Molecular Insights into the Dual Mechanism of Bepristat 2a on Protein Disulfide Isomerase (PDI) Activity

Protein disulfide isomerase (PDI) is a multifunctional thiol isomerase with extracellular activity crucial for thrombus formation. Inhibition of extracellular PDI suppresses thrombosis without impairing normal hemostasis, highlighting it as a promising antithrombotic target. We previously identified bepristats, a class of small molecules that selectively and reversibly inhibit the substrate oxidoreductase activity of PDI while paradoxically enhancing its ability to reduce GSSG. Here, we elucidate the molecular basis for this dual regulation by bepristat 2a (bep2a) through an integrated strategy combining molecular simulations and site-directed mutagenesis. Our results reveal that bep2a targets the major substrate-binding site with H256 identified as a critical residue for ligand recognition. This interaction is stabilized through a water-mediated hydrogen bond and induces compaction of the protein. In addition, bep2a binding allosterically triggered a conformational rearrangement of W396 and partially unwound the active-site helix. This structural change increases solvent exposure of catalytic cysteines C397 and C400, thereby enhancing the cleavage activity of the a' domain by facilitating nucleophilic attack. Thus, these findings uncover the structural basis for the simultaneous inhibition of PDI substrate oxidoreductase activity and enhancement of its catalytic disulfide cleavage function of bep2a and offer new insights for the design of highly selective PDI modulators.