Development of selective inhibitors for the PDIA3 protein for pharmacological use in glioblastoma

PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as inflammation, neurodegeneration and cancer. In particular, PDIA3 expression level has been considered as a biomarker in glioblastoma, the most aggressive and common tumor of the central nervous system in adults and so proposed as a potential new pharmacological target. In my PhD project, starting from scaffolds of compounds known as PDIA inhibitors, we developed new molecules and evaluated their potential selective inhibition on PDIA3 compared to PDIA1, following different strategies. We initially analyzed punicalin, a smaller punicalagin derivative lacking the hexahydroxydiphenic acid moiety, which has been shown to be a more selective inhibitor of PDIA3. This suggested the possibility of developing ellagitannin scaffold-based molecules with a smaller structure with better affinity and selectivity for PDIA3. In collaboration with the research group of Prof. Roberto Di Santo, a group of new structural derivatives of polyphenols and 16F16 molecules were designed and synthesized. The analysis of these new compounds led to the identification of two new molecules, RDS 1554 and RDS 4310, which showed a greater ability to bind and inhibit the PDIA3 protein. DSF studies indicated that RDS 1554, like punicalin, showed a greater change in thermal stability than PDIA3. RDS 4310, a non-selective inhibitor of PDIA3, showed a destabilizing effect against PDIA3, but less than that induced by RDS 1554. Unlike RDS1554, which caused a cellular redistribution of PDIA3, RDS 4310 at the same concentration of 20 uM, caused a high cytotoxic effect in T98G glioblastoma cells. Finally, proteomic and computational analyzes reveal a mode of action that has not yet been observed for covalent inhibitors, thus offering valuable mechanistic insights into isoform selectivity. Based on these results obtained from my doctoral work, information has been obtained that can be used to develop new potential more selective inhibitors towards the PDIA3 protein.