Abstract
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 cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3.