Isoform Specific Synthetic Glutathione Peroxidase Mimetics in Redox Regulation of Cell Death and Diseases

Abstract

Reactive oxygen species (ROS)-induced oxidative stress has been implicated in various pathologies and cell death processes. Cells have antioxidant systems to neutralize the harmful effects of excess ROS. Glutathione peroxidases (GPXs) are the key antioxidant enzymes that catalyze the reduction of H2O2 or organic hydroperoxides to water or the corresponding alcohols using glutathione (GSH). In humans, eight GPX isoforms are found, of which GPX1, GPX2, GPX3, GPX4, and GPX6 are selenoproteins containing selenocysteine (Sec) in their active site. Each isoform has unique characteristics and specific tissue localization that determines the precise biological role. Insufficiency of the GPX antioxidant system has been implicated in various diseases, including neurodegenerative disorders, stroke, pulmonary embolism, ischemia/reperfusion injury (IRI), brain damage, cancer, organ failure, and cell death processes such as apoptosis and ferroptosis. My work entails the development of isoform-specific small molecule GPX mimetics and the validation of GPX mimetics in cell death and disease models where insufficiency of GPX activity is the underlying cause of cell death and disease progression. Blood platelets, primary mediators of homeostasis and thrombosis, play a crucial role in angiogenesis, repair of the endothelium, and wound healing. Platelets can be targets of ROS produced within or by the vasculature, which dramatically affects platelet physiology. A decrease in the circulating platelet population leads to a condition referred to as thrombocytopenia, which can be life-threatening due to trouble stopping bleeding. Reduced intracellular antioxidant capacity in platelets contributes to thrombocytopenia via ROS mediated apoptotic signalling pathways. Our study demonstrates that compd.1, a selenium-based small molecule having GPX1/3-like activity protects platelets from apoptosis by regulating ROS-induced ASK1 (Apoptosis Signal-Regulating kinase 1) activation. GPX deficiency has also been associated with an increased risk of platelet-dependent thrombosis and vascular dysfunction which results in vessel occlusion leading to stroke, pulmonary embolism, and heart attack leading cause of death worldwide. We found that Compd.1, a GPX1/3 mimetic, inhibits platelet activation in response to various agonists such as collagen, thrombin, and ADP. We further show that Compd.1 effectively suppresses platelet activation-mediated platelet neutrophil aggregates and neutrophil extracellular trap formation, which are associated with inflammatory and thrombotic disorders such as sepsis, vascular inflammation, and COVID-19. Importantly, we show that Compd.1 potently inhibits platelet-dependent thrombosis in mouse models without causing bleeding complications. Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death caused by aberrant accumulation of phospholipid hydroperoxides in cellular membranes. Ferroptosis has been implicated in several pathological conditions, including neurodegenerative disorders, ischemia/reperfusion injury (IRI), brain damage, cancer, and organ failure. The selenoenzyme glutathione peroxidase 4 (GPX4) is the master regulator of ferroptosis suppresses ferroptosis by reducing phospholipid hydroperoxides to their corresponding alcohol. We have synthesized a novel organoselenium compound (Compd.5) that effectively mimics the activity of GPX4 and suppresses ferroptosis in primary neurons. Compd.5 actively reduces lipid hydroperoxides and suppresses ferroptosis induced by classic ferroptosis inducers such as RSL3, FIN56, FINO2 Erastin, and HCA in primary cortical neurons. Overall, our studies led to the development of isoform-specific synthetic GPX mimetics that can be harnessed to combat platelet activation-mediated thrombotic diseases, ROS-mediated thrombocytopenia in platelets, and neurological disorders associated with ferroptosis.