Flavonoids as Protein Disulfide Isomerase Inhibitors: Key Molecular and Structural Features for the Interaction

Abstract

Quercetin-3-rutinoside (rutin) is a bioflavonoid that is common in foods. The finding that quercetin-3-rutinoside inhibits protein disulfide isomerase (PDI) and potently blocks thrombosis in vivo has enabled the evaluation of PDI inhibition in multiple animal models of thrombus formation and has prompted clinical studies of PDI inhibition in thrombosis. Nonetheless, how quercetin-3-rutinoside blocks PDI activity remains an unanswered question. Combining NMR spectroscopy, site-directed mutagenesis, and biological assays, we identified H256 as the key residue for PDI interacting with quercetin-3-rutinoside. Quercetin-3-rutinoside inhibited the activity of PDI (WT) but not PDI (H256A). Molecular dynamic simulations indicated that the flavonoid skeleton, but not the rutinoside conjugate, is embedded in the major binding pocket on the b′ domain. Among several quercetin-3-rutinoside analogues tested, only compounds with a phenoxyl group at position 7 showed direct binding to PDI, further supporting our molecular model. Studies using purified coagulation factors showed that quercetin-3-rutinoside inhibited the augmenting effects of PDI (WT), but not PDI (H256A), on tissue factor (TF) activity. Quercetin-3-rutinoside also inhibited chemotherapy-induced TF activity enhancement on endothelial cells. Together, our studies show that residue H256 in PDI and the phenoxyl group at position 7 in quercetin-3-rutinoside are essential for inhibition of PDI by quercetin-3-rutinoside. These results provide new insight into the molecular mechanism by which flavonoids block PDI activity.

Enhanced inhibition of protein disulfide isomerase and anti-thrombotic activity of a rutin derivative: rutin:Zn complex

“…Moreover, we also developed a probe-based assay using a molecular substrate smaller than insulin. 8 Our results showed that adding either rutin:Zn complex or rutin inhibited PDI cleavage for the GSSG probe at comparable levels (Fig. S6b † ).…”

“…The addition of different concentrations of PDI increased the uorescence intensity of rutin and caused a red shi of the maximal emission wavelength to about 550 nm, demonstrating the direct molecular interaction of rutin with PDI. 8 Our results showed that the presence of PDI in either rutin or rutin:Zn complex signicantly increased the rutin's uorescence in a dose-dependent manner (Fig. 2a), demonstrating the direct molecular interaction of PDI to rutin or rutin:Zn complex.…”

“…Recently, we delineated the direct molecular interaction of PDI with rutin and employed NMR spectroscopy, mutagenesis, function assay, and molecular dynamics to show that PDI residue H256 is the key mediating the interaction between PDI and rutin, and the analogs without a phenoxy group at 7-position showed no binding to PDI, which demonstrated an essential role of the 7-position phenoxy group for binding to PDI. 8 Moreover, the avonoid analogs without a phenoxy group at the 7-position showed no binding to PDI, which demonstrated an essential role of the 7-position phenoxy group for binding to PDI. In this study, we did not choose a sodium rutin formulation (NaR), because the sodium of NaR chelates at the 7position of rutin, 16 which may intervene with rutin interaction with PDI.…”

“… 7 Recently, we identified the molecular binding site of rutin on PDI using both structural biology and mutagenesis techniques, and demonstrated that rutin occupies the major substrate binding site located at the b′ domain of PDI with residue H256 as the key residue. 8 Rutin was shown to significantly reduce thrombin generation in humans without inducing bleeding side effects. 6 Despite their wide biological functions, flavonoids have low bioavailability and low druggability, partly due to limited gastrointestinal absorption, rapid degradation and excretion, leading to low the requirement of a high dose to induce an anti-thrombotic effect in clinical trials.…”

Targeting of non-apoptotic cancer cell death mechanisms by quercetin: Implications in cancer therapy

“…Natural products are an essential source of anticancer lead molecules due to their multi-targeting efficacy and low toxicity, especially flavonoids ( Yang H. et al, 2022a ; Liao et al, 2022 ). As a flavonoid with various biological activities, quercetin is abundantly present in plants, fruits, and vegetables, mainly in the form of glycosides, such as onions, apples, blueberries, cauliflower, etc.…”