Structural and biophysical characterization of novel GSK-3β inhibitors

The deregulation of GSK-3β (glycogen synthase kinase-3β) is involved in the pathogenesis of diverse diseases, such as cancer, diabetes and neurodegenerative disorders. Therefore, GSK-3β has become an attractive target for the design and development of new inhibitors for pathologies that present many limitations in therapeutic treatment. In the present thesis, we report a fast and efficient protocol for the expression of GSK-3β in insect cells with the baculovirus system, and employ purified GSK-3β in two different drug discovery projects. The first project describes the perspective validation of a novel computational method based on adiabatic bias molecular dynamics (ABMD) that aims to simulate protein-ligand unbinding events. Surface plasmon resonance (SPR) experiments performed on a series of GSK-3β inhibitors confirmed a coherence between predicted and experimental koff, highlighting the potential of this method for the calculation of residence times in hit-to-lead and lead optimization phases of drug discovery programs. Three novel X-ray crystal structures of pyrazine inhibitors in complex with GSK-3β are reported, providing further details in the binding mode of ATP-competitive GSK-3β inhibitors. The second project involves the structural and biophysical characterization of known GSK-3β allosteric inhibitors, and, in parallel, the discovery of novel allosteric modulators through computational and biophysical techniques. Herein, we describe the identification of promising molecules that have been selected by virtual screening and microscale thermophoresis (MST) analysis that display affinity to GSK-3β in the micromolar range. Taken together, our results provide useful insights for future rational drug design and discovery of small selective GSK-3β inhibitors.