Molecular mechanisms of protein disulfide isomerase antagonism by punicalagin

Punicalagin, an ellagic acid polyphenol from pomegranate, has been proposed as an antagonist of protein disulfide isomerase (PDI) and endoplasmic reticulum resident protein 57 (ERp57)-thiol isomerases that regulate protein folding and extracellular thrombotic signaling. Here, biochemical oxidase and reductase assays on PDI showed that punicalagin inhibits both activities with micromolar potency, thereby extending earlier work that described disulfide reductase inhibition. Using purified domains to determine potential interaction sites indicated that punicalagin antagonizes both the N-terminal a and C-terminal a' domains similarly. Broader profiling of the PDI family demonstrated that punicalagin selectively inhibits the oxidase activity of multiple thiol isomerases while leaving ERp72 unaffected. In parallel, jump-dilution experiments revealed a reversible mechanism of inhibition. Thiol labeling of PDI's catalytic cysteines detected no change in the redox state, supporting a noncovalent, allosteric mechanism. Complementary spectrophotometric and fluorometric assays showed that punicalagin coordinates with zinc to further inhibit PDI, suggesting metal complexation as an additional feature that may shape its interaction with thiol isomerases. Extensive molecular docking and molecular dynamics simulations showed that punicalagin binds stably and preferentially to defined sites on both the N- and C-terminal domains through extensive hydrogen bonding and van der Waals contacts. Finally, artificial intelligence-driven network analysis identified PDI as a high-confidence target of punicalagin and related galloylated polyphenols, alongside additional signaling proteins. Together, these findings provide further mechanistic framework for punicalagin-mediated antagonism of PDI and highlight galloylated polyphenols as promising scaffolds for PDI-targeted therapeutics.