Received for publication September 28, 2006.
Revised December 15, 2006.
Accepted for publication December 18, 2006.

Structural requirements for optimized delivery, inhibition of oxidative stress and anti-apoptotic 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 Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) and its analogs combine several important features, including recyclability, electron acceptance from respiratory complexes, SOD mimicry, and radical scavenging. While successful in antioxidant protection, their effective concentrations are too high for successful in vivo applications. Recently (JACS 2005, v.127, pp 12460), we reported that 4-amino TEMPO (4-AT), covalently conjugated to a 5-residue segment of gramicidin S (GS), was integrated into mitochondria and blocked actinomycin D (ActD) induced superoxide generation and apoptosis. Using a model of ActD induced 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 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 (CD, 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 ^DPhe-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 nitroxide-peptide and -peptide isostere conjugates are instrumental for development of new mechanism-based therapeutically effective agents.

Key words: 4-amino TEMPO, actinomycin D, apoptosis, cardiolipin, gramicidin S, superoxide

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