Oxidative stress has also been associated with diverse diseases, including cancer, renal disease, and neurodegenerative disorders such as Alzheimer's and Parkinson's disease. All vascular cells produce ROS, which contribute to many of the abnormalities associated with vascular diseases, including atherosclerosis and hypertension. Although plants and animals maintain the level of antioxidants, such as glutathione (GSH), vitamins C, A and E as well as enzymes such as catalase, superoxide dismutase and various peroxidases, insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress.
Figure 1. Diseases/disorders Caused by Reactive Oxygen Species and Oxidative Stress.
Figure 2. Synthetic Compounds with GPx-like Activity
Structure-activity relationship studies in our laboratory led to the development of synthetic compounds with 10-20-fold enhancements in the catalytic activities (Figure 2). The mechanistic investigations of functional mimics were useful not only for the understanding the complex chemistry at the active site of GPx but also for development of novel redox modulators and anti-inflammatory agents (Bhabak, K. P.; Mugesh, G. Acc. Chem. Res. 2010, 43, 1408). In a preliminary study, compound 5 reduces atherosclerosis in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse.
Very recently, we described the synthesis, GPx and peroxiredoxin (Prx)-like activity of several novel isoselenazoles (Bhowmick, D.; Srivastava, S.; D’Silva, P.; Mugesh, G. Angew. Chem. Int. Ed. 2015, Click here for the article). The high GPx activity of 11a-d as compared to that of ebselen led us to investigate the
Funding for Research
It should be noted that some antioxidant supplements may promote disease and increase mortality in humans. The reason for this unexpected behaviour is that antioxidants with strong reducing ability can act as pro-oxidants and actually increase oxidative stress. Therefore, it is important to develop synthetic antioxidants without pro-oxidant acvitivy. In this regard, our group is working on the development of enzyme mimetics redox modulators that can combat oxidative stress without downregulating the cellular antioxidant systems.
(a) Development of Redox Modulators based on Glutathione Peroxidase Activity
Glutathione peroxidase (GPx), a selenoenzyme expressed in every mammalian cell, plays a key role in protecting the organism from oxidative damage by catalyzing the reduction of harmful hydroperoxides with thiol cofactors. GPx modulates the effects of cellular oxidants on cell signaling and cell growth. Lower plasma GPx level was observed in patients with type 2 diabetes with macroalbuminuria and was correlated to the stage of diabetic nephropathy. In pathological situation, the cellular antioxidant machinery is insufficient to balance the elevated ROS resulting from the aberrant metabolic pathways; thus leading to disorders such as neurodegeneration, cancer, diabetes, atherosclerosis, arthritis, kidney failure and aging. The GPx-mimetic-based redox modulators offer a clear advantage over the conventional antioxidants as such compounds can scavenge ROS without showing any pro-oxidant activity. Mugesh and co-workers showed that several small-molecule selenium compounds having suitable substituents exhibit GPx-like antioxidant activity (Bhabak, K. P.; Mugesh, G. Acc. Chem. Res. 2010, 43, 1408-1419; Click here to read the article).
Our group worked extensively on the molecular mechanism for the antioxidant and anti-inflammatory function of ebselen (1, 2-phenyl-1,2-benzoselenazol-3-one), a compound which is undergoing clinical trials for a number of disease states, including stroke and hearing loss and is an excellent scavenger of ROS such as peroxynitrite (PN). The toxicity of ebselen is very low at pharmacologically active concentrations, and therefore, this compound is part of the National Institutes of Health (NIH) Clinical Collection, a chemical library of drugs considered clinically safe. Ebselen is bioavailable and can cross blood-brain barrier. We showed that the inhibition of a number of enzymes by ebselen and its analogues is due to the formation of a stable selenenyl sulfide bond (-Se-S-), which is not generally reversed by cellular thiols (Sarma, B. K.; Mugesh, G. J. Am. Chem. Soc. 2005, 127, 11477; Chem. Eur. J. 2008, 14, 10603).
Reactive Oxygen Species (ROS), Oxidative Stress and Redox Modulators
Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the biological system's ability to detoxify these reactive intermediates. It is well known that oxidative stress is responsible for several disease states (Figure 1). Both Type I and II diabetics display increased levels of ROS such as free radicals and the onset of diabetes is closely associated with oxidative stress.
antioxidant potential in human cell lines. The human kidney derived HEK293T cells pretreated with hydrogen peroxide were incubated with the selenium compounds. Treatment of cells with 40 µM of compounds 11a-d led to a decrease in the fluorescence intensity (Figure 3B), indicating the ROS scavenging effect of the selenium compounds. To understand the effect on elevated levels of hydrogen peroxide inside the cells, the antioxidant enzyme catalase, was inhibited using 3-amino,1,2,4 triazole (3-AT). At this elevated peroxide level, the ROS scavenging activity of 11a-d was much higher than that of ebselen (1) (Figure 3C). To test the ability of ebselen and 11a-d to combat oxidative stress in other mammalian cell
types, experiments were carried out with HeLa cell lines. Enhancement of ROS level either upon treatment of cells with peroxide or by inhibiting catalase activity was studied by fluorescence microscopic analysis. As shown in Figure 3D, the ROS scavenging activity of 11a is significantly higher than that of ebselen under oxidative stress conditions. The HEK293T cells were treated with peroxide followed by test compounds and the extent of DNA damage was probed with anti- γH2AX antibody. While ebselen provided about 70% protection, compound 11a exhibited remarkable activity with more than 95% protection (Figure 3E). For the first time, we showed that the cytotoxicity of many of the selenium compounds is mainly due to their ability to inhibit glutathione reductase (GR) in the cells, an enzyme that recycles GSH from GSSG by using NADPH (Figure 3F). The toxicity of 11a was found to be remarkably lower than that of ebselen, suggesting that compound 11a is a potential candidate for further in vivo studies, which is in progress in our laboratory.