (Meisner et al 2007). We used MS-444 as a positive control, and we did not observe inhibition of dimerization by TMs at least at the used dosages. Data are reported in Supplementary Figure S6B of the revised manuscript.

    3.6. HuR does alter M2-associated targets like IL-10 and this should be addressed more directly. Fig. 3 suggests that IL-10 is reduced by TM7nox but the variance is so high that the statistics show NS. HuR regulates IL-10 in other cellular contexts and this would be important to determine for TM7 in the long run.

    R3.6 – Although we acknowledge its relevance, however, we did not investigate this gene directly. The variance becomes significant in the RIP-seq experiment (Supplementary Figure 9D). Therefore, we confirm that Il10 is among the 47/82 genes that show the same behavior as Cxcl10, Il1b and many other cytokines as Ccl12, Ccl7, Fas, Il1a, Il33; in conclusion, it is among the restricted list of genes modulated by TM7nox according to the presence of less AU rich sequences than average.

    3.7. Fig. 5-10 um of the TM used here produces significant toxicity to BMDM according to fig. S7. This may distort the ELISA/qPCR results as the RNA levels may be lower due to toxicity. The authors should address this or use a lower dose that is not toxic.

    R3.7 – The viability curves mentioned by the reviewer are run at 24-48 hours, and no toxic effects have been observed using TMs after 6 hours of treatment.

    3.8. In Fig 6 the immunocytochemistry is difficult to interpret as the magnification is too small to appreciate the N/C ratio. The investigators should provide higher magnification. A nuclear/cytoplasmic western blot is recommended as well to confirm that TM does not impair HuR shuttling (or NFkb shifts). This is an important area as there is a suggestion that TM blocks dimerization (Fig. 2) which does impair shuttling. Also, the modeling data suggest that TMs appear to sit in a similar groove between RRM1 and 2 as other HuR inhbitors that block shuttling.

    R3.8 – This point has also been raised by other reviewers, and we replied in R2.3 and R1.11. We understand the reviewer’s points, and we agree with the observation. However, we do not observe a change in HuR nuclear/cytoplasmic shuttling by immunofluorescence, neither we see an effect on HuR dimerization.

    3.9. IL-6 does not appear to be affected by TM treatment after LPS stimulation in vivo or in vitro -either mRNA or protein. However, DHTS did suppress this cytokine. The authors should address this discrepancy. Likewise, TNFa data here show no change and possibly a trend upward (Fig 3,4 and 7). This is in contrast to the effect of DHTS on TNF-a reported by the authors in a prior publication (D'Agnistino et al). The authors should address this discrepancy. There are reports suggesting that HuR is a translational inhibitor of TNFa in macrophages--Katsanou V, Papadaki O, Milatos S, Blackshear PJ, Anderson P, Kollias G, Kontoyiannis DL. HuR as a negative posttranscriptional modulator in inflammation (PMID 16168373)

    R3.9 – The reviewer’s comments are correct, but we do not have an explanation for this. In theory, there could be several possibilities such as 1) a DHTS effect on NFkB, 2) the fact that previously mentioned experiments with DHTS are not run with the same cells-at the same doses and timing as our current TM experiments, and 3) that HuR silencing is only partially overlapping with TMs treatment also in our recent experiments. Irrespective of specific transcripts, we think we have shown that TMs’ mechanism of action involves the modulation of HuR binding at the transcriptional level in our experimental condition.

    Review Cross-commenting

    I think the other reviewers' comments are pertinent and well thought out. I have no further suggestions.

    Reviewer #3 (Significance):

    The characterization of novel HuR inhibitors derived from tanshinones is an important advancement to the field which is rapidly growing. This complements other work with small molecule inhibitors and will allow the field to better understand the role of HuR in different disease contexts (cancer, neuroinflammatory etc) and cell types (e.g. macrophages, microglia, astrocytes). The ultimate significance is the clinical application of the inhibitors and the more options the better, particularly if there are toxic effects of some. My expertise is in post-trasnscriptional regulation of cytokines and we have already characterized some potent effects in cancer.

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    Referee #3

    Evidence, reproducibility and clarity

    In this study, the authors investigated the modulation of HuR by tanshinone mimics and how it mitigates LPS response in murine macrophages. This represents a nice integration of synthetic chemistry, molecular simulations, and in vitro as well as in vivo experimental validations. Overall, this is an interesting study, and will add to the growing interest in HuR in inflammatory-mediated disease. The paper contains a lot of data (actually several papers in one) which may bog down the reader and distract from the overall message. it is suggested that they condense the data and simplify the figures and use more supplemental figures.

    Major Comments:

    1. The authors have shown the dose response and cytotoxicity effect of tanshinone mimics; The authors show that TMs affect the overall HuR mRNA but they don't show protein levels.
    2. It is unclear the timing of certain experiments for LPS vs TMs (whether macrophages were pre-treated with TMs before LPS)-e.g fig 5. The authors should clarify for all experiments as the long-term clinical paradigm would be treatment after inflammation has been established.
    3. They have also identified differentially expressed genes which are RNA binding ligands of HuR by RIP-Seq. However, it would be necessary to check whether TM7nox affects the stability of those targets before conclusions can be made that TMs don't inhibit the primary transcriptional response (as mentioned in the Discussion section). Transcriptional effects of HUR chemical inhbiition or genetic silencing has been reported previously inother cell systems.
    4. HuR competes with many RBPs (e.g. TTP and KSRP) as well as microRNAs (including miR-21 and miR-122) to regulate the stability/translational efficiency of several AU-rich transcripts. Does TM binding to HuR lead to increase access of these RBPs/microRNA to the transcripts? This could be addressed by RNA IP with antibodies to TTP or KSRP.
    5. Another aspect of HuR functioning is the dimerization of HuR. HuR dimerization has been linked with many pathophysiologic conditions. The authors should show the effect of TM7nox on HuR dimerization. In figure 2, for example, there is a suggestion of this in the representative EMSAs where an intermediate shifted band is seen with the addition of TMs. Also, the legend should make clear which ligand is being tested in the modeling (purple structure) versus the RNA probe in the EMSAs. It would help the reader to identify the RNA probe used-e.g. "5′-DY681-labeled ARE RNA probe.
    6. HuR does alter M2-associated targets like IL-10 and this should be addressed more directly. Fig. 3 suggests that IL-10 is reduced by TM7nox but the variance is so high that the statistics show NS. HuR regulates IL-10 in other cellular contexts and this would be important to determine for TM7 in the long run.
    7. Fig. 5-10 um of the TM used here produces significant toxicity to BMDM according to fig. S7. This may distort the ELISA/qPCR results as the RNA levels may be lower due to toxicity.The authors should address this or use a lower dose that is not toxic.
    8. In Fig 6 the immunocytochemistry is difficult to interpret as the magnification is too small to appreciate the N/C ratio. The investigators should provide higher magnification and provide examples of ActD, LPS and LPS + drug. A nuclear/cytoplasmic western blot is recommended as well to confirm that TM does not impair HuR shuttling (or NFkb shifts). This is an important area as there is a suggestion that TM blocks dimerization (Fig. 2) which does impair shuttling. Also, the modeling data suggest that TMs appear to sit in a similar groove between RRM1 and 2 as other HuR inhbitors that block shuttling.
    9. IL-6 does not appear to be affected by TM treatment after LPS stimulation in vivo or in vitro -either mRNA or protein. However, DHTS did suppress this cytokine. The authors should address this discrepancy. Llikewise, TNFa data here show no change and possibly a trend upward (Fig 3,4 and 7). This is in contrast to the effect of DHTS on TNF-a reported by the authors in a prior publication (D'Agnistino et al). The authors should address this discrepancy. There are reports suggesting that HuR is a translational inhbitor of TNFa in macrophages--Katsanou V, Papadaki O, Milatos S, Blackshear PJ, Anderson P, Kollias G, Kontoyiannis DL. HuR as a negative posttranscriptional modulator in inflammation (PMID 16168373)

    Review Cross-commenting

    I think the other reviewers' comments are pertinent and well thought out. I have no further suggestions.

    Significance

    The characterization of novel HuR inhibitors derived from tanshinones is an important advancement to the field which is rapidly growing. This complements other work with small molecule inhibitors and will allow the field to better understand the role of HuR in different disease contexts (cancer, neuroinflammatory etc) and cell types (e.g. macrophages, microglia, astrocytes). The ultimate significance is the clinical application of the inhibitors and the more options the better, particularly if there are toxic effects of some. My expertise is in post-trasnscriptional regulation of cytokines and we have already characterized some potent effects in cancer.

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript entitled "HuR modulation with tanshinone mimics impairs LPS response in murine
    macrophages" the authors have described the synthesis and application of small molecule mimics of the naturally occurring compound tanshinone, which is known to inhibit the binding of the RBP HuR to a class of its mRNA targets. The authors have shown that the tanshinone mimics (TMs) used by them block the binding of RRM1-2 of HuR to ARE-containing RNA in vitro, and reduce the interaction of HuR with a set of ARE-containing mRNAs in LPS-treated mouse macrophage cells. This reduction of interaction of HuR with some of these mRNAs correlates with the reduction in their level in the cells treated with the TMs, and in the secreted level of their proteins in the serum of animals with LPS-induced peritonitis. Together, the study demonstrates the role of these TMs as modulators of the LPS-induced inflammatory response by blocking the binding of HuR to a subset of LPS-induced inflammatory mRNAs and thereby downregulating their mRNA and protein levels in inflammatory cells.

    The manuscript describes a comprehensive study, ranging from chemical synthesis of TMs, MD simulations to demonstrate the binding site of the TMs to the cleft formed by the RRM1-linker-RRM2 domains of HuR, which has been shown in crystal structure to be the main binding site of A/U-rich RNA molecules, in vitro studies showing the ability of the TMs to hinder ARE-containing RNA binding to HuR RRM1-2, whole transcriptome analysis to show the effect of the TMs on LPS-induced differential gene expression in murine macrophages, and on HuR binding to target mRNAs, and animal studies to show the effect of the TMs on the level of some inflammatory mediators in the serum of mice with LPS-induced peritonitis. The results are quite convincing and is in line with what is generally known about the effect of HuR on the expression of a large number of genes encoding pro-inflammatory proteins, and the ability of tanshinone derivatives/mimics in inhibiting HuR binding to target mRNAs. The authors put these two information together in this study and the results are on expected lines. While the results are convincing and quite comprehensive, I would suggest the following in order to substantiate and strengthen the results:

    1. The experiments do not have any "positive control", such that the performance of the TMs can be compared with that of a molecule with known HuR binding inhibition activity, such as DHTS. It would be good to have such a comparison, to understand whether the TMs work similar to DHTS or differently, both qualitatively in terms of the mRNA targets which they affect and the extent of their anti-inflammatory activity.
    2. It is not clear to me whether the mRNAs which show differential expression in the RNAseq analysis of cells treated with LPS and TMs are exactly the ones which show difference in binding with HuR in the RIPseq analysis in presence of the TMs. This analysis is important for a number of reasons: all the mRNA binding targets of HuR are not affected by HuR at the level of mRNA stability, many are affected at the level of translation, without change in mRNA level. These mRNAs should therefore show change in binding of HuR in the RIPseq assay in presence of TM, but not show change in expression. Secondly, there may be mRNAs which show a change in expression in presence of TMs, but do not show binding of HuR, suggesting pleiotropic roles of the TMs. Therefore, instead of an overall correlation between differential expression and change in HuR binding of mRNAs, a table comparing the RIPseq status of individual mRNAs with that of their differential expression status, in presence and absence of LPS/TMs would be useful, further designating the different groups of mRNAs based on these differential status (change in HuR binding/change in expression, change in HuR binding/no change in expression etc.).
    3. Nuclear/cytoplasmic localization of HuR and NFkb is impossible to discern at the magnification of the immunofluorescence images in Fig 6 B and C. Higher magnification images are required to understand changes in localization.
    4. It has been shown that DHTS-I increases the binding of HuR to the mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, whereas it reduces the interaction of HuR with the mRNAs having shorter 3'UTR and with low density of U/AU-rich elements (Lal et al., NAR, 2017). It is not clear if the same is observed in case of the TMs or not, and such a comparative analysis would be useful to address this point.

    I think that the above suggested points are feasible as most of them really involve re-analysis of existing data. Only the suggestion to add DHTS or tanshinone as a positive/comparison control will require experimentation and addition of new data.

    Review Cross-commenting

    I think most of the reviewers' comments coincide in the evaluation of the manuscript. I would especially like to draw attention to the fact that all three reviewers found that the content and form of data presented in the paper is very dense and bogs down the reader and distracts from the overall focus of the manuscript.

    Significance

    The work described in the manuscript is comprehensive as it ranges from chemical synthesis and in vitro evaluation of the TMs to the characterization of their effects in vivo. Although the effect of tanshinone derivatives on HuR mRNA target binding is known, and the effect of HuR on inflammatory gene expression is also known, the manuscript is significant as it brings these two information together and tests the effect of these TMs on HuR-mediated regulation of inflammatory gene expression.
    However the extensiveness of the work also makes it quite dense, and I feel that the focus of the paper is often lost in the details. Also, the text of the manuscript is dense and verbose and uses many irregular grammatical and phraseological usages, for eg "their
    modulation or mis-localization lead to the insurgence of complex phenotypes and diseases". It appears to me that it would be ideal if the chemical synthesis, MD simulation studies and in vitro studies are presented in an independent manuscript. Also, that would allow a more exhaustive referencing of the known studies in literature where tanshinone derivatives, and other small molecules, have been used to modulate HuR binding to mRNA targets.
    This work would be of interest to molecular cell biologists in general and RNA biologists in particular, especially those who are studying RNA-protein interactions, and scientists who are interested in drug development using RNA-protein interactions as drug targets.
    My interest in the work lies in my expertise in studying RNA-protein interactions, especially of RNA-binding proteins such as HuR involved in regulating the translation of mRNAs encoded by inflammatory genes. I do not have expertise in chemical synthesis and am therefore not qualified to evaluate the first set of results describing the chemical synthesis of TMs.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The Authors report on the synthesis and characterization of a class of small molecules, the tanshinone mimics (TMs), which interfere with binding of the RNA binding protein (RBP) HuR to its mRNA targets. HuR is an important regulator of mRNA stability and translation of genes involved in key homeostatic (cell cycle, stress response) and pathologic process (inflammation, carcinogenesis). In particular, the first part of the study describes the compounds' chemical synthesis and some pharmacokinetic parameters (i.e., definition of molecular binding, solubility, bioavailability, prodrug generation etc). The second part undertakes, in in vitro and ex-vivo model of LPS-induced mouse macrophage activation, the identification of HuR-bound mRNA targets, which is then evaluated within the global LPS-induced transcriptome; finally, the study evaluates the ability of TMs to inhibit HuR-mediated proinflammatory gene regulation, indicating their use and potential value as therapeutic anti-inflammatory strategy.

    Major comments:

    The manuscript contains a wealth of data generated from different experimental systems, spanning from synthetic chemistry to preclinical models of gene regulation, requiring cultural backgrounds in chemistry and biology as well. The key conclusions are well supported by the data, but it takes a great effort to get to the core results and thus critically read and evaluate their interpretation. Although the complexity and sheer size of data sets generated lends itself to a hard read, this is further complicated by data presentation, which especially in the second part needs to be significantly improved to gain clarity and focus. For ease of referral, specific comments will be addressed related to Figures whenever possible.

    • Page 15: To measure TM7nox disrupting ability of HuR:mRNA complex for the HTRF assay (Figure 2G) and for biotin pull down assay (Figure 5C), it was chosen a biotinylated probe containing the AU rich elements of the TNFα, as known HuR target. Please comment on the rationale, and whether could it be relevant reevaluate these parameters post-hoc, based on the sequences identified in HuR targets more susceptible of modulation by TM compound (listed in table 1, Figure 5 A/B) and based on the absence of regulation of TNF (Figures 3D, 4D, 7A) found in the tested systems.
    • Page 16-18: Description of the RNAseq data shown in Figure 3 should be more centered around the main findings regarding the effect of TMnox that are further pursued in the study: that is, (Figure 3B), the 249 downregulated DEGs found modulated by TM7nox in presence of LPS, where was observed a strong enrichment of categories related to the inflammatory response: cytokines (Il1b, Cxcl10, Il10, Il19, Il33), immune cell chemotaxis (Ccl12, Ccl22, Ccl17, Ccl6) and innate immune response. The description of the GO for the remaining data should be shortened to main points, perhaps reporting what described in the results with each section of the Venn in a table, while referring to the whole list in the supplements as already done. This could replace Figures 3D, E which do not add substantially to what provided in the supplementary table 2 and to which they can be added as further visualization.
    • Page 18-19: Description of the results of the RIP-seq shown in Figure 4 set is very confusing: onward from the line "477 HuR-bound transcripts (log2 FC > 3) were also upregulated by LPS at the transcriptional level..." the numbers do not match or reconcile with those shown in the Venn diagram (Fig. 4B) nor with those listed in the figure legend of Figure S8. Moreover, as previously remarked for Figure 3 (and even more for this dataset in which initial description of Venn in 4B is unclear), panel 4E does not add as much to the info provided in Table 1/supplementary Table 1, where they can eventually be added as further data visualization; Instead, Figure S8 displays very informative data merging together the results obtained in RNAseq (Fig. 3) and RIP-Seq (Fig.4) and should be displayed in Figure 4, as in the result section they are indeed presented together.
    • Page 19-20: Description of the modulation by TM7nox of HuR binding to specific consensus sequences is summarized at the end of the relative paragraph as follows: "TM7nox reshapes HuR binding to target genes in presence of LPS by disrupting the binding of HuR towards target genes containing a lower number of HuR consensus sequences than the average observed in the HuR-bound transcripts". Understanding of these data through the provided text and the Supplementary Figure 9 is very laborious and referring of an entire dataset to a supplementary figure makes it even harder. It would be best to show this as main figure, not supplemental, either adding a Venn diagram as in 3B/4B showing to which dataset each part of the analysis refers, or even more efficaciously, extrapolate a representative gene set for the main analyses showing TM7nox activity in target genes with higher vs lower consensus sequences; same approach for the analysis in Figure 9B, where the effect on a gene with sequence #1 or #10 could be compared with one bearing sequence #3 for example.
    • Page 21: Description of the effect of three TMs (TM6, TM7nox and TM7nred) on LPS response in macrophages at the single gene level (Figure 5 and Figure 6): TM6 and TM7nox were used in exps in Fig. 5 A and E, while only TM7nred was used for CXCL10 secretion analysis (fig.5 D and F): please describe the compound choices' rationale (as done for experiments in Figure 6).
    • Page 21-22: The effect on HuR expression of siRNA silencing and, more importantly, of TMs shown in Figure 6A, first panel, should be visualized at protein level by western blot. This is an important point as for CXCL10 and iL1there seems to be an additive effect between decreased HuR levels and pharmacological blocking.
    • Page 24: please rephrase the statement 'These observations suggest the utilization of TMs in autoinflammatory and autoimmune diseases' as 'These observations suggest the evaluation of TMs in specific preclinical models for autoinflammatory and autoimmune diseases'.
    • In the discussion, please include a paragraph with study limitation and possible biases (for example, the choice of RNP-IP without crosslinking has pros and cons).
    • The data and the methods are correctly presented for reproducibility, replicates and statistical analysis are adequate.

    Minor comments:

    • At least in the single gene validation experiments (Fig.5), a negative control (such as recombinant HuR with mutated RRMs in trans-, or ARE-less/non-HuR targetable sequence in cis, or inactive TM) would be advisable.
    • Figure 6B/C: for immunofluorescence panels, zooming on a smaller number of cells will render more visible HuR and NFB nucleocytoplasmic shuttling, given that quantification and statistics are provided by imaging software. Negative control stainings (secondary Abs only) should be included.
    • Figure 7A: in the X axis LPS+8n is indicated: is it a typo for LPD+6n or was compound TM8n indeed used?
    • In the Methods section please include protocols and materials for immunofluorescence (results shown in Fig. 6B/C).
    • There are some typos and repetition in figure legends (legend Figure S9).
    • Prior studies are referenced appropriately.

    Review Cross-commenting

    I fully agree with the Reviewer's remarks. I would add that a general concern expressed is that this manuscript in its present form has a readership issue: the first part is for chemistry/pharmacology audience, the second is biology-based. Splitting the work has been suggested; or, the Authors may decide which part is more impactful and present the other in a streamlined version.

    Significance

    This is a large study reporting progress in the development of synthetic antagonists of HuR function, which is the Authors' well-established line of research. The TM compounds are small molecules with anti-inflammatory effects with strong potential for therapeutic use due to selected inhibition of HuR-mediated upregulation of proinflammatory molecules. The physicochemical and early biological characterization done in this study will allow further testing of their efficacy and of the overall role of HuR-mediated regulation as targetable mechanism in several preclinical human disease models.

    Targeting of the RNA-binding protein HuR has been tackled as therapeutic approach in cancer, less in chronic immune and inflammatory diseases despite many common mechanisms and mediators.

    This study could be well received by researchers involved in basic science and drug development (chemistry, biochemistry/biophysics, pharmacology, computational modeling) and biologists/physician scientists interested in testing these compounds in translational research settings where HuR-driven functions can be relevant (cancer, chronic inflammation), though the chemical part would be less accessible to the latter audience.

    Reviewer's background is in preclinical human models of chronic inflammation with interest in posttranscriptional gene regulation with familiarity with RNAseq and RIPseq dataset and analysis. For the part of the manuscript regarding the synthesis and physicochemical characterization of the TN compound I requested assistance to a faculty from the chemistry department with expertise in that field, who did not request any specific clarification or addendum.

  6. Version published to 10.1101/2023.01.16.524289v1 on bioRxiv