Peptide Nucleic Acids as RNA mimics in HuR-Ligand studies: insights from STD-NMR and protein-based techniques into HuR-ligand interactions

Hu (or ELAV-L) proteins belong to the RNA-Binding Protein (RBP) family, playing a crucial role in post-transcriptional gene regulation by forming complexes with mRNAs and modulating protein synthesis. 1 Among them, HuR is particularly relevant due to its involvement in key cellular processes, including cell growth, tumorigenesis, angiogenesis, inflammation, invasion, and metastasis. Notably, HuR overexpression is associated with high-grade malignancies and poor prognosis across multiple tumour types. 2 In previous studies3[FV3.1], we investigated the interaction of rationally designed ligands4 with HuR using Saturation Transfer Difference (STD) NMR, confirming their binding to the RNA-binding site through competition studies. To address challenges related to RNA handling in NMR experiments, we employed Peptide Nucleic Acid (PNA) fragments as RNA mimics, successfully demonstrating their viability as substitutes in STD assays. Starting from these findings, we are now working to validate and improve previous results using protein-based NMR techniques with ¹⁵N-labeled HuR, to provide direct structural and dynamic insights into ligand binding. ¹⁵N-HSQC titration experiments is useful to monitor residue-specific chemical shift perturbations upon ligand addition, confirming direct interactions and identifying the binding domain. Furthermore, relaxation-based experiments can provide additional evidence of binding affinity, ligand-induced conformational changes, and insights into the dynamical behaviour of the protein. This strategy is complementary to the previous reported approach and allow to assess ligand binding with higher resolution. Moreover, the data presented here will aid in identifying novel molecules that can modulate HuR-RNA complexes as potential pharmacological agents. Furthermore, this approach can be extended to other RNA-binding proteins, contributing to the broader field of drug discovery targeting RBPs. References: [1] S. Hong, 2017, J. Cancer Prev. 22 (4), 203–210. [2] C. Barreau, L. Paillard and H.B. Osborne, 2005, Nucleic Acids Research, 33, 7138–7150. [3] S. Della Volpe, R. Listro, M. Parafioriti, M. Di Giacomo, D. Rossi, F. A. Ambrosio, G. Costa, S. Alcaro, F. Ortuso, A. K. H. Hirsch, F. Vasile, and S. Collina, 2020, ACS Med. Chem. Lett., 11, 883−888 [3] S. Della Volpe, P. Linciano, R. Listro, E. Tumminelli, M. Amadio, I. Bonomo, W.A.M. Elgaher, S. Adam, A.K.H. Hirsch, F.M. Boeckler, F. Vasile, D. Rossi, and S. Collina, 2021, Bioorganic Chemistry 116, 105305 [3] I. Gado, M. Garbagnoli, F.A. Ambrosio, R. Listro, M. Parafioriti, S. Cauteruccio, D, Rossi, P, Linciano, G. Costa, S. Alcaro, F. Vasile, and S. Collina, 2024, ACS Omega, 9, 45, 45147–45158 [4] S. Della Volpe, R. Listro, F.A. Ambrosio, M. Garbagnoli, P. Linciano, D. Rossi, G. Costa, S. Alcaro, F. Vasile, A.K. H. Hirsch, and S. Collina, 2023, ACS Med. Chem. Lett., 14, 1509−1516