Identification of Antithrombotic Natural Products Targeting the Major Substrate Binding Pocket of Protein Disulfide Isomerase

Abstract

Protein disulfide isomerase (PDI) is a vital oxidoreductase. Extracellular PDI promotes thrombus formation but does not affect physiological blood hemostasis. Inhibition of extracellular PDI has been demonstrated as a promising strategy for antithrombotic treatment. Herein, we focused on the major substrate binding site, a unique pocket in the PDI b′ domain, and identified four natural products binding to PDI by combining virtual screening with tryptophan fluorescence-based assays against a customized natural product library. These hits all directly bound to the PDI-b′ domain and inhibited the reductase activity of PDI. Among them, galangin showed the most prominent potency (5.9 μM) against PDI and as a broad-spectrum inhibitor for vascular thiol isomerases. In vivo studies manifested that galangin delayed the time of blood vessel occlusion in an electricity-induced mouse thrombosis model. Molecular docking and dynamics simulation further revealed that the hydroxyl-substituted benzopyrone moiety of galangin deeply inserted into the interface between the PDI-b′ substrate-binding pocket and the a′ domain. Together, these findings provide a potential antithrombotic drug candidate and demonstrate that the PDI b′ domain is a critical domain for inhibitor development. Besides, we also report an innovative highthroughput screening method for the rapid discovery of PDI b′ targeted inhibitors.

Discovery of 5-Hydroxy-1,4-naphthoquinone (Juglone) Derivatives as Dual Effective Agents Targeting Platelet-Cancer Interplay through Protein Disulfide Isomerase Inhibition

“… 17 Second, the substrate binding pocket in the PDI b′ domain was revealed by point mutation, which is related to the activity of PDI substrate identification and binding. 59 , 60 Third, the pocket in the hinge region of PDI between the a′ and b′ domain was revealed by molecular dynamics, and the binding of molecules might restrict the conformation of PDI. 61 The results showed that monosubstituted JUG derivatives exhibited better binding affinity to the substrate binding pocket of PDI than the hinge binding pocket and the active site, indicating that the inhibition mechanism of monosubstituted JUG derivatives might start with the binding to the substrate binding site and induce conformational change to make the compounds attach to Cys397 covalently ( Figure 7 b).…”