Mitochondrial targeting of electron scavenging antioxidants: Regulation of selective oxidation vs random chain reactions

Abstract

Effective regulation of highly compartmentalized production of reactive oxygen species and peroxidation reactions in mitochondria requires targeting of small molecule antioxidants and antioxidant enzymes into the organelles. This review describes recently developed approaches to mitochondrial targeting of small biologically active molecules based on: (i) preferential accumulation in mitochondria because of their hydrophobicity and positive charge (hydrophobic cations), (ii) binding with high affinity to an intra-mitochondrial constituent, and (iii) metabolic conversions by specific mitochondrial enzymes to reveal an active entity. In addition, targeted delivery of antioxidant enzymes via expression of leader sequences directing the proteins into mitochondria is considered. Examples of successful antioxidant and anti-apoptotic protection based on the ability of targeted cargoes to inhibit cytochrome c-catalyzed peroxidation of a mitochondria-specific phospholipid cardiolipin, in vitro and in vivo are presented. Particular emphasis is placed on the employment of triphenylphosphonium- and hemi-gramicidin S-moieties as two effective vehicles for mitochondrial delivery of antioxidants.

Figure 1

Increased survival of 32D cl 3 cells with elevated mitochondrial SOD activity. Cells from 32D cl 3, 32D-Cu/ZnSOD, 32D-MnSOD, 32D-MnSOD-ML, and 32D-ML-Cu/ZnSOD were irradiated with doses ranging from 0 to 8 Gy. The cells were plated in 4% methylcellulose and colonies of > 50 cells were counted 7 days later. The data were analyzed using linear-quadratic and single-hit, multitarget models. 32D-MnSOD and 32D-ML-Cu/ZnSOD cells having increased mitochondrial SOD activity were more resistant to radiation [D₀ = 2.10 ± 0.10 (P = 0.001) and 1.97 ± 0.17 (P = 0.0127) Gy, respectively] than 32D cl 3, 32D-Cu/ZnSOD, or 32D-MnSOD-ML cells (D₀ = 1.15 ± 0.11, 0.89 ± 0.01, and 1.08 ± 0.02 Gy, respectively) (P < 0.195 and P < 0.673). Results are presented as the means ± SEM with an n of 3. P values were determined using a Student’s t test. *Represents a statistically significant difference from 32D cl 3 cells. (Reproduced from M.W. Epperly et al., Radiat Res 160 (2003) 568–78, with permission of the publisher.)

Figure 2

TPP and rhodamine localize in mitochondria due to the negatively charged membrane potential across the inner mitochondrial membrane.

Figure 3

A charge-targeted peptide antioxidant.

Figure 4

The neutral, 4-AT conjugated mitochondrial targeting agent XJB-5-131 was designed based on the molecular structure of the antibiotic gramicidin S.

Figure 5

The radioprotective and anti-inflammatory agent JP4-039 was designed based on the minimum pharmacophore of XJB-5-131.

Figure 6

Nitroxide radicals such as 4-AT can redox cycle between several states.

Figure 7

TPP- comjugates of aminoxyls.

Figure 8

Cartoon representation of cytochrome c (cyt c) with the predicted binding sites for cardiolipin (CL), free fatty acid hydroperoxides (FFA-OOH), adenosine triphosphate (ATP), tert-butyl hydroperoxide (t-BuOOH), and Phosphate (PO₄⁻). The helices of cyt c are colored in red and loops in green. The predicted binding site residues that are within 5Å distance in each case are represented by arrows and rendered in spheres. The site for CL is colored in green, for FFA-OOH and ATP in blue, and for the t-BuOOH and PO₄⁻ binding sites in magenta.

Figure 9

Microphotographs of intracellular distribution of NBD-labeled phospholipids in mouse embryonic cells obtained by confocal fluorescence microscopy. C₁₂-NBD-CL (A) and C₁₂-NBD-phosphotidylcoline (B) were delivered in liposomes. Mitochondria were stained with Mitotracker Red CMX Ros (red color); NBD-labeled phospholipids are shown in green color. A – note a punctuate pattern of intracellular distribution of C₁₂-NBD-CL characteristic for endosomes; NBD-CL and mitochondrial staining are not colocalized. B – diffuse staining with C₁₂-NBD-phosphotidylcoline.

Scheme 1. illustrates the major mitochondria-targeted compounds with anti-oxidant and anti-apoptotic effects described in the review

The triphenylphosphonium (TPP) moiety is targeted (“electrophoresed”) into mitochondria due to their lipophilic nature and positive charge. Triphenylphosphonium oleic acid ester (TPP-OA) accumulates in mitochondria and is acted upon by esterase releasing oleic acid at the site of cardiolipin (CL) synthesis and remodeling. Through activation by oleic acid-CoA synthase, followed by integration into CL by monolyso-cardiolipin transferase, CL unsaturation decreases. This limits both oxidation and further downstream apoptotic events. 2-hydroxyamino-vinyl-triphenylphosphonium (TPP-aminoxyl) interacts with cytochrome c/CL complexes liberating nitric oxide, which interacts with reactive peroxidase intermediates thus preventing CL peroxidation and apoptosis. Gramicidin S-TEMPO conjugates (GS-TEMPO·), preferentially accumulates in mitochondria due to high-affinity GS binding with the inner mitochondrial membranes; the nitroxide cargo of TEMPO· undergoes cyclic reduction and oxidation by acting as a scavenger of electrons thus preventing superoxide generation.