Small molecules modulating RNA splicing: a review of targets and future perspectives.

In eukaryotic cells, RNA splicing is crucial for gene expression. Dysregulation of this process can result in incorrect mRNA processing, leading to aberrant gene expression patterns. Such abnormalities are implicated in many inherited diseases and cancers. Historically, antisense oligonucleotides, which bind to specific RNA targets, have been used to correct these splicing abnormalities. Despite their high specificity of action, these oligonucleotides have drawbacks, such as lack of oral bioavailability and the need for chemical modifications to enhance cellular uptake and stability. As a result, recent efforts focused on the development of small organic molecules that can correct abnormal RNA splicing event under disease conditions. This review discusses known and potential targets of these molecules, including RNA structures, trans-acting splicing factors, and the spliceosome - the macromolecular complex responsible for RNA splicing. We also rely on recent advances to discuss therapeutic applications of RNA-targeting small molecules in splicing correction. Overall, this review presents an update on strategies for RNA splicing modulation, emphasizing the therapeutic promise of small molecules.

Molecular basis of RNA-binding and autoregulation by the cancer-associated splicing factor RBM39.

Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy but requires high levels of the adaptor protein DCAF15. Consequently, novel approaches to deplete RBM39 in an DCAF15-independent manner are required. Here, we uncover that RBM39 autoregulates via the inclusion of a poison exon into its own pre-mRNA and identify the cis-acting elements that govern this regulation. We also determine the NMR solution structures of RBM39's tandem RNA recognition motifs (RRM1 and RRM2) bound to their respective RNA targets, revealing how RRM1 recognises RNA stem loops whereas RRM2 binds specifically to single-stranded N(G/U)NUUUG. Our results support a model where RRM2 selects the 3'-splice site of a poison exon and the RRM3 and RS domain stabilise the U2 snRNP at the branchpoint. Our work provides molecular insights into RBM39-dependent 3'-splice site selection and constitutes a solid basis to design alternative anti-cancer therapies.

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1.

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a ß-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

Revisiting the Molecular Interactions between the Tumor Protein TCTP and the Drugs Sertraline/Thioridazine.

TCTP protein is a pharmacological target in cancer and TCTP inhibitors such as sertraline have been evaluated in clinical trials. The direct interaction of TCTP with the drugs sertraline and thioridazine has been reported in vitro by SPR experiments to be in the ∼30-50 µM Kd range (Amson et al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells. However, the molecular details of the interaction remain elusive although they are crucial to improve the efforts of on-going medicinal chemistry. In addition, TCTP can be phosphorylated by the Plk-1 kinase, which is indicative of poor prognosis in several cancers. The impact of phosphorylation on TCTP structure/dynamics and binding with therapeutical ligands remains unexplored. Here, we combined NMR, TSA, SPR, BLI and ITC techniques to probe the molecular interactions between TCTP with the drugs sertraline and thioridazine. We reveal that drug binding is much weaker than reported with an apparent ∼mM Kd and leads to protein destabilization that obscured the analysis of the published SPR data. We further demonstrate by NMR and SAXS that TCTP S46 phosphorylation does not promote tighter interaction between TCTP and sertraline. Accordingly, we question the supported model in which sertraline and thioridazine directly interact with isolated TCTP in tumor cells and discuss alternative modes of action for the drugs in light of current literature.

Drimane Derivatives as the First Examples of Covalent BH3 Mimetics that Target MCL-1.

Drimane sesquiterpenoid dialdehydes are natural compounds with antiproliferative properties. Nevertheless, their mode of action has not yet been discovered. Herein, we demonstrate that various drimanes are potent inhibitors of MCL-1 and BCL-xL, two proteins of the BCL-2 family that are overexpressed in various cancers, including lymphoid malignancies. Subtle changes in their structure significantly modified their activity on the target proteins. The two most active compounds are MCL-1 selective and bind in the BH3 binding groove of the protein. Complementary studies by NMR spectroscopy and mass spectrometry analyses, but also synthesis, showed that they covalently inhibit MCL-1 though the formation of a pyrrole adduct. In addition, cytotoxic assays revealed that these two compounds show a cytotoxic selectivity for BL2, a MCL-1/BCL-xL-dependent cell line and induce apoptosis.

Conformational Ensemble and Biological Role of the TCTP Intrinsically Disordered Region: Influence of Calcium and Phosphorylation.

The translationally controlled tumor protein (TCTP) is a multifunctional protein that may interact with many other biomolecules, including itself. The experimental determinations of TCTP structure revealed a folded core domain and an intrinsically disordered region, which includes the first highly conserved TCTP signature, but whose role in the protein functions remains to be elucidated. In this work, we combined NMR experiments and MD simulations to characterize the conformational ensemble of the TCTP intrinsically disordered loop, in the presence or not of calcium ions and with or without the phosphorylation of Ser46 and Ser64. Our results show that these changes in the TCTP electrostatic conditions induce significant shifts of its conformational ensemble toward structures more or less extended in which the disordered loop is pulled away or folded against the core domain. Particularly, these conditions impact the transient contacts between the two highly conserved signatures of the protein. Moreover, both experimental and theoretical data show that the interface of the non-covalent TCTP dimerization involves its second signature which suggests that this region might be involved in protein-protein interaction. We also show that calcium hampers the formation of TCTP dimers, likely by favoring the competitive binding of the disordered loop to the dimerization interface. All together, we propose that the TCTP intrinsically disordered region is involved in remodeling the core domain surface to modulate its accessibility to its partners in response to a variety of cellular conditions.

Structural Insights into TCTP and Its Interactions with Ligands and Proteins.

The 19-24 kDa Translationally Controlled Tumor Protein (TCTP) is involved in a wide range of molecular interactions with biological and nonbiological partners of various chemical compositions such as proteins, peptides, nucleic acids, carbohydrates, or small molecules. TCTP is therefore an important and versatile binding platform. Many of these protein-protein interactions have been validated, albeit only few received an in-depth structural characterization. In this chapter, we will focus on the structural analysis of TCTP and we will review the available literature regarding its interaction network from a structural perspective.