Functional Evaluation of Novel G6PD Inhibitors Using a SCAR Laser Spectroscopy–Based Radioglucose Metabolic Assay

This thesis focuses on validating SCAR laser technology (Saturated-absorption Cavity Ring-down) as an innovative bioanalytical tool for studying cellular metabolism and screening new inhibitors of the G6PD enzyme. G6PD is a crucial therapeutic target, as its upregulation in cancer supports cell survival and chemoresistance via the pentose phosphate pathway (PPP). The research led to the development of the MW24Assay, an optimized protocol using 14C-labeled glucose to measure the metabolic flux of the PPP. Results demonstrate that SCAR technology offers over 20 times greater sensitivity than traditional Liquid Scintillation Counting (LSC), allowing for a drastic reduction in the radioactivity required for assays. The method was validated by monitoring PPP alterations induced by metabolic stress and by testing the efficacy of novel molecules synthesized at the University of Pisa. Specifically, studies on G6PD-overexpressing cells confirmed that metabolic dependency can be exploited to identify more effective inhibitors, such as the reference compound G6PDi-1 and the new analogs AB148 and AB196.