Novel Insights into the inhibition of phosphorylating enzymes: A unique mechanism for glyceraldehyde 3-phosphate dehydrogenase inhibition and characterization of truncated phosphoinositide 3-kinase δ for inhibitors identification

This doctoral project, carried out in collaboration with Chiesi Farmaceutici, includes two complementary research lines, both centered on the characterization of structurally complex proteins of high pharmaceutical relevance, investigated as potential drug targets. The first line of research focused on glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a tetrameric enzyme best known for its central role in glycolysis but also endowed with a wide range of non-canonical, “moonlighting” functions, including roles in oxidative stress responses, apoptosis and cancer progression. Within this project, a novel inhibition mechanism was identified involving a spirocyclic 3-bromo-4,5-dihydroisoxazole derivative, which selectively destabilizes the quaternary structure of the enzyme. Due to the inherent complexity of GAPDH, characterized by dynamic conformational and oligomeric states, its study required a broad spectrum of analytical methodologies, ranging from advanced mass spectrometry approaches (HDX-MS, native MS, top-down and bottom-up MS) to spectroscopic and biophysical techniques such as circular dichroism, fluorescence spectroscopy, and size-exclusion chromatography coupled with light scattering. The second line of research was devoted to phosphoinositide 3-kinase delta (PI3Kδ), a lipid kinase that plays a role in B and T lymphocytes function, whose dysregulation has been linked to immune disorders and hematological malignancies. Given its pharmacological importance, PI3Kδ represents an attractive therapeutic target. The project developed a recombinant expression strategy for the expression of the catalytic subunit p110δ in E. coli and P. pastoris, providing a biotechnological platform alternative to insect cell systems and enabling the production of material suitable for structural, biochemical, and enzymatic assays with isoform-selective inhibitors. Together, these two research lines exemplify the necessity of employing an integrated and versatile instrumentation platform with orthogonal biochemical and biophysical techniques to investigate complex proteins that combine structural plasticity with diverse biological functions.