6.1. Delivery of GS-nitroxides and TPP-nitroxides
Triphenylphosphonium (TPP) salts offer a general platform to shuttle antioxidants into the mitochondrial matrix and have been conjugated to ubiquinols, tocopherols, lipoic acid, nitroxides and other electron- and radical scavengers [94
]. A useful extension of this concept were cationic, arginine-rich oligopeptides such as the Szeto-Schiller tetrapeptides () [100
]. The peptide sequences Dmt-D-Arg-Phe-Lys-NH2
have been shown to be very effective in inhibiting mitochondrial swelling, oxidative cell death, and reperfusion injury [101
]. Notably, these small peptides were concentrated 1000-fold across the inner mitochondrial membrane and also readily crossed the cell membrane. This work supports the hypothesis that small peptides, or peptide mimetics, that contain appropriate antioxidants can be used to counteract the overproduction of ROS.
A charge-targeted peptide antioxidant.
Several “classical” lipophillic antioxidants - vitamin E, coenzyme Q, plastoquinones – have been utilized as cargoes for mitochondrial targeting and demonstrated useful protective propensities [98
]. TPP-conjugated homologues of vitamin E were among the first mitochondria-targeted lipid antioxidants that were shown to be effectively taken up and protect mitochondrial functions from oxidative damage far more effectively than vitamin E itself [98
]. Another lipid-soluble antioxidant, MitoQ represents a mixture of 10-(6′-ubiquinolyl)-decyltriphenylphosphonium bromide and 10-(6′-ubiquinonyl)-decyltriphenyl-phosphonium bromide and was found to exert biochemical evidence of protection against oxidative stress in vitro
] and to lessen dysfunction and augmented mitochondrial membrane potential in a rat model of lipopolysaccharide-peptidoglycan-induced organ failure [85
]. Currently, MitoQ is under development by Antipodean Pharmaceuticals, Inc in phase II clinical trials for Parkinson’s disease and liver damage associated with HCV infection (reviewed by [103
]). However, it should be noted that multifunctional modes of action of coenzyme Q and a risk of its involvement in the generation of oxygen radicals have been also associated with its potential to cause damage to mitochondria and cells [104
Similar quinone-based mitochondria-targeted protectors - SkQs - employs cationic plastoquinone derivatives. These compounds were demonstrated to be effective in preventing CL peroxidation and apoptosis in cell culture models [96
]. Moreover, SkQs prolonged lifespan in different species, being especially effective at early and middle stages of aging in different species. Most notably, in mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc [96
An alternative concept to targeting ROS scavengers to mitochondria was based on the affinity of certain antibiotics to microbial cell membranes [105
]. Due to the close relationship between bacterial membrane and the mitochondrial inner membrane structure, in particular their lipid composition, the antibacterial membrane disruptor, gramicidin S (GS), could be re-engineered as a mitochondrial targeting agent (). The uncharged XJB-5-131 contains a hemi-GS sequence, specifically the pentapeptide Leu-D-Phe-Pro-Val-Orn, attached to a stable nitroxide, 4-amino-Tempo (4-AT). The peptide bond between Leu and D-Phe was replaced with an alkene peptide isostere in order to increase the metabolic stability of the compound.
The neutral, 4-AT conjugated mitochondrial targeting agent XJB-5-131 was designed based on the molecular structure of the antibiotic gramicidin S.
The major advantage of sterically hindered free radicals such as 4-AT is their ability to accept and donate electrons depending on the redox potential of the environment [106
]. Upon loss of an electron, the nitroxide radical is converted to an oxoammonium cation, which is readily reduced by ascorbate (in biofluids) or via NAD(P)H-driven electron transport (in cells) to the hydroxylamine (). Nitroxides can be also directly reduced by ascorbate and electron transporting enzymes to hydroxylamines [107
]. The hydroxylamine is by itself a powerful reducing agent, and can regenerate the nitroxide radical or scavenge a reactive oxygen species by electron or hydrogen atom transfer, respectively. The 4-AT subunit acts as a scavenger of electrons and antioxidant, whereas the peptidic component of XJB-5-131 targets the mitochondrial lipid, possibly CL, and thus enriches the agent ca. 600-fold over the cytosol [65
]. XJB-5-131 was shown to prolong survival in a rodent model of hemorrhagic shock, mitigate oxidative stress, and preempt the mitochondrial pathway toward apoptosis [110
]. Furthermore, a related compound in this series, XJB-5-125, demonstrated substantative in vivo
radioprotective effects [113
]. Detailed structure-activity studies revealed that the presence of GS-moiety is a necessary but not sufficient pre-requisite for the effective prevention of ROS formation, oxidative stress and anti-apoptotic effects of the GS-nitroxide conjugates in mitochondria [109
]. Monte Carlo simulations showed that active
nitroxide conjugates of hemi-GS peptides with intact β-turn structures were positioned at the interface between polar and nonpolar regions of the lipid membrane. Thus not only the presence but also optimized localization in the membrane need to be taken into consideration to design hemi-GS-nitroxide conjugates that can successfully compete with O2
for electrons from ETC to prevent O2•−
The radioprotective and anti-inflammatory agent JP4-039 was designed based on the minimum pharmacophore of XJB-5-131.
In the course of optimizing the pharmacokinetic properties of XJB-5-131 and XJB-5-125, JP4-039 was identified as a small molecular weight analog with impressive anti-inflammatory and radioprotective properties () (Pierce et al, unpublished data). While JP4-039 does not have the complete hemi-GS derived mitochondrial targeting sequence and therefore does not reach the high mitochondrial enrichment factor of XJB-5-131, it is similarly uncharged and therefore readily passes through mammalian cell membranes.
Nitroxide radicals such as 4-AT can redox cycle between several states.
As an alternative to hemi-GS as a vehicle used for delivery into mitochondria, we and others have also employed TPP to deliver nitroxide cargoes into mitochondria using “electrophoresis” due to a gradient of membrane potential [114
]. We tested the potential of several TPP-nitroxide homologues to affect CL peroxidation and inhibit apoptosis in cells. Interestingly, of several nitroxide homologues studied, only TPEY-Tempo () was taken up by cells, concentrated in mitochondria and exhibited both anti-apoptotic and radioprotective properties (Jiang et al., 2009, submitted). However, the inability of other homologues to accumulate in cells indicates that it is an important a pre-requisite for the process is penetration of TPP-nitroxide’s effective integration into mitochondria and anti-apoptotic protection. Further studies delineating containing molecules into cells, a process possibly dependent on chemical structure of structural requirements and the role of linkers connecting TPP with nitroxides in their penetration into cells are essential. Interestingly, several nitroxide homologues studied, only TPEY-Tempo was taken up by cells, concentrated in mitochondria and exhibited both anti-apoptotic and radioprotective properties (Jiang et al., 2009, submitted).
Another important issue is appropriate topography of mitochondria-targeted cargoes and their proximity to the cyt c
/CL complex and its catalytic domains participating in CL oxidation and subsequent release of pro-apoptotic factors [65
]. The peroxidase activity of cyt c
is triggered by binding of CL itself, probably involving residues K72, K73, K86, K87 [115
] which is believed to partially unfold, hence activate the protein. Free fatty acid hydroperoxides (FFA-OOH) are 102
times more effective as sources of oxidizing equivalents for the peroxidase activity of cyt c
/CL than H2
or other small organic hydroperoxides (Belikova et al., 2009, submitted). This suggests that FFA-OOH rather than H2
may be endogenous substrates for CL peroxidation. Computer modeling including docking experiments implicated residues V20, L32, H33, L35, F36, R38, L98, K99, K100, A101, T102 and N103 in the FFA-OOH binding. In contrast, small organic hydroperoxides such as tert-
butyl hydroperoxide (t
-BuOOH) were predicted to associate with cyt c
near residues V11, C14, T19, V20, E21, L22, G23, and Y97 (). Thus, the enhanced peroxidase activity in the presence of FFA-OOH as compared to small organic hydroperoxides is likely related to differences in binding site. Furthermore, it is clear from these studies that the CL and FFA-OOH interaction sites on cyt c
are also distinct. This indicates that cyt c
contains multiple binding pockets, interaction with which results in different effects on cyt c
structure and activity. Furthermore, ATP may act as a regulatory effector in modulating structural transitions of cyt c
and its activity [117
]. Since ATP can displace the binding of oleic acid to cyt c
, it has been proposed that FFAs can reverse a non-native conformation (induced by CL) of the protein to the native (non-lipid bound) one. However, the peroxidase activity of cyt c
is retained in the presence of ATP [117
] suggesting that the cyt c
native structure was not recovered in the presence of excess ATP. It is possible that the ATP-bound state of the protein is different from its native conformation, albeit not as extensively altered as the lipid-bound form. Docking simulations indicate that ATP was preferentially bound at two sites, one involving residues Q16, T19, K22, and K27 and another composed of residues F36, K60, E61, E62, T89, E92, D93, I95, A96, K99, K100. Of the two predicted binding sites, the latter was the preferred, based on energetic considerations as well as the largest number of docked conformations at this site. This preferred ATP binding site and the FFA-OOH predicted binding site slightly overlap with each other, most importantly at the positively charged residues K99, K100. The predicted overlap in the binding site region thus provides a plausible explanation for the experimental finding why ATP interferes with oleic acid interaction with cyt c
]. These studies further suggest that ATP can also be used as a regulator of the binding of FFA-OOH to cyt c
and may be used to avoid or decrease peroxidase activity of cyt c
. These structural studies indicate new important strategies in optimizing mitochondria-targeted regulators of peroxidase function of cyt c
/CL complexes as potential inhibitors of CL oxidation with anti-apoptotic protective properties.
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 (more ...)
In summary, we utilized three major approaches for mitochondria targeting of antioxidant agents (enzymes, small molecule electron scavengers/antioxidants) to achieve significant protection against apoptosis induced by either chemical agents or physical factors (irradiation): i) creation of protein constructs with mitochondria leader sequences, ii) synthesis of conjugates in which the targeting moiety – TPP - facilitated “electrophoresis” of the cargo into mitochondria, and iii) use of fragments of natural compounds, antibiotics such as hemi-GS, with high affinity to one or more intramitochondrial constituents. In all cases, we were able to localize/enrich the vehicle/cargo conjugates in mitochondria and achieve significant protective effects against ROS production and CL oxidation that associated with anti-apoptotic action. As exemplified by radiation protection, all of these mitochondria-targeted molecules (enzymes, nitroxides) demonstrated superior effect in vitro and in vivo compared to non-targeted counterparts (enzymes without mitochondria leader sequences, non-conjugated 4-AT). Since mitochondrial targeted transgene products including MnSOD and catalase, as well as small molecule nitroxides, are effective radioprotectors, we suggest that enhanced methods for the smart delivery of these agents and/or combinations thereof to mitochondria should further improve their effectiveness in both clinical radiation protection in cancer patients as well as systemic radiation protection and radiation damage mitigation in individuals exposed to accidental or radiological terrorism mediated total body irradiation exposure.
6.4. Targeted avoidance of specific compartments such as mitochondria: delivery of NBD-CL
Liposomes are common vehicles for the delivery of various compounds into cells. In particular, liposomes have been successfully used for the incorporation of phospholipids in cell membranes. Liposomes with signaling phospholipids, such as phosphatidylserine (PS), are known to be selectively taken-up by professional phagocytes, such as macrophages, dendritic cells and microglial cells and are utilized for selective delivery of their contents into phagocytozing cells [127
]. Recently, PS has been successfully utilized as a vector for delivery of PS-coated nano-particles with other types of cargo into phagocytozing cells [128
]. After integration into cells, PS-containing liposomes are found in plasma membrane as well in endosomes from which they slowly exchange with other intracellular membranes. An interesting opportunity may be associated with organelle-specific phospholipids. Cardiolipin is not normally found in any other intracellular organelle or plasma membranes [129
]. This suggests that specific mechanisms restricting CL translocation to other membranes are implemented precluding its intracellular distribution. Surprisingly, incubation of cells with fluorescently labeled CL – 7-nitro-2-1,3-benzoxadiazol (NBD) group (NBD-CL) – exogenously added to cells revealed its avoidance of mitochondria (). More detailed analysis of intracellular trafficking of exogenous NBD-CL showed that in contrast to other NBD-labeled phospholipids, NBD-CL, when delivered to mouse embryonic cells by using liposomes, was internalized via the endocytotic pathway, but was not transferred between cell membranes. Even endosomal disruptor chloroquine did not facilitate redistribution of NBD-CL to other intracellular membranes. Moreover, CL and a combination of CL with cationic phospholipids (e.g., dioleoyl-ethylphosphocholine) drastically enhanced up-take of the liposomes by cells. It is tempting to speculate that CL is recognized by specific receptors, which enhance up-take of CL-containing vehicles, and that CL trafficking in cells is tightly controlled.
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 (more ...)
These unique properties of CL can be used for the delivery of liposomes into cells and for the targeted avoidance of the unwanted compartmentalization of the liposomal content. This approach may be particularly useful for the delivery of cationic drugs such antracycline antibiotics. One of the antracyclines, doxorubicin, has found outstanding clinical utility as an anticancer drug. The antitumor activity of doxorubicin is mainly attributed to a direct binding to DNA. In addition, this compound causes cytotoxic effects via free radical generation, metal chelation, bioalkylation and disordering of the membrane (reviewed in [130
]). The major side effect of doxorubicin that significantly limits its application is cardiotoxicity, which is mainly due to the targeting of cardiomyocyte mitochondria. In mitochondria, doxorubicin binds with high affinity to cardiolipin, inhibits Complex I, deviates electron flow from the respiratory chain to molecuar oxygen yielding ROS (O2•−
) and stimulating peroxidation of lipids [131
]. Therefore, targeting of doxorubicin to other compartments than mitochondria is of importance. Cationic anthracyclines form high affinity complexes with anionic phospholipids. For example, the affinity constant for the interaction between doxorubicin and cardiolipin is 1.6×106
]. Application of CL-containing liposomes for doxorubicin delivery can limit its free diffusion through the cell membranes and avoid integration of the drug into mitochondria.
The further fate of the drug in the liposomes is likely to be governed by changes in pH and by CL metabolism. The pH in late endosomes and lysosomes is acidic. At acidic conditions, the headgroup of CL bears only one negative charge (pK2
>8.0), while the amino group of doxorubicin is deprotonated (pK 8.2) and, hence, less than 1% of drug molecules is in the cationic state when the pH drops form below 6. This should lead to the loss of electrostatic interactions between doxorubicin and CL that are important for the formation of CL/doxorubicin complex [133
]. The fast decomposition of CL in liposomes (that we recently documented by analyzing NBD-CL and its metabolites in cells) is the second factor, which will effectively liberate doxorubicin from lysosomes to cytosol. Overall, employment of CL containing liposomes with inclusions of antitumor tetracycline antibiotics, such as doxorubicine, may be viewed as a useful idea in the development of approaches to targeted avoidance in selective drug delivery to specific intracellular compartments.