Molecules Inhibiting Translation in Cancer cells

Informazioni relative al progetto

MITiC

ID dell’accordo di sovvenzione: 895024

DOI

10.3030/895024

Progetto concluso il 30 Settembre 2022

Data della firma CE 6 Aprile 2020

Data di avvio 1 Novembre 2020

Data di completamento 31 Ottobre 2022

Finanziato da

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Costo totale

€ 184 707,84

Contributo UE

€ 184 707,84

Coordinato da

UNIVERSITE D'EVRY-VAL D'ESSONE
France

Periodic Reporting for period 1 - MITiC (Molecules Inhibiting Translation in Cancer cells)

Periodo di rendicontazione: 2020-11-01 al 2022-10-31

Sintesi del contesto e degli obiettivi generali del progetto

While targeting Protein:Protein interactions has served as basis for the development of new drugs, RNA:Protein interactions, which are as important in human pathologies, notably cancer, is very promising but remains largely unexploited. Several challenges arise from the drug discovery process such as finding a druggable pocket in RNA-binding interfaces, the quality of the computational models, the strategies used in the in silico screening, and the lack of experimental feedback and validation of computationally predicted inhibitors essential to orient the rational drug design procedure toward the most relevant molecules. Besides the above listed issues, new experimental assays must be developed to screen molecules targeting RPIs which ideally would work in a cellular context and be amenable to high content screening (HCS). In this project, we address the lack of methods able to score RPIs in a cellular context by adapting the microtubule bench assay (MT bench) to score protein interactions with endogenous mRNAs in cells and implemented a robust HCS-based detection scheme. We have also developed a drug screening approach that integrates chemical, structural and cellular data from both advanced computational and experimental techniques for the development of small molecules that target RPIs. This is done by developing an innovative approach that would integrate a) molecular modeling data (Drug design, Molecular Dynamics and Free Energy Simulations using a sufficiently accurate computational model that is computationally efficient), b) NMR Spectroscopy data, c) together with an experimental validation in cells with a new HCS technology, “MT Bench”, that quantifies RNA:protein interactions at the single cell level. The major advantage is providing cellular and structural data to feed, with little delay, the computational approach to propose efficient and specific ligands that target translation regulation in vitro and in cancer cells. As an application, we chose to target YB-1 (YBX1 gene), a mRNA-binding protein relevant target in cancer, notably owing to its role in cancer resistance and cancer cell plasticity. YB-1 has been recently considered as a therapeutic target for the treatment of cancer and drug-resistant cancer.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

In this project, we introduce an integrative approach that leads to the identification of several effective YB-1 inhibitors in the low micromolar range selected computationally and validated in vitro by NMR spectroscopy and in cells using the MT bench assay. Here, the MT bench was adapted to score small molecules targeting RBP interactions with endogenous mRNA in cells. The MT bench assays can notably fill the gap between in vitro and functional assays by probing whether the interaction of a selected RBP with mRNAs is affected in a cellular context but not that of other RBPs. Our results validated the reliability of the MT bench assay in detecting and scoring YB-1 interactions with mRNA in 96-well plates (SSMD >8). The results presented here, show that the physics-based in silico approach allowed the identification of 22 potential hits that we subsequently tested in vitro by nuclear magnetic resonance (NMR) spectroscopy and in cells using the adapted MT bench assay by scoring the interaction of YB-1 with mRNA in the cytoplasm. Of these 22 potential YB-1 inhibitors, 15 compounds were found to bind YB-1 in vitro and 11 of them were found to efficiently interfere with the interaction of YB-1 with mRNA in cells at low micromolar concentrations; and with a notable specificity when compared with two other RBPs, Human antigen R (HuR) and fused in sarcoma (FUS). The potency of the selected compounds was further demonstrated by in depthMD and NMR analyses. The results also validate that theMT bench allows to score RBP-specific interactions robustly and automatically with endogenous mRNAs by using high-resolution HCS imagers.
Interestingly, compound P1, an FDA-approved poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor, was found to interact with YB-1 with higher selectivity compared to the other hits. The efficiency of P1 in targeting RPIs and interfering with YB-1 cellular functions was then challenged with functional assays. Together, these results demonstrate the validity of our integrative approach and the efficacy of the MT bench assay that critically complements tional and structural approaches to identify compounds targeting RPIs in cells.


The project results were disseminated in scientific publications and were also deposited in open access repositories (bioRxiv). All of them include reference to EU funding:
1- K El Hage*, et al. Targeting RNA:Protein Interactions with an Integrative Approach Leads to the Identification of Potent YB-1 Inhibitors. bioRxiv. 2022. https://doi.org/10.1101/2022.04.08.487452
This paper is under review at eLife.
2- K Budkina, et al. YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells. Nucleic acids research 2021, 49 (17) : 10061-10081. https://doi.org/10.1093/nar/gkab748
3- A Samsonova, et al. Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain. Communications Biology, 2020, 4(1) :1-16. https://doi.org/10.1038/s42003-021-01862-3

The results were also disseminated in international conferences and workshops:
1) The ISQBP President’s meeting 2022 in Innsbruck, Austria “The International Society of
Quantum Biology and Pharmacology”. The researcher gave an oral contribution on the first day of the meeting entitled “Targeting RNA:Protein Interactions using an integrative approach: Identification of potent YB-1 inhibitors.”; and the talk was tweeted on social media.
2) EMBO: Advances and challenges in BioMolecular Simulations (18-21 October 2021, Virtual). The researcher presented a Poster.
3) The ISQBP President’s meeting 2021 (29 June - 1st July 2021, Virtual). The researcher presented a Poster.
4) 2021 Virtual Workshop on free energy methods in drug design (June 15-June 17). The researcher presented a Poster.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

In summary, this integrative approach allowed us to identify 22 potential hits, of which 15 are active in vitro and 11 are active in cancer cells at 10 micromolar; and one of our leads is Niraparib, an FDA-approved PARP-1 inhibitor, with a Kd ~ 6 microM in vitro and an IC50 ~ 10 microM in cells. And we were able to identify 2 scaffolds that are now being optimized to increase affinity and selectivity for YB-1 to be synthetized and tested. We also have now access to a Zebra fish model to test our predictions. As for the computational approach that we developed it is efficient, balancing accurarcy and computational cost. However it needs to be optimized to improve the ranking and reduce the errors.
This approach is transferable and can be used to target other systems such as Lin28, TDP43 and COVID-19 RNA binding proteins.

The work carried out creates a new venue in drug discovery by providing robust methods capable of targeting RNA:Protein Interactions. We have developed a unique method compatible with HCS to assess RPIs in cells, which, combined with experimental and in silico tructural approaches, can be used to target RPIs involved in human pathologies. And, here, in this work we demonstrated the usefulness of our integrative approach by successfully targeting mRNA:YB-1 complexes in cells.

Illustration of the integrative approach targeting RNA:protein interactions