Structural Requirements for Optimized Delivery, Inhibition of Oxidative Stress, and Antiapoptotic Activity of Targeted Nitroxides

Abstract

Suppression of mitochondrial production of reactive oxygen species is a promising strategy against intrinsic apoptosis typical of degenerative diseases. Stable nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL) and its analogs combine several important features, including recycleability, electron acceptance from respiratory complexes, superoxide dismutase mimicry, and radical scavenging. Although successful in antioxidant protection, their effective concentrations are too high for successful in vivo applications. Recently (J Am Chem Soc 127:12460, 2005), we reported that 4-amino 2,2,6,6-tetramethyl-1-piperidinyloxy, covalently conjugated to a five-residue segment of gramicidin S (GS), was integrated into mitochondria and blocked actinomycin D (ActD)-induced superoxide generation and apoptosis. Using a model of ActDinduced apoptosis in mouse embryonic cells, we screened a library of nitroxides to explore structure-activity relationships between their antioxidant/antiapoptotic properties and chemical composition and three-dimensional (3D) structure. High hydrophobicity and effective mitochondrial integration are necessary but not sufficient for high antiapoptotic/antioxidant activity of a nitroxide conjugate. By designing conformationally preorganized peptidyl nitroxide conjugates and characterizing their 3D structure experimentally (circular dichroism and NMR) and theoretically (molecular dynamics), we established that the presence of the ␤-turn/␤-sheet secondary structure is essential for the desired activity. Monte Carlo simulations in model lipid membranes confirmed that the conservation of the D-Phe-Pro reverse turn in hemi-GS analogs ensures the specific positioning of the nitroxide moiety at the mitochondrial membrane interface and maximizes their protective effects. These new insights into the structure-activity relationships of nitroxidepeptide and -peptide isostere conjugates are instrumental for development of new mechanism-based therapeutically effective agents.Poorly controlled and excessive generation of reactive oxygen species (ROS), coupled with their ability to cause oxidative damage to phospholipids, proteins, and DNA, has been associated with the pathogenesis of a number of major human cardiovascular (Ambrosio and Tritto, 1999) and neurodegenerative diseases (Andersen, 2004) as well as cancer (Szatrowski and Nathan, 1991). Not surprisingly, significant efforts have been directed toward the use of radical scavengers and antioxidants in preventive and therapeutic strategies, albeit with limited success. The search for new protective remedies has been focused on molecules combining antioxidant utilities with recycling capacities (Mitchell et al.,

Interplay between bax, reactive oxygen species production, and cardiolipin oxidation during apoptosis

“…Recently, we reported that mitochondria targeted electron scavengers -hemigramicidin S conjugated nitroxides -prevented both superoxide accumulation and CL peroxidation and apoptosis initiated by ActD or irradiation without affecting Bax translocation [27,28]. Combined with the results of this work showing that Bax/Bak translocation precedes superoxide production and CL peroxidation and that genetic ablation of Bax/Bak inhibited ActD and rotenone induced CL oxidation and apoptosis, we speculate that CL peroxidation not only correlates but is likely causatively and time-dependently related to mitochondrial relocation of Bax/Bak.…”

Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential

“…The activity is presumably due to the nitroxide being reduced by ubiquinol within respiring mitochondria (90,318,319). Jiang et al (163) proposed peptidyl nitroxide conjugates for targeting mitochondria. With anticipated higher efficacy, smaller amounts of nitroxide would be required.…”

A Mitochondria-Targeted Nitroxide/Hemigramicidin S Conjugate Protects Mouse Embryonic Cells Against Gamma Irradiation

“…Although found to be effective, the required high millimolar concentrations, partially because of its poor partitioning into cells and mitochondria, set a limit for broader applications of TEM-POL (30). Recent developments in targeted delivery of antioxidants, including nitroxides, to mitochondria offer new opportunities in radioprotection of cells and tissues (31,32). We showed that conjugation of 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-AT) with a segment of gramicidin S (GS) dramatically increased intracellular and mitochondrial accumulation of the nitroxide conjugates, resulting in decreased levels of mitochondrial superoxide, inhibition of CL peroxidation, and protection of mouse embryonic cells against actinomycin D-induced apoptosis (31,32).…”

“…Recent developments in targeted delivery of antioxidants, including nitroxides, to mitochondria offer new opportunities in radioprotection of cells and tissues (31,32). We showed that conjugation of 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-AT) with a segment of gramicidin S (GS) dramatically increased intracellular and mitochondrial accumulation of the nitroxide conjugates, resulting in decreased levels of mitochondrial superoxide, inhibition of CL peroxidation, and protection of mouse embryonic cells against actinomycin D-induced apoptosis (31,32). Therefore, we hypothesized that mitochondria-targeted hemigramicidin S conjugated 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl hemi-GS-TEMPO may be effective in protecting cells against IR-induced cell death, thus offering a convenient therapeutic window for radiomitigation.…”