Mitochondrial targeting of radioprotectants using peptidyl conjugates

Abstract

Ionizing radiation activates a mitochondrial nitric oxide synthase, leading to inhibition of the respiratory chain, generation of excess superoxide, peroxynitrite production and nitrosative damage. We have measured the radioprotective effects of a nitric oxide synthase antagonist (AMT) versus a free radical scavenger (4-amino-TEMPO) using electrochemical detection of nitric oxide and peroxynitrite. To enhance their efficacy, we have conjugated these compounds to peptides and peptide isosteres-derived from the antibiotic gramicidin S-that target the mitochondria. The targeting ability of these peptidyl conjugates was measured using quantitative mass spectrometry.

Effect of total body irradiation on late lung effects: Hidden dangers

“…Although earlier results from our own group have suggested that it is the “perpetual cascade” of cytokines and chemokines that is the critical element in the development of radiation pneumonitis and, ultimately, late radiation fibrosis (Rubin et al 1995, Johnston et al 1996), other investigators have suggested that chronic dysregulation may be due to other mechanisms, such as the maintenance of damage within the mitochondria, leading to an activated inflammatory site as a result of chronic free radical production (Epperly et al 2003, Kanai et al 2007, Zhao & Robbins 2009). Therefore, observation of persistent periods of increased cytokine expression may not be a requirement for confirmation of the presence of ongoing pulmonary damage.…”

Targeted Delivery of Radioprotective Agents to Mitochondria

“…Critical amide bonds in these compounds were replaced with (E)-alkene peptide isosteres which improved their bioavailability, possibly due to increased resistance against protease action (42—44), and the analogs could be structurally altered to modulate cell membrane passage relative to mitochondrial targeting. ESR spectroscopy and mass spectroscopy confirmed that the gramicidin S–based compounds successfully delivered and concentrated both TEMPO and AMT to mitochondria, whereas the presence of free drugs was not observed (18, 45). Harmful ONO 2 − production in irradiated cells was reduced or eliminated upon treatment with conjugated TEMPO and AMT, respectively (18).…”

“…ESR spectroscopy and mass spectroscopy confirmed that the gramicidin S–based compounds successfully delivered and concentrated both TEMPO and AMT to mitochondria, whereas the presence of free drugs was not observed (18, 45). Harmful ONO 2 − production in irradiated cells was reduced or eliminated upon treatment with conjugated TEMPO and AMT, respectively (18). The ability of rats to survive lethal hemorrhagic shock was significantly increased by intravenous injection of the TEMPO-conjugated compound (46).…”

“…Although nitroxides are cell membrane–permeable, they may not get into mitochondria. Using mass spectroscopy, we have shown that the nitroxide 4-amino-TEMPO is unable to penetrate mitochondrial membranes and requires targeting for effective antioxidant activity (18). Despite these findings, some nitroxides, including TEMPO, show radioprotective properties in vitro and in vivo in mice (when administered intraperitoneally [see (19)]).…”

“…These gramicidin S analogs were used to target mitochondria with TEMPO (the SOD mimic discussed above) and the potent NOS inhibitor AMT (2-amino-6-methylthiazine) (18). Critical amide bonds in these compounds were replaced with (E)-alkene peptide isosteres which improved their bioavailability, possibly due to increased resistance against protease action (42—44), and the analogs could be structurally altered to modulate cell membrane passage relative to mitochondrial targeting.…”