Development of small molecule mitochondria-targeted antioxidants for radioprotection
Authors: Michael W. Epperly, Valerian E. Kagan, Anthony J. Kanai, Peter Wipf, Andrew A. Amoscato, Joel S. Greenberger
Journal: Cancer Research (2006)

4410  We have previously demonstrated that overexpression of MnSOD protects cells from irradiation damage by reduction of superoxide and inhibition of subsequent production of peroxynitrite resulting in stabilization of the mitochondria. To develop new compounds for radioprotection we have attached a gramicidin (GS)-peptide isotere linker that facilitates transportation of proteins into the mitochondria to the antioxidant 4-amino-Tempo (4-AT) and the nitric oxide synthase (NOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) to concentrate the inhibitions in the mitochondria. Initial experiments were performed with two GS-Tempo derivatives (XJB-5-125 or XJB-5-131) or two AMT derivatives (XJB-5-127 or XJB-5-133). To demonstrate concentration of the compounds into the mitochondria, 32D cl 3 cells were incubated in the presence of 100 μM of XJB-5-125, XJB-5-127, XJB-5-131, XJB-5-133, tempol or AMT for 1 hour at which time the mitochondria were isolated and analyzed by mass spectrometry. There was no detectable uptake of 4-AT or AMT into the mitochondria but there was significant uptake of XJB-5-125, 127, 131 or 133 in the mitochondria. Localization of 4-AT to the mitochondria using the mitochondrial leader sequence resulted in increased radiation resistance. Irradiation survival curves were performed on 32D cl 3 cells incubated in 100 μM XJB-5-125, 10 μM XJB-5-131 or 100 μM 4-AT for 1 hour and irradiated to doses ranging from 0 to 800 cGy. The cells were plated in methylcellulose and colonies of greater than 50 cells were counted 7 days later. Cells incubated in XJB-5-125 or XJB-5-131 had increased radiation resistance as shown by an increased shoulder on the survival curve (n=18.24) compared to 32D cl 3 cells only or cells incubated in 4-AT (n=7.47 or 5.82, respectively). The mitochondrial localized AMT (XJB-5-127 or XJB-5-133) also exhibited increased radiation resistance. Irradiation survival curves were performed by incubating 32D cl 3 cells for 1 hour in 10 or 100 μM AMT, XJB-5-127 or XJB-5-133 and irradiating the cells at doses ranging from 0 to 800 cGy. Radiation survival curves at 10 μM revealed no significant change in the D0 between AMT and the mitochondrial linked XJB-5-125 or XJB-5-133. At 100 μM there was a significant increase in the D0 of 1.925 Gy for AMT compared to 1.281 for control 32D cl 3. The two AMT conjugates with the mitochondria linker had a higher D0 of 2.771 or 3.317 Gy compared to the AMT of 1.925 and the 32D cl 3 at 1.281. Using probes specific for peroxynitrite, 32D cl 3 cells incubated in 10 μM or 100 μM of XJB-5-127 or XJB-5-133 produced no peroxynitrite following irradiation while control cells or cells incubated in AMT produced peroxynitrite. The mitochondrial localization was necessary in preventing production of peroxynitrite. These results demonstrate the importance of localization of antioxidants or NOS inhibitors to the mitochondria in protecting cells from irradiation.