Strong Protection by Bazedoxifene Against Chemically-Induced Ferroptotic Neuronal Death In Vitro and In Vivo

Ferroptosis is a form of regulated cell death characterized by excessive iron-dependent lipid peroxidation. Ferroptosis can be induced in cultured cells by exposure to certain chemicals (e.g., erastin and RSL3). Recently it was shown that protein disulfide isomerase (PDI) is a mediator of chemically-induced ferroptosis and also a target for ferroptosis protection. In this study, we find that bazedoxifene (BAZ), a selective estrogen receptor modulator with reported neuroprotective actions in humans, can inhibit PDI function and also strongly protect against chemically-induced ferroptosis in cultured neuronal cells. We find that BAZ can directly bind to PDI in vitro and in intact neuronal cells, and also can inhibit PDIs catalytic activity. Computational modeling analysis reveals that BAZ forms a hydrogen bond with PDI-His256. Inhibition of PDI by BAZ markedly reduces nNOS and iNOS dimerization and NO accumulation, which have recently been shown to play a crucial role in mediating chemically-induced ferroptosis. In addition, the direct antioxidant activity of BAZ may also partially contribute to its protective effect against chemically-induced ferroptosis. Behavioral analysis shows that mice treated with BAZ are strongly protected against kainic acid-induced memory deficits and hippocampal neuronal damage in vivo. In conclusion, the results of this study demonstrate that BAZ is an inhibitor of PDI and can strongly prevent chemically-induced ferroptosis in hippocampal neurons both in vitro and in vivo. These observations offer a novel, estrogen receptor-independent mechanism for the recently-reported neuroprotective actions of BAZ in humans. SIGNIFICANCE STATEMENTFerroptosis is an iron- and lipid peroxidation-dependent form of regulated cell death. Recent evidence has shown that protein disulfide isomerase (PDI) is an important mediator of chemically-induced ferroptosis and also a new target for ferroptosis protection. We find that bazedoxifene is an inhibitor of PDI, which can strongly protect against chemically-induced ferroptotic neuronal death in vitro and in vivo. Additionally, the molecular mechanism of PDI{square}bazedoxifene binding interaction is defined. This work provides evidence for an estrogen receptor-independent, PDI-mediated mechanism of neuroprotection by bazedoxifene.