Glycogen Synthase Kinase 3Beta as Target for Neurodegenerative Disease Drug Discovery: Proteomic Approaches to Characterize its Activity in Vitro

The work described in this thesis was performed in order to develop advanced analytical methods suitable to select and characterize GS- 3β inhibitors in vitro. GSK-3β is recognized as a key target for the development of new therapeutic agents for Alzheimer disease (AD). We validated an UHPLC-UV-Vis diode arrays detector (DAD) method for the very fast identification (resolution in less than 2 min) and determination of ADP and ATP in enzyme-based assay containing GSM-S syntetic peptide, ATP and GSK-3β. By using this method, selected inhibition hits will be characterized by defining their competitive mode of action with the substrate rather than with the ATP cofactor, in view of the discovery of compounds endowed of an increased GSK-3β selectivity over other protein-kinases. Next we hypothesized that GSK-3β could be directly involved in the regulation of histone acetylation through HDAC protein. Our hypothesis accounts that inhibition of GSK-3β, which leads to reduced HDAC activity, could restores the acetylation level in histones, protecting against neurodegeneration as both aging and AD pathology are associated with loss of histone acetylation (H4/H3) at N-terminus. Therefore, quantification of histone modifications on individual lysine residues is of crucial importance to understand their role in cell biology. We developed a targeted liquid chromatography mass spectrometry (LC-MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 from murine macrophage-like cell line RAW 264.7, with the perspective to apply the method on neuronal cells upon administration of GSK-3β inhibitors. The analytical strategy we developed shows that careful optimization of chemical derivatization steps at the protein and at the peptide level, combined with a more extensive digestion using chymotrypsin and trypsin, allows to differentiate between acetylation levels of each lysine residues.