TARGETING SPLICEOSOME AS A THERAPEUTIC STRATEGY: DESIGN AND SYNTHESIS OF NOVEL SF3B MODULATORS AND PRP4 KINASE INHIBITORS

A crucial stage in the expression of eukaryotic genes is the RNA splicing, in which the exons are combined to generate mature RNA products (such as protein-coding mRNAs or long non-coding RNAs (lnc-RNAs)) whereas the introns are removed from the precursor RNA (pre-RNA). Pre-RNA splicing is performed with remarkable accuracy by the spliceosome, a sophisticated ribonucleoprotein complex composed of five different snRNPs (U1, U2, U4, U5, and U6) in turn made up of one RNA each and several proteins. The spliceosome doesn’t consist in a permanent complex, but it assembles and disassembles every time the pre-mRNA splicing process occurs, known as the splicing cycle. This extremely large molecular machine requires essential regulation to ensure that all its parts function precisely and correctly at each stage of the cycle. More than 200 diseases such as genetic, neurodegenerative and several types of cancer are associated with dysregulated splicing. Therefore, the identification of small molecules that specifically modulate the spliceosome components offers a promising therapeutic avenue. Hence, the purpose of this thesis project is to develop new pharmacological strategies for modulating spliceosome activity exploring molecular targets relevant to its function. The first part of the research dealt with the synthesis and enantiomeric separation of a candidate modulator of the spliceosome factor SF3b (named AOAM216), which proved promising in preliminary in vitro assays as a racemic mixture and will therefore be further investigated. A second topic was the creation of a large virtual library of compounds analogue of AOAM216 with varied components and the most diverse substituents, to computationally explore the chemical space within the putative binding site of AOAM216 in SF3b and prioritize the synthesis of new chemical entities with increased affinity. The third part of the project dealt with the synthesis of another splicing modulator (FRB04), targeting Prp4 kinase, a known regulator of the spliceosome activity. This molecule has already been tested in vitro with promising results; therefore, the aim was to increase the scale of its production, optimizing the synthetic methodologies. FRB04 will be employed to carry out in vivo tests, validate the pharmacological target and proceed with the "hit-to-lead" optimization phases of the drug discovery process.