Targeting RNA:protein interactions with an integrative approach leads to the identification of potent YBX1 inhibitors
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
Protein-RNA interactions are involved in many diseases and targeting them with drugs can be valuable. Because protein-RNA complexes are considered difficult to target both computationally and experimentally, an integrated computational-experimental approach to solve this limitation is introduced. The approach is demonstrated by targeting the mRNA-binding protein YB-1, which works remarkably well. Inhibitors in the micromolar range are detected, including a previously approved drug. The main strength here is the proof of concept that protein-RNA interactions are targetable. However, additional data are required to support the central claims of the paper.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Structural Biology and Molecular Biophysics (eLife)
Abstract
RNA-protein interactions (RPIs) are promising targets for developing new molecules of therapeutic interest. Nevertheless, challenges arise from the lack of methods and feedback between computational and experimental techniques during the drug discovery process. Here, we tackle these challenges by developing a drug screening approach that integrates chemical, structural and cellular data from both advanced computational techniques and a method to score RPIs in cells for the development of small RPI inhibitors; and we demonstrate its robustness by targeting Y-box binding protein 1 (YB-1), a messenger RNA-binding protein involved in cancer progression and resistance to chemotherapy. This approach led to the identification of 22 hits validated by molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy of which 11 were found to significantly interfere with the binding of messenger RNA (mRNA) to YB-1 in cells. One of our leads is an FDA-approved poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor. This work shows the potential of our integrative approach and paves the way for the rational development of RPI inhibitors.
Article activity feed
-
-
Author Response
Reviewer #1 (Public Review):
A novel approach is introduced for targeting Protein-RNA interactions. The approach (presented in Figure 1) integrates computational techniques with cellular assays, and is applicable, in principle, whenever the protein-RNA complex has a druggable binding pocket. It is demonstrated with the discovery of inhibitors of YB-1's interaction with its mRNA target. Of 22 putative hits, discovered based on virtual screen, 11 come out as very strong hits. Far beyond the 5-10 percent success rate that one often sees in drug discovery. The main strength here is the proof of concept that protein-RNA interactions are targetable.
We agree with the reviewer that large computational screens to identify potential inhibitors generally lead to dead ends. This is why we have rationally designed this …
Author Response
Reviewer #1 (Public Review):
A novel approach is introduced for targeting Protein-RNA interactions. The approach (presented in Figure 1) integrates computational techniques with cellular assays, and is applicable, in principle, whenever the protein-RNA complex has a druggable binding pocket. It is demonstrated with the discovery of inhibitors of YB-1's interaction with its mRNA target. Of 22 putative hits, discovered based on virtual screen, 11 come out as very strong hits. Far beyond the 5-10 percent success rate that one often sees in drug discovery. The main strength here is the proof of concept that protein-RNA interactions are targetable.
We agree with the reviewer that large computational screens to identify potential inhibitors generally lead to dead ends. This is why we have rationally designed this integrative approach where predictions are experimentally validated with different tools and the obtained results feed/orient the computational approach. The workflow illustrated in Figure 1 creates a vivid exchange between computational and experimental data and allows a back-and-forth between both to enhance and refine the computational screen. We have also put in place a refined physics-based computational approach to increase our chances in avoiding these dead-end screens (details are in Computational Methods and in Appendix 2). The high predictive power of our computational approach comes from a rationally designed workflow combining the following:
1- Understanding the dynamic behavior of the target, the binding pocket, and identification of key residues using MD simulations.
2- The starting 3D structures used and refined using MD simulations.
3- The prior identification and validation of the binding site and the identification of F1 and F4 as hits by NMR spectroscopy. F1 was then used in the pharmacophore screen.
4- The statistical mechanics-based filter played an important role in orienting and refining this selection. For example, the use of ligand-water interactions to qualitatively estimate the residence of the ligand in the binding site.
Nevertheless, the high success rate also comes from human intervention, where visual inspection and rational selection of structurally promising candidates (sometimes intuition-driven) also played an important role in selecting the 111 molecules issued from the static virtual screen (pharmacophore screens). We now clarify this point on pages 5 and 6 of the revised manuscript and give more details on the selection criteria used. We also specify that the large computational screen we implemented was mandatory to validate the MT bench.
Reviewer #2 (Public Review):
In the manuscript "Targeting RNA-Protein Interactions with an Integrative Approach Leads to the Identification of Potent YB-1 Inhibitors" the authors have tried to integrate computational, structural, and cellular imaging approaches to identify small molecule inhibitors of RNA-protein interactions. They take up as their target YB-1, an abundant RNA-binding protein (RBP) involved in regulating the translation and/or processing of multiple mRNAs, many of which encode genes involved in tumorigenesis and tumor progression. Firstly, the authors find a binding pocket in the cold shock domain (CSD) of YB-1, for the flavonoid fisetin, and more so for the analog quercetin, by NMR spectroscopy, which they name the "quercetin pocket". They then delineate and refine the RNA-binding characteristics of this pocket by MD simulations. Further, they conduct a computational screen of a large library of small molecules to find candidates which bind to this pocket. They then check the selected candidates as inhibitors of YB1-mRNA interaction using the microtubule bench (MT-bench) method. They find 11 molecules as significant hits with this approach, including one FDA-approved PARP-inhibitor drug (P1). P1 is shown to bind YB-1 by MD-simulation and NMR spectroscopy and was also shown to interfere with YB-1-mRNA interaction by NMR and in cells by the MT-bench assay. Finally, they showed that the molecule P1 reduced cellular translation by a puromycin incorporation assay and this effect was not observed in cells depleted of YB-1.
Together, these multifarious approaches appear to establish a workflow useful for scoring for inhibitors of RNA-protein interactions. The workflow is rationally designed, moving from the identification of a binding pocket to the identification of binding molecules and then selecting molecules that inhibit protein-mRNA interactions. This workflow may be useful for other researchers attempting to screen libraries of compounds targeting RNA interactions by other RNA-binding proteins. However, as many RNA-binding proteins have large intrinsically disordered regions or no recognizable RNA-binding domains, it is to be seen whether such a structural "binding-pocket"-based approach can be generalizable to all RNA-binding proteins.
We agree with the reviewer that this is not sufficient to generalize to all RBPs. Performing a complete study for other RBPs would require a separate paper. In the current work, we did show that we can detect mRNA-RBP interactions with two other RBPs HuR and FUS and used them as a control to show the specificity of the tested small molecules towards YB-1 (Figures 3d and 4b,c). We have now tuned down the statements about the generality of the method (page 20).
In the discussion, we now also explain that YB-1, because it has a single cold-shock domain and a druggable pocket, is an “ideal” target. We also explain that many RNPs harbors many RNA-binding domains, which may reduce the sensitivity of our method when a specific domain is targeted by small molecules because the other domains would contribute to the binding to mRNA. However, a single RNA-binding domain may be isolated and used as bait for the MT bench assay to overcome this obstacle. Developing molecules what would target a specific domain may be sufficient to modulate the biological function exerted by the full length protein.
While the data presented in the paper is coherent and generally supports the demonstration of an inhibition of RNA-binding by YB-1, what appears to be lacking is evidence that the observed effect is specific to inhibition of YB-1-mediated regulation of translation and whether the expression of transcripts specifically regulated by YB-1 is affected. Secondly, it is not clear what is the effect of the putative inhibitor on cellular activity and behaviour, which is important to judge both specific phenotypic effects as well as non-specific cytotoxic effects.
Overall the work is interesting and instructive, but the lack of the above observations detracts from its significance.
We thank the reviewer for his feedback and for raising these interesting points. As indicated in the manuscript, it is very difficult to find functional cellular assays that would reveal a phenotype specific to a general RBP such as YB-1. This is even more difficult with YB-1 since it binds nonspecifically to most mRNAs as shown from CLIP analysis1. This was one of the reasons to develop a specific cellular assay such as the MT bench assay. YB-1 originates from cold shock proteins in bacteria which preserve global mRNA translation during cold stress, presumably by removing secondary structures. YB-1 in contrast with many RBPs has only a single structured RNA-binding domains, which is not favorable for a specific binding to some mRNA sequences/structures. As noticed by the reviewers, YB-1 is indeed not a general translation factor but is a general protein that binds to most non polysomal mRNA 2. mRNAs, even those highly translated, switch from a polysomal state (active) to a non polysomal state (dormant) from time to time. In a recent work, we showed that YB-1 prepared non polysomal mRNAs in a way to facilitate the translation from dormant to active state. We also showed that, accordingly, decreasing the expression of YB-1 reduces global mRNA translation rates in HeLa cells3. Consistent with this trend, a global decrease of mRNA translation as observed with Niraparib P1 that targets YB-1 makes sense. We have no knowledge of established 3’UTRs which would be highly specific to YB-1. YB-1 binds non specifically to both mRNA coding sequences and 3’UTRs (YBX1 data1, YBX3 data4). Large scale and in depth analysis should be performed to find out whether specific structures/sequences increase significantly the YB-1 dependency in mRNA translations. However, the expression of some proteins associated to malignancy have been associated to YB-1 expression level notably Vimentin and E-cadehrin3. For this we performed a new experiment where we measured the expression levels of these two proteins after silencing YB-1 expression in HeLa cells, in the absence and in the presence of Niraparib P1 and Olaparib P2 (used as a negative control). Results show that P1, but not P2, decrease the dependence on YB-1 of Vimentin expression level (significant) and that of E-cadherin (non-significant). Other proteins such as eIF5a and RPL36, used here as negative controls, did not show a similar behavior. These results were thus in agreement with a specific effect of Niraparib on YB-1-mediated translation. In agreement with these results, we now add a result from a recent report showing the down regulation of Vimentin expression in ovarian cancer cells when treated with Niraparib5. This is now discussed on pages 16 and 17 of the revised manuscript and the new data are included as a new figure Figure 8-Figure supplement 3.
Wu, S.-L. et al. Genome-wide analysis of YB-1-RNA interactions reveals a novel role of YB-1 in miRNA processing in glioblastoma multiforme. Nucleic acids research 43, 8516-8528 (2015).
Singh, G., Pratt, G., Yeo, G.W. & Moore, M.J. The clothes make the mRNA: past and present trends in mRNP fashion. Annual review of biochemistry 84, 325 (2015).
Budkina, K. et al. YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells. Nucleic acids research 49, 10061-10081 (2021).
Van Nostrand, E.L. et al. A large-scale binding and functional map of human RNA-binding proteins. Nature 583, 711-719 (2020).
Zhen Zeng, Jing Yu, Zhongqing Jiang, Ningwei Zhao, "Oleanolic Acid (OA) Targeting UNC5B Inhibits Proliferation and EMT of Ovarian Cancer Cell and Increases Chemotherapy Sensitivity of Niraparib", Journal of Oncology, vol. 2022, 12 pages, 2022. https://doi.org/10.1155/2022/5887671
As for the effect of the putative inhibitor on cellular activity and behaviour, which is important to judge both specific phenotypic effects as well as non-specific cytotoxic effects. We agree with the reviewer on this remark. YB-1 is associated with the high proliferation rate of cancer cells (and silencing YB-1 does not induce apoptosis). Therefore, we performed cell proliferation assays using cells treated with siRNA and siNEG allowing us to manipulate the endogenous YB-1 expression level rather than a more artificial rescue experiment. These assays were performed in the presence of 3 PARP-1 inhibitors at low concentrations: Niraparib P1 our hit, and two negative controls Olaparib P2 and Talazoparib P3. We used a 48 h incubation time which allows to observe effects at lower concentration of compounds. All PARP-1 inhibitors decrease cell proliferation, albeit to a higher extent with P3. However, P2 or P3 further decrease cell proliferation in siRNA-treated cells compared to siNEG-treated cells (significant differences at 5 µM)). In contrast, Niraparib rather further decreases cell proliferation in siNEG-treated cells when YB-1 levels are high (non-significant variations but opposite to those observed with P2 and P3). This new result is now presented as new Figure 8a. In addition, we show that the separation distance between cells increases significantly in YB-1-rich cells treated with P1, in contrast to P2 and P3 (significant differences) (new figure Figure 8-Figure supplement 1). A short distance of separation between cells may be due to colony formation when cells were plated at low density and allowed to grow for 48 h. Again, it means that Niraparib better inhibits cell proliferation in YB-1-rich cells when compared with what is observed with the two other PARP inhibitors Talazoparib and Olaparib. The text on page 17 was rewritten to include these new results and put this in evidence.
Reviewer #3 (Public Review):
The authors introduce an integrative platform for identifying small molecule ligands that can disrupt RNA-protein interactions (RPIs) in vitro and in cells. The screening assay is based on prior work establishing the MT bench assay (Boca et al. 2015) for evaluating protein-protein interactions in cells by utilizing microtubules as a platform to recruit and detect PPIs in cells. In the current manuscript, the authors adapted this methodology to evaluate small molecules targeting RNA-binding protein (RBPs) interactions with mRNA in cells. By combining the MT bench assay with computational docking/screening and ligand-binding evaluations by NMR, the authors discover inhibitors of the RBP YB-1, which included FDA-approved PARP-1 inhibitors. The impact of this work could be high given the critical roles of RNA-binding proteins in regulating the function and fate of coding and non-coding RNA. While the presented data are promising, the ability to generally apply this method beyond YB-1 and to RBPs in general remains to be addressed.
We agree with the reviewer on his comments. In the revised version of the manuscript, we have tuned down the statements about the generality of the method. In addition, we elaborate about the potential of our assays and how to deal with RBPs that often have more than one RNA-binding domain. If many RNA-binding domains participate to the binding of a given RBP to mRNA, we may lose the sensitivity of the MT bench assays. However, one point is to use as bait to target isolated RNA-binding domain which could be enough to impair/correct the function of the full length RBP target. A statement has been added on page 20 of the revised manuscript to discuss this point.
-
Evaluation Summary:
Protein-RNA interactions are involved in many diseases and targeting them with drugs can be valuable. Because protein-RNA complexes are considered difficult to target both computationally and experimentally, an integrated computational-experimental approach to solve this limitation is introduced. The approach is demonstrated by targeting the mRNA-binding protein YB-1, which works remarkably well. Inhibitors in the micromolar range are detected, including a previously approved drug. The main strength here is the proof of concept that protein-RNA interactions are targetable. However, additional data are required to support the central claims of the paper.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to …
Evaluation Summary:
Protein-RNA interactions are involved in many diseases and targeting them with drugs can be valuable. Because protein-RNA complexes are considered difficult to target both computationally and experimentally, an integrated computational-experimental approach to solve this limitation is introduced. The approach is demonstrated by targeting the mRNA-binding protein YB-1, which works remarkably well. Inhibitors in the micromolar range are detected, including a previously approved drug. The main strength here is the proof of concept that protein-RNA interactions are targetable. However, additional data are required to support the central claims of the paper.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
A novel approach is introduced for targeting Protein-RNA interactions. The approach (presented in Figure 1) integrates computational techniques with cellular assays, and is applicable, in principle, whenever the protein-RNA complex has a druggable binding pocket. It is demonstrated with the discovery of inhibitors of YB-1's interaction with its mRNA target. Of 22 putative hits, discovered based on virtual screen, 11 come out as very strong hits. Far beyond the 5-10 percent success rate that one often sees in drug discovery.
The main strength here is the proof of concept that protein-RNA interactions are targetable.
-
Reviewer #2 (Public Review):
In the manuscript "Targeting RNA:Protein Interactions with an Integrative Approach Leads to the Identification of Potent YB-1 Inhibitors" the authors have tried to integrate computational, structural, and cellular imaging approaches to identify small molecule inhibitors of RNA-protein interactions. They take up as their target YB-1, an abundant RNA-binding protein (RBP) involved in regulating the translation and/or processing of multiple mRNAs, many of which encode genes involved in tumorigenesis and tumor progression.
Firstly, the authors find a binding pocket in the cold shock domain (CSD) of YB-1, for the flavonoid fisetin, and more so for the analog quercetin, by NMR spectroscopy, which they name the "quercetin pocket". They then delineate and refine the RNA-binding characteristics of this pocket by MD …
Reviewer #2 (Public Review):
In the manuscript "Targeting RNA:Protein Interactions with an Integrative Approach Leads to the Identification of Potent YB-1 Inhibitors" the authors have tried to integrate computational, structural, and cellular imaging approaches to identify small molecule inhibitors of RNA-protein interactions. They take up as their target YB-1, an abundant RNA-binding protein (RBP) involved in regulating the translation and/or processing of multiple mRNAs, many of which encode genes involved in tumorigenesis and tumor progression.
Firstly, the authors find a binding pocket in the cold shock domain (CSD) of YB-1, for the flavonoid fisetin, and more so for the analog quercetin, by NMR spectroscopy, which they name the "quercetin pocket". They then delineate and refine the RNA-binding characteristics of this pocket by MD simulations. Further, they conduct a computational screen of a large library of small molecules to find candidates which bind to this pocket. They then check the selected candidates as inhibitors of YB1-mRNA interaction using the microtubule bench (MT-bench) method. They find 11 molecules as significant hits with this approach, including one FDA-approved PARP-inhibitor drug (P1). P1 is shown to bind YB-1 by MD-simulation and NMR spectroscopy and was also shown to interfere with YB-1-mRNA interaction by NMR and in cells by the MT-bench assay. Finally, they showed that the molecule P1 reduced cellular translation by a puromycin incorporation assay and this effect was not observed in cells depleted of YB-1.
Together, these multifarious approaches appear to establish a workflow useful for scoring for inhibitors of RNA-protein interactions. The workflow is rationally designed, moving from the identification of a binding pocket to the identification of binding molecules and then selecting molecules that inhibit protein-mRNA interactions. This workflow may be useful for other researchers attempting to screen libraries of compounds targeting RNA interactions by other RNA-binding proteins. However, as many RNA-binding proteins have large intrinsically disordered regions or no recognizable RNA-binding domains, it is to be seen whether such a structural "binding-pocket"-based approach can be generalizable to all RNA-binding proteins.
While the data presented in the paper is coherent and generally supports the demonstration of an inhibition of RNA-binding by YB-1, what appears to be lacking is evidence that the observed effect is specific to inhibition of YB-1-mediated regulation of translation and whether the expression of transcripts specifically regulated by YB-1 is affected. Secondly, it is not clear what is the effect of the putative inhibitor on cellular activity and behaviour, which is important to judge both specific phenotypic effects as well as non-specific cytotoxic effects.
Overall the work is interesting and instructive, but the lack of the above observations detracts from its significance.
-
Reviewer #3 (Public Review):
The authors introduce an integrative platform for identifying small molecule ligands that can disrupt RNA-protein interactions (RPIs) in vitro and in cells. The screening assay is based on prior work establishing the MT bench assay (Boca et al. 2015) for evaluating protein-protein interactions in cells by utilizing microtubules as a platform to recruit and detect PPIs in cells. In the current manuscript, the authors adapted this methodology to evaluate small molecules targeting RNA-binding protein (RBPs) interactions with mRNA in cells. By combining the MT bench assay with computational docking/screening and ligand-binding evaluations by NMR, the authors discover inhibitors of the RBP YB-1, which included FDA-approved PARP-1 inhibitors. The impact of this work could be high given the critical roles of …
Reviewer #3 (Public Review):
The authors introduce an integrative platform for identifying small molecule ligands that can disrupt RNA-protein interactions (RPIs) in vitro and in cells. The screening assay is based on prior work establishing the MT bench assay (Boca et al. 2015) for evaluating protein-protein interactions in cells by utilizing microtubules as a platform to recruit and detect PPIs in cells. In the current manuscript, the authors adapted this methodology to evaluate small molecules targeting RNA-binding protein (RBPs) interactions with mRNA in cells. By combining the MT bench assay with computational docking/screening and ligand-binding evaluations by NMR, the authors discover inhibitors of the RBP YB-1, which included FDA-approved PARP-1 inhibitors. The impact of this work could be high given the critical roles of RNA-binding proteins in regulating the function and fate of coding and non-coding RNA. While the presented data are promising, the ability to generally apply this method beyond YB-1 and to RBPs in general remains to be addressed.
-
