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Hemigramicidin–TEMPO conjugates: Novel mitochondria-targeted anti-oxidants
biochemical pharmacology 74 (2007) 801–809
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/biochempharm
Hemigramicidin–TEMPO conjugates: Novel mitochondriatargeted anti-oxidants Mitchell P. Fink a,*, Carlos A. Macias a, Jingbo Xiao b, Yulia Y. Tyurina c, Jianfei Jiang c, Natalia Belikova c, Russell L. Delude a, Joel S. Greenberger d, Valerian E. Kagan c, Peter Wipf b a
Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, United States c Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, United States d Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, PA, United States b
article info
abstract
Article history:
Oxidative damage to various cellular constituents (such as, proteins and lipids) mediated by
Received 7 March 2007
reactive oxygen species (ROS) is thought to be an important mechanism underlying the
Accepted 23 May 2007
pathogenesis of a variety of acute and chronic diseases. Mitochondria are the main source of ROS within most cells. Accordingly, there is increasing interest in the development of pharmacological ROS scavengers, which are specifically targeted to and concentrated within
Keywords:
mitochondria. Numerous compounds with these general characteristics have been synthe-
Superoxide
sized and evaluated in a variety of in vitro and in vivo models of redox stress. Among the more
Oxidant stress
promising of these mitochondria-targeted anti-oxidants are those that employ various
TEMPOL
peptides (or peptide-like moieties) derived from the antibiotic, gramicidin S, as the targeting
Hemorrhagic shock
construct and employ the stable free radical, 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl
LPS
(4-NH2–TEMPO), as the ROS scavenging ‘‘payload.’’ One of these hemigramicidin–TEMPO
Caspase
conjugates, XJB-5-131, has been shown to ameliorate intestinal mucosal injury and prolong survival in rats subjected to lethal hemorrhage. # 2007 Elsevier Inc. All rights reserved.
1. Mitochondria are an important source of reactive oxygen species Reactive oxygen species (ROS) are intermediates formed during one-electron reduction of molecular oxygen (dioxygen, O2). Some important ROS in biological systems include superoxide radical anion (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH). ROS are implicated as being important in the pathogenesis in a wide range of diseases or pathological processes, including various forms of cancer, type 2 diabetes mellitus, atherosclerosis, chronic inflammatory conditions,
ischemia/reperfusion injury, sepsis and some neurodegenerative diseases [1]. Additionally, ROS are important under both physiological and pathophysiological conditions as key mediators in a number of different intracellular signaling cascades [1]. The general processes whereby ROS cause pathological changes in cells and tissues are collectively referred to as oxidative stress. Since ROS are continuously being produced in cells, oxidative stress occurs not as a result of the production of ROS per se, but rather when the biosynthesis of ROS exceeds the capacity of various intrinsic anti-oxidant defense systems
* Corresponding author at: 616A Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, United States. Tel.: +1 412 647 6965; fax: +1 412 647 5258. E-mail address: [email protected] (M.P. Fink). 0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2007.05.019
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to detoxify these reactive species. Although many enzymatic processes can lead to the formation of ROS, the mitochondrial electron transport chain is thought to be the principal source of partially reduced forms of oxygen, at least in cell types, such as neurons, cardiomyocytes and hepatocytes, which are richly endowed with these organelles. Indeed, it has been estimated that about 0.2% of the O2 consumed by cells is converted into ROS, and about 90% of the ROS generated are produced within mitochondria [2]. Nevertheless, estimates about the absolute amounts or ROS produced strongly depend on the analytical methods applied. For example, estimates for the basal rate of H2O2 production range from 1–6 nmol/min/mg [3] to 0.1 nmol/ min/mg [4] and estimates for the basal rate of O2 formation range from 0.1–0.2/min/mg [5] to 0.003 nmol/min/mg [6]. Estimates made using highly indirect detection methods may be particularly prone to errors. Mitochondria, the intracellular organelles, which are responsible for carrying out the synthesis of adenosine triphosphate (ATP) via oxidative phosphorylation, contain numerous redox-active proteins and protein complexes that can leak single electrons to O2, leading to the formation of O2. Although O2 is relatively stable, this free radical is a key reactant in the formation of numerous other more toxic moieties, such as H2O2, OH and peroxynitrite (ONOO). Because the redox potential for the superoxide/dioxygen couple (E1/2 = 0.16 V) is relatively modest, the one-electron reduction of O2 is thermodynamically favorable for numerous mitochondrial oxidoreductases [7]. Furthermore, since O2 is rapidly removed from the equilibrium, the reaction is virtually irreversible and kinetically controlled. In addition to the enzymes and enzyme complexes that lead to O2 formation, mitochondria also contain proteins that catalyze the direct generation of the more toxic compound, H2O2. Some of the potential sources for O2 or H2O2 production in mitochondria include cytochrome b5 reductase (located in the outer mitochondrial membrane), monoamine oxidases isoforms A and B (located in the outer mitochondrial membrane), dihydroorotate dehydrogenase (outer surface of the inner mitochondrial membrane), glycerol-3-phosphate dehydrogenase (outer surface of the inner mitochondrial membrane), succinate:ubiquinone oxidoreductase (Complex II; inner surface of the inner mitochondrial membrane), aconitase (matrix), and alpha-ketoglutarate dehydrogenase complex (inner surface of the inner mitochondrial membrane) [7]. The most important mitochondrial sources for ROS generation, however, are Complex III (inner mitochondrial membrane) and Complex I (inner mitochondrial membrane) [8,9]. O2 is released into the matrix in the case of Complex I and to both the matrix and the intermembranous space by Complex III. O2 is formed in Complex III during the redox cycling of the electron acceptor ubiquinone, which can donate electrons to molecular oxygen at both the internal and the external faces of the inner mitochondrial membrane [10].
2. Mitochondrial ROS production increases during hypoxia During various forms of critical illness, such as hemorrhagic shock, myocardial ischemia or stroke, microvascular perfusion to key tissues is compromised, leading to cellular
hypoxia. Although the idea seems paradoxical, there are good data to suggest that mitochondrial O2 production increases under hypoxic conditions [11–13]. Based on purely thermodynamic considerations, one would not predict that a decrease in substrate (O2) concentration would cause an increase in the rate of a simple first-order reaction. However, other factors apparently are involved. Guzy and Schumacher [14] have identified at least two potential mechanisms, which conceivably can contribute to this paradoxical response. One hypothesis suggests that the interaction of molecular oxygen with proteins or lipids at Complex III modulates the lifetime of the free radical, ubisemiquinone, at this site. By prolonging the lifetime of the unstable ubisemiquinone molecule, the opportunity for O2 production increases, despite the decreased availability of substrate (O2). Of note, the radical anion intermediate, ubisemiquinone, was only recently detected for the first time [15]. Another hypothesis suggests that hypoxia might increase the access of O2 to ubisemiquinone at Complex III. Thus, if the molecular structure of one or more of the proteins in Complex III is affected by the O2 concentration in the membrane such that the ability of O2 to react with ubisemiquinone is improved under low-O2 conditions, then this change would increase ROS production despite the decrease in the availability of O2.
3. Mitochondria-targeted anti-oxidants might be useful therapeutic agents Given the apparent importance of oxidant stress in the pathogenesis of many diseases, there has been considerable interest among scientists and clinical researchers in the development of effective ROS (or electron) scavengers as therapeutic agents. Many of the most widely studied compounds in this general category fail to localize within mitochondria or even to enter the intracellular milieu. For example, when used as therapeutic agents, proteins like superoxide dismutase (SOD) and catalase fail to penetrate cell membranes and are therefore ineffective against ROS that are produced intracellularly. Vitamin E and Coenzyme Q are very lipophilic. Therefore, these compounds tend to be retained within the cytosolic membrane, and may fail to achieve pharmacologically significant intracellular concentrations. Prompted by the recognition that mitochondria are the major intracellular source of ROS under pathological conditions, there is increasing interest in the notion of developing mitochondria-targeted anti-oxidants as therapeutic agents [16,17]. The most common method for targeting compounds to mitochondria makes use of the potential gradient across the mitochondrial inner membrane, which renders the matrix side of the membrane negatively charged. Because of the electrical polarization of the inner membrane, lipophilic cations tend to accumulate within the mitochondrial matrix. Thus, compounds, which consist of the lipophilic and positively charged moiety, triphenylphosphonium (TPP+) covalently linked to an ROS scavenger, can function as mitochondria-targeted anti-oxidants. Several such compounds have been described, including [2(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran2-yl)ethyl] triphenylphosphonium bromide (i.e., TPP+ linked to
biochemical pharmacology 74 (2007) 801–809
vitamin E; MitoVit E) [18,19], 10-(60 -ubiquinolyl)decyl triphenylphosphonium bromide (i.e., TPP+ linked to co-enzyme Q; Mitoquinol or MitoQ) [20–23], [4-[4-[[(1,1-dimethylethyl)-oxidoimino]methyl]phenoxy]butyl] triphenylphosphonium bromide (TPP+ linked to a nitrone; MitoPBN) [24], 2-[4-(4-triphenylphosphoniobutoxy) phenyl]-1,2-benzisoselenazol)-3(2H)-one iodide (TPP+ linked to ebsalen; MitoPeroxidase) [25], and TPP+ linked to nitroxides, such as (Mito-CP) [26] and others [27]. These compounds have been extensively evaluated in a variety of in vitro models of redox stress. For example, MitoVit E was shown to protect isolated rat liver mitochondria against oxidative damage induced by tert-butylhydroperoxide [28] and MitoQ was shown to protect Jurkat cells from hydrogen peroxide-induced apoptosis [23]. Importantly, Adlam et al. recently reported that pre-treating rats for 14 days with oral MitoQ resulted in preservation of myocardial function when hearts were studied ex vivo, using a Langendorff constant pressure system, and subjected to transient coronary ischemia and reperfusion [29]. In another recent study, Esplugues and colleagues showed that pre-treating rats with oral MitoQ for 14 days blunted the development of tolerance to the vasodilating properties of nitroglycerin when aortic rings were studied ex vivo [30]. In contrast to these positive studies, Covey et al. recently reported that directly infusing MitoVit E into the brain (using an osmotic pump) failed to provide protection against striatal injury in a neonatal rat model of cerebral hypoxia and ischemia [31]. The authors of this study interpreted their findings as showing that mitochondrial oxidative damage does not contribute significantly to the death of striatal neurons after perinatal hypoxia– ischemic injury, but an equally plausible interpretation is that MitoVitE was ineffective (for some reason) as a scavenger of mitochondria-derived oxidants in this in vivo model system. As pointed out recently by Szeto [17], drugs like MitoQ and MitoVit E may have only limited utility because their selective uptake by mitochondria depends on the electrical polarization of the organelles. During sepsis and shock as well as in some more chronic diseases, mitochondrial function is impaired, leading (at least in some cases) to mitochondrial depolarization [32–34]. Furthermore, the uptake of triphenylphosphonium-conjugated ROS scavengers is reduced at concentrations greater than 50 mM [28], possibly because mitochondria depolarize when cations accumulate in the matrix. Another problem with at least one of the compounds in this class, MitoQ, is that it can undergo autoxidation in mitochondria, leading to O2 generation and promoting H2O2 efflux from mitochondria [17]. In addition to compounds like MitoQ and MitoVit E, another novel class of small cell-permeable peptide anti-oxidants that target mitochondria was recently described [35]. The essential structural motif of these peptides consists of alternating aromatic residues and basic amino acids [35]. Tyrosine- or dimethyltyrosine-containing analogs are capable of scavenging H2O2, OH, and ONOO and can inhibit lipid peroxidation in vitro. These peptides contain an amino acid sequence that allows them to freely penetrate cells despite carrying a 3+ net charge at physiologic pH [36], and they are highly concentrated in mitochondria [37,35]. Membrane depolarization only slightly inhibits mitochondrial uptake [35]. Administration to rats of one of these compounds (20 ,60 -dimethyltyrosine-DArg-Phe-Lys-NH2) has been shown to ameliorate myocardial
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dysfunction after transient occlusion of the left anterior descending coronary artery followed by reperfusion [38]. One of these novel peptide-based mitochondria-targeted ROS scavengers, SS-31 (D-Arg-Dmt-Lys-Phe-NH2, where Dmt stands for 20 ,6-dimethyltyrosine), is capable of reducing infarct volume when administered to mice immediately after reperfusion following transient acute cerebral ischemia [39]. SS-31 also has been shown to protect cultured N2a murine neuroblastoma neuron-like cells against death induced by exposure to H2O2 in vitro [40]. Furthermore, daily intraperitoneal injections of SS-31 (5 mg/kg), starting at 30 days of age, improve the survival and motor performance of G93A transgenic mice, an animal model of the degenerative neurological disease, amyotrophic lateral sclerosis [40]. Yet another approach has been to use manganese-centered SOD mimics. For example, Asayama et al. covalently coupled a mitochondria-targeting peptide leader sequence, Met-LeuSer-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Gly-Cys, to a water soluble manganese-centered porphyrin SOD mimic, 5-(4aminophenyl)-10,15,20-tris(N-methyl-4-pyridyl)porphine [41]. When certain Mn-centered porphyrin-based SOD mimics are employed, it may not be necessary to deliberately include structural features, which are designed to improve mitochondrial localization. Thus, when mice are injected intraperitoneally with 10 mg/kg of the ortho isomer of Mn(III) mesotetrakis(N-ethylpyridinium-2-yl)porphyrin, which is a potent water soluble SOD mimic (and peroxynitrite scavenger), pharmacologically relevant concentrations of the compound can be detected in rat heart mitochondria [42].
4. Hemigramicidin-dragged mitochondriatargeted ROS scavengers prevent oxidant-induced apoptosis of cultured cells Another distinct and novel class of mitochondria-targeted electron and ROS scavengers was recently described by the authors of the present report. These molecules consist of a ‘‘payload’’ – the portion of the molecule with electron – and ROS-scavenging activities – and a ‘‘targeting moiety’’ – the portion of the molecule, which promotes selective accumulation within mitochondria. For the payload portion, these molecules utilize stable nitroxide radicals, such as 4-hydroxy2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL; Fig. 1), which have been extensively investigated as cytoprotectors in a number of experimental models of oxidative stress [43–46]. By accepting one-electron, nitroxide radicals are converted to the corresponding hydroxylamine. Hydroxylamines act as effective ROS scavengers and in the process are converted back into nitroxides. In other words, these compounds undergo redox recycling [47]. Furthermore, nitroxide radicals exert SOD mimetic activity [48,49], which contributes to the prevention of the reaction of O2 with nitric oxide, thereby inhibiting formation of the highly toxic species, ONOO. Thus, nitroxides combine several important anti-oxidant and electron-scavenging properties in a single functional moiety. Nevertheless, it must be pointed out that stable nitroxide radicals like TEMPOL can have some deleterious effects as well. Specifically, TEMPOL-like compounds can be reduced by anti-oxidants and by ubiquinol in the respiratory chain [50,51], thereby
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Fig. 1 – Chemical structures of XJB-5-131 and XJB-5-208.
consuming cellular defenses against redox stress and interfering with mitochondrial respiration. Maximizing the beneficial effects of TEMPOL-like compounds while minimizing their deleterious effects probably depends, at least to some extent, on optimizing the concentration of the drug at the site(s) of action. An important aspect of the design of these molecules was the use of a targeting sequence that delivered the scavenging agent preferentially to the mitochondrial membrane. In accordance with this concept, we synthesized a series of compounds that consist of 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-NH2–TEMPO) conjugated to fragments of the membrane-active cyclopeptide antibiotic, gramicidin S [52]. The gramicidin segments were used to target the 4-NH2–TEMPO payload to mitochondria because antibiotics of this group have a high affinity for bacterial membranes [53] and because of the close relationship between bacteria and mitochondria [54]. We selected the Leu-D-Phe-Pro-Val-Orn fragment of gramicidin S as the targeting sequence, because it encompasses the betaturn motif that directs most of the polar functionality of the peptide strand into the core, and acylated the amino functions
of Leu and Orn in order to reduce gramicidin S-related cytotoxicity [55]. We sought to determine whether various conjugates of 4NH2–TEMPO and derivatives of gramicidin S (‘‘hemigramicidin– TEMPO conjugates’’) are concentrated within mitochondria [56]. Because of the presence of an unpaired electron, nitroxides, such as 4-NH2–TEMPO, display a distinctive and characteristic triplet signal (with hyperfine splitting constants of 16.6 G) when analyzed by electron paramagnetic resonance (EPR) spectroscopy. Accordingly, we employed EPR spectroscopy to monitor the cellular delivery and metabolic fate of hemigramicidin– TEMPO conjugates, such as XJB-5-131 (Fig. 1). Mouse embryonic cells (MECs) were incubated with 10 mM XJB-5-131 and cytosolic and mitochondrial fractions were prepared (Fig. 2A). The cells elicted the characteristic nitroxide EPR signal as did the mitochondrial preparation from the cells. In contrast, the cytosolic fraction did not elicit the EPR signature of nitroxide radicals, indicating that [1] XJB-5-131 is incorporated into MECs and [2] the compound localizes within the mitochondria of these cells. Similar results were observed with another conjugate, XJB-5-125 (data not shown). In contrast, 4-NH2–
Fig. 2 – EPR-based analysis of the integration and reduction of hemigramicidin–TEMPO conjugates in MECs. Cells (10 million/ mL) were incubated with 10 mM 4-NH2–TEMPO or 10 mM XJB-5-131 (labeled 5a) for 15 min. Recovered nitroxide radicals in whole cells, mitochondria, or cytosol fractions were resuspended in PBS in the presence or absence of 2 mM K3Fe(CN)6 Panel A depicts representative EPR spectra of XJB-5-131 (5a) in different fractions of MECs in the presence of K3Fe(CN)6. Note that distinctive EPR signals were detectable from cells and mitochondria but not from the cytosol. 4-NH2–TEMPO, the non-GSconjugated parental compound, did not significantly partition into cells. Panel B depicts the presence of nitroxides (n = 3); * p < 0.01 vs. K3Fe(CN)6; #p < 0.01 vs. XJB-5-131 (labeled 5a) under the same conditions. Reprinted from Reference [56] with permission.
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TEMPO did not effectively partition into either cells or mitochondria. Incubation of MECs in the presence of XJB-5131 resulted not only in its uptake into the cells but also in its one-electron reduction, as evidenced by a significant increase in the magnitude of the EPR signal intensity upon addition of a one-electron oxidant, ferricyanide (Fig. 2B). Using flow cytometry to monitor the oxidation of dihydroethidium to the fluorescent compound, ethidium, we carried out experiments to determine whether XJB-5-131 and XJB-5-125 are capable of preventing intracellular generation of O2 following incubation MECs with actinomycin D (ActD). Both XJB-5-131 and XJB-5-125 (but not 4-NH2–TEMPO) completely inhibited ActD-induced O2 production in MECs. Additionally, XJB-5-131 dramatically inhibited the induction of apoptosis (as assessed using three different assays) when MECs were incubated with ActD. Importantly, 4-NH2–TEMPO failed to inhibit ActD-induced apoptosis. We also tested another conjugate of 4-NH2–TEMPO, which is highly lipophilic (like XJB-5-131) but lacks the gramicidin S-based mitochondrial targeting motif. This conjugate also failed to block ActDinduced apoptosis. In more recent studies designed to better characterize the structure–activity relationships for hemigramicidin–TEMPO conjugates, ActD-induced apoptosis was again used as the primary measure of activity [57]. In these studies, we determined that a high degree of hydrophobicity and effective mitochondrial integration are necessary but not sufficient conditions for high anti-apoptotic/anti-oxidant activity for these nitroxide-based compounds. The presence of a betaturn/beta-sheet secondary structure is essential for optimal activity. Based on Monte-Carlo simulations in model lipid membranes, conservation of the D-Phe-Pro reverse turn in hemi-gramicidin analogs is important to ensure positioning of the nitroxide moiety at the mitochondrial membrane interface in order to maximize the protective effects of these compounds (at least in a reductionist in vitro assay system using cultured MECs).
5. XJB-5-131 and XJ B-5-208 ameliorate intestinal mucosal barrier dysfunction following hemorrhagic shock in rats Recognizing the inherent limitations of any cell-based screening assay, we sought to develop an in vivo assay system that would avoid the necessity to administer the compounds systemically to experimental animals, but instead would permit assessments of the test compounds in a local milieu. Because hemorrhagic shock in rats leads to marked derangements in intestinal mucosal barrier function [58,59], we used ileal mucosal permeability to fluorescein-labeled dextran (4 kDa average molecular mass) as a read-out. For the assay, the ileum was divided (like links of sausage) into a series of well-vascularized compartments. The lumen of each compartment was filled with the same volume (0.3 ml) of test solution. In every case, two of the compartments were filled with vehicle alone and thus served as internal controls to account for animal-to-animal variations in the severity of shock or the response of the mucosa to it. Using this assay system, we evaluated eight compounds: TEMPOL, one dipeptidic TEMPO analog, three hemigramici-
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din–TEMPO conjugates, and three hemigramicidin compounds lacking the TEMPO payload. As expected, the mucosal permeability of intestinal segments from hemorrhaged animals was significantly greater than the permeability of segments from normal rats. TEMPOL was previously shown to ameliorate organ damage [60,46] when used to treat rats subjected to hemorrhagic shock. Accordingly, we used intraluminal TEMPOL as a ‘‘positive control’’ for the gut mucosal protection assay. As anticipated, TEMPOL concentrations 1 mM in the gut lumen ameliorated hemorrhagic shock-induced ileal mucosal hyperpermeability [61]. Two of the TEMPO conjugates, namely XJB-5-208 and XJB-5-131, also significantly ameliorated hemorrhagic shock-induced ileal mucosal hyperpermeability. The lowest effective concentration for XJB-5-208 and XJB-5-131 was 1 mM; i.e., both of these compounds were 1000-fold more potent than TEMPOL. Two other compounds carrying the TEMPO payload, XJB-5-125 and XJB-5-197, failed to provide protection against gut barrier dysfunction induced by hemorrhage. XJB-5-133 has the same (hemigramicidin-based) mitochondrial-targeting moiety as XJB-5-131 but lacks the TEMPO payload. It is noteworthy, therefore, that XJB-5-133 did not afford protection from the development of ileal mucosal hyperpermeability. Ineffective as well were the two other hemigramicidin-based compounds that also lacked the TEMPO payload. Of the compounds screened, XJB-5-131 appeared to be the most effective, reducing hemorrhagic shock-induced mucosal hyperpermeability to approximately 60% of the control value.
6. XJB-5-131 ameliorates peroxidation of mitochondrial phospholipids and activation of Caspases 3/7 in intestinal mucosal samples from hemorrhaged rats Cardiolipin (CL) is an anionic phospholipid found exclusively in the inner mitochondrial membrane of eukaryotic cells [62]. Under normal conditions, the pro-apoptotic protein, cytochrome c, is anchored to the mitochondrial inner membrane by its specific and stoichiometric association with CL [63]. The acyl chains of CL are unsaturated and, therefore, susceptible to peroxidation by ROS. When ROS are generated within mitochondria in excessive quantities, cytochrome c bound to CL can function as an oxidase and induce extensive peroxidation of CL in the inner mitochondrial membrane [64,65]. Peroxidation of CL has two important consequences. First, peroxidized CL fails to bind cytochrome c tightly [66], leading to release of this protein into the intermembrane space. Second, peroxidation of CL is important for opening of the mitochondrial permeability transition pore [67,68]. Opening of the mitochondrial permeability transition pore promotes mitochondrial swelling and release of cytochrome c into the cytosol. Thus, peroxidation of CL promotes the release of cytochrome c and, on this basis, apoptosis [62]. In view of the preceding, we carried out another series of in vivo experiments. Again, isolated segments of intestine were prepared and filled with either vehicle or a 10 mM solution of XJB-5-131, the most active of the hemigramicidin–TEMPO conjugates examined in the previous series of experiments. The rats were subjected to 2 h of hemorrhagic shock, and at
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the end of this period, samples of ileal mucosa from the gut sacs filled with vehicle or XJB-5-131 solution were obtained. Samples of ileal mucosa from completely normal rats were also obtained for purposes of comparison. The samples were assayed for caspase 3/7 activity (an index of the extent of mucosal cell aptoptosis) as well as peroxidation of CL and several other phospholipids. When mucosal samples from normal animals were compared to those from vehicle-treated segments from shocked rats, it was apparent that hemorrhage was associated with significant peroxidation of key phospholipids, including phosphatidylcholine (PC; Fig. 3A), phosphatidylethanolamine (PE; Fig. 3B), phosphatidylserine (PS; Fig. 3C) and CL (Fig. 3D) [61]. Treatment with XJB-5-131 significantly ameliorated hemorrhagic shock-induced peroxidation of CL, the only one of the four phospholipids found exclusively in mitochondria. In contrast, treatment with XJB-5-131 had only a small effect on PE peroxidation and no effect on peroxidation of PC and PS. These data indicate that HS is associated with substantial oxidative stress even in the absence of resuscitation (reperfusion). Furthermore, these data support the view that XJB-5-131 is an effective ROS scavenger, which localizes predominantly in mitochondria where it is capable of protecting CL from peroxidation. Relative to the activity measured in samples from normal animals, the activity of caspases 3 and 7 was markedly increased in vehicle-treated mucosal samples from hemorrhaged rats. However, when the ileal segments were filled with XJB-5-131 solution instead of its vehicle, the level of caspases 3
and 7 activity after hemorrhagic shock was significantly decreased. Consistent with previously reported findings [69,70], our observations support the view that hemorrhagic shock is associated with activation of pro-apoptotic pathways in gut mucosal cells. Moreover, our data support the view that this process is significantly ameliorated following (local) treatment with XJB-5-131.
7. Treatment with XJB-5-131 prolongs survival of rats subjected to lethal hemorrhagic shock Whereas the previous experiments tested the activity of a solution of XJB-5-131 applied directly to the intestinal mucosa, the next series of experiments tested the therapeutic efficacy of this compound when administered systemically by intravenous infusion. These studies were designed to determine whether systemic administration of this compound could prolong the survival of rats subjected to massive blood loss, even in the absence of standard resuscitation with blood and crystalloid solution. Sixteen rats were used. The rats were hemorrhaged over 60 min, according to a strict protocol, which resulted in the removal of almost 60% of the estimated blood volume. Three rats died during the hemorrhage phase of the protocol (i.e., during the first 60 min) and were excluded from the analyses of data. Thirteen survived for at least 60 min and received the full dose of either XJB-5-131 solution or vehicle [61]. Six of seven animals in the vehicle-treated (control) group died
Fig. 3 – (A–D) Effect of intraluminal XJB-5-131 on hemorrhagic shock-induced peroxidation of phospholipids in intestinal mucosa. Tissue samples were harvested after 2 h of shock and assayed for oxidation products in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and cardiolipin (CL). Samples were obtained from intestinal segments of normal rats not subjected to hemorrhagic shock (control; n = 3), intestinal segments filled with vehicle from rats subjected to hemorrhagic shock (n = 5), and intestinal segments filled with 10 mM XJB-5-131 from rats subjected to hemorrhagic shock (n = 5). Data are means W S.E.; # indicates p < 0.05 vs. control; * indicates p < 0.05 vs. HS + vehicle. Reprinted from Reference [61] with permission.
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pharmacologically relevant concentrations of ROS scavengers within mitochondria. The field has matured to the point that investigators are now moving rapidly from purely in vitro studies to in vivo proof-of-principle experiments, and the early results suggest that mitochondria-targeted anti-oxidants could prove to be valuable therapeutic agents for a variety of acute and chronic medical conditions. At the present time, little is known about the toxicology of agents in this general class, and targeting ROS scavengers to mitochondria might prove to have deleterious effects. This cautionary note notwithstanding, the next several years should provide valuable new insights into the therapeutic possibilities for this fascinating new pharmacological approach.
Acknowledgements Fig. 4 – Effects of intravenous treatment with XJB-5-131 on survival of rats subjected to volume-controlled hemorrhagic shock, Rats were treated with 2.8ml/kg of vehicle or the same volume of XJB-5-131 solution during the final 20 min of the bleeding protocol. The total dose of XJB-5-131 infused was 2 mmol/kg. The difference in survival time between the two groups was statistically significant ( p < 0.01). Reprinted from Reference [61] with permission.
within 1 h after the end of the bleeding protocol and all were dead within 125 min (Fig. 4). The rats treated with XJB-5-131 survived significantly longer ( p < 0.01). Three of six survived for longer than 3 h after completion of the hemorrhage protocol and one rat survived for the whole 6 h post-bleeding observation period. These findings suggest that treatment with XJB-5131 might be able to prolong the period of time that patients can survive after losing large quantities of blood due to traumatic injuries or other catastrophes (e.g., rupture of an abdominal aortic aneurysm). By extending the treatment window before irreversible shock develops, treatment in the field with XJB-5131 might extend survival time enough to allow transport of more badly injured patients to locations where definitive care, including control of bleeding and resuscitation with blood products and asanguinous fluids, can be provided. Our findings using a rodent model of hemorrhagic shock also open up the possibility that drugs like XJB-5-131 might be beneficial in other conditions associated with marked tissue hypoperfusion, such as stroke and myocardial infarction. Although previous studies have shown that treatment with TEMPOL is beneficial in rodent models of HS [60,46], a relatively large dose (175 mmol/kg bolus + 175 mmol/kg per h) of the compound was required. In contrast, treatment with a dose of XJB-5-131 (about 2 mmol/kg) that was more than 80-fold lower was clearly beneficial. The greater potency of XJB-5-131 as compared to TEMPOL presumably reflects the tendency of the former compound to localize in mitochondrial membranes.
8.
Summary
During the past decade or so, medicinal chemists and biochemists have developed a variety of strategies for achieving
Defense Advanced Research Projects Administration (DARPA contract W81XWH-05-2-0026) as well as U.S. Public Health Service National Institutes of Health (GM067082). Figs. 1, 3 and 4 were used with permissions from Lippincott Williams & Wilkins: Ann Surg 2007 Feb; 245(2):305–14.
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Hemigramicidin–TEMPO conjugates: Novel mitochondriatargeted anti-oxidants Mitchell P. Fink a,*, Carlos A. Macias a, Jingbo Xiao b, Yulia Y. Tyurina c, Jianfei Jiang c, Natalia Belikova c, Russell L. Delude a, Joel S. Greenberger d, Valerian E. Kagan c, Peter Wipf b a
Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, United States c Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, United States d Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, PA, United States b
article info
abstract
Article history:
Oxidative damage to various cellular constituents (such as, proteins and lipids) mediated by
Received 7 March 2007
reactive oxygen species (ROS) is thought to be an important mechanism underlying the
Accepted 23 May 2007
pathogenesis of a variety of acute and chronic diseases. Mitochondria are the main source of ROS within most cells. Accordingly, there is increasing interest in the development of pharmacological ROS scavengers, which are specifically targeted to and concentrated within
Keywords:
mitochondria. Numerous compounds with these general characteristics have been synthe-
Superoxide
sized and evaluated in a variety of in vitro and in vivo models of redox stress. Among the more
Oxidant stress
promising of these mitochondria-targeted anti-oxidants are those that employ various
TEMPOL
peptides (or peptide-like moieties) derived from the antibiotic, gramicidin S, as the targeting
Hemorrhagic shock
construct and employ the stable free radical, 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl
LPS
(4-NH2–TEMPO), as the ROS scavenging ‘‘payload.’’ One of these hemigramicidin–TEMPO
Caspase
conjugates, XJB-5-131, has been shown to ameliorate intestinal mucosal injury and prolong survival in rats subjected to lethal hemorrhage. # 2007 Elsevier Inc. All rights reserved.
1. Mitochondria are an important source of reactive oxygen species Reactive oxygen species (ROS) are intermediates formed during one-electron reduction of molecular oxygen (dioxygen, O2). Some important ROS in biological systems include superoxide radical anion (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH). ROS are implicated as being important in the pathogenesis in a wide range of diseases or pathological processes, including various forms of cancer, type 2 diabetes mellitus, atherosclerosis, chronic inflammatory conditions,
ischemia/reperfusion injury, sepsis and some neurodegenerative diseases [1]. Additionally, ROS are important under both physiological and pathophysiological conditions as key mediators in a number of different intracellular signaling cascades [1]. The general processes whereby ROS cause pathological changes in cells and tissues are collectively referred to as oxidative stress. Since ROS are continuously being produced in cells, oxidative stress occurs not as a result of the production of ROS per se, but rather when the biosynthesis of ROS exceeds the capacity of various intrinsic anti-oxidant defense systems
* Corresponding author at: 616A Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, United States. Tel.: +1 412 647 6965; fax: +1 412 647 5258. E-mail address: [email protected] (M.P. Fink). 0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2007.05.019
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to detoxify these reactive species. Although many enzymatic processes can lead to the formation of ROS, the mitochondrial electron transport chain is thought to be the principal source of partially reduced forms of oxygen, at least in cell types, such as neurons, cardiomyocytes and hepatocytes, which are richly endowed with these organelles. Indeed, it has been estimated that about 0.2% of the O2 consumed by cells is converted into ROS, and about 90% of the ROS generated are produced within mitochondria [2]. Nevertheless, estimates about the absolute amounts or ROS produced strongly depend on the analytical methods applied. For example, estimates for the basal rate of H2O2 production range from 1–6 nmol/min/mg [3] to 0.1 nmol/ min/mg [4] and estimates for the basal rate of O2 formation range from 0.1–0.2/min/mg [5] to 0.003 nmol/min/mg [6]. Estimates made using highly indirect detection methods may be particularly prone to errors. Mitochondria, the intracellular organelles, which are responsible for carrying out the synthesis of adenosine triphosphate (ATP) via oxidative phosphorylation, contain numerous redox-active proteins and protein complexes that can leak single electrons to O2, leading to the formation of O2. Although O2 is relatively stable, this free radical is a key reactant in the formation of numerous other more toxic moieties, such as H2O2, OH and peroxynitrite (ONOO). Because the redox potential for the superoxide/dioxygen couple (E1/2 = 0.16 V) is relatively modest, the one-electron reduction of O2 is thermodynamically favorable for numerous mitochondrial oxidoreductases [7]. Furthermore, since O2 is rapidly removed from the equilibrium, the reaction is virtually irreversible and kinetically controlled. In addition to the enzymes and enzyme complexes that lead to O2 formation, mitochondria also contain proteins that catalyze the direct generation of the more toxic compound, H2O2. Some of the potential sources for O2 or H2O2 production in mitochondria include cytochrome b5 reductase (located in the outer mitochondrial membrane), monoamine oxidases isoforms A and B (located in the outer mitochondrial membrane), dihydroorotate dehydrogenase (outer surface of the inner mitochondrial membrane), glycerol-3-phosphate dehydrogenase (outer surface of the inner mitochondrial membrane), succinate:ubiquinone oxidoreductase (Complex II; inner surface of the inner mitochondrial membrane), aconitase (matrix), and alpha-ketoglutarate dehydrogenase complex (inner surface of the inner mitochondrial membrane) [7]. The most important mitochondrial sources for ROS generation, however, are Complex III (inner mitochondrial membrane) and Complex I (inner mitochondrial membrane) [8,9]. O2 is released into the matrix in the case of Complex I and to both the matrix and the intermembranous space by Complex III. O2 is formed in Complex III during the redox cycling of the electron acceptor ubiquinone, which can donate electrons to molecular oxygen at both the internal and the external faces of the inner mitochondrial membrane [10].
2. Mitochondrial ROS production increases during hypoxia During various forms of critical illness, such as hemorrhagic shock, myocardial ischemia or stroke, microvascular perfusion to key tissues is compromised, leading to cellular
hypoxia. Although the idea seems paradoxical, there are good data to suggest that mitochondrial O2 production increases under hypoxic conditions [11–13]. Based on purely thermodynamic considerations, one would not predict that a decrease in substrate (O2) concentration would cause an increase in the rate of a simple first-order reaction. However, other factors apparently are involved. Guzy and Schumacher [14] have identified at least two potential mechanisms, which conceivably can contribute to this paradoxical response. One hypothesis suggests that the interaction of molecular oxygen with proteins or lipids at Complex III modulates the lifetime of the free radical, ubisemiquinone, at this site. By prolonging the lifetime of the unstable ubisemiquinone molecule, the opportunity for O2 production increases, despite the decreased availability of substrate (O2). Of note, the radical anion intermediate, ubisemiquinone, was only recently detected for the first time [15]. Another hypothesis suggests that hypoxia might increase the access of O2 to ubisemiquinone at Complex III. Thus, if the molecular structure of one or more of the proteins in Complex III is affected by the O2 concentration in the membrane such that the ability of O2 to react with ubisemiquinone is improved under low-O2 conditions, then this change would increase ROS production despite the decrease in the availability of O2.
3. Mitochondria-targeted anti-oxidants might be useful therapeutic agents Given the apparent importance of oxidant stress in the pathogenesis of many diseases, there has been considerable interest among scientists and clinical researchers in the development of effective ROS (or electron) scavengers as therapeutic agents. Many of the most widely studied compounds in this general category fail to localize within mitochondria or even to enter the intracellular milieu. For example, when used as therapeutic agents, proteins like superoxide dismutase (SOD) and catalase fail to penetrate cell membranes and are therefore ineffective against ROS that are produced intracellularly. Vitamin E and Coenzyme Q are very lipophilic. Therefore, these compounds tend to be retained within the cytosolic membrane, and may fail to achieve pharmacologically significant intracellular concentrations. Prompted by the recognition that mitochondria are the major intracellular source of ROS under pathological conditions, there is increasing interest in the notion of developing mitochondria-targeted anti-oxidants as therapeutic agents [16,17]. The most common method for targeting compounds to mitochondria makes use of the potential gradient across the mitochondrial inner membrane, which renders the matrix side of the membrane negatively charged. Because of the electrical polarization of the inner membrane, lipophilic cations tend to accumulate within the mitochondrial matrix. Thus, compounds, which consist of the lipophilic and positively charged moiety, triphenylphosphonium (TPP+) covalently linked to an ROS scavenger, can function as mitochondria-targeted anti-oxidants. Several such compounds have been described, including [2(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran2-yl)ethyl] triphenylphosphonium bromide (i.e., TPP+ linked to
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vitamin E; MitoVit E) [18,19], 10-(60 -ubiquinolyl)decyl triphenylphosphonium bromide (i.e., TPP+ linked to co-enzyme Q; Mitoquinol or MitoQ) [20–23], [4-[4-[[(1,1-dimethylethyl)-oxidoimino]methyl]phenoxy]butyl] triphenylphosphonium bromide (TPP+ linked to a nitrone; MitoPBN) [24], 2-[4-(4-triphenylphosphoniobutoxy) phenyl]-1,2-benzisoselenazol)-3(2H)-one iodide (TPP+ linked to ebsalen; MitoPeroxidase) [25], and TPP+ linked to nitroxides, such as (Mito-CP) [26] and others [27]. These compounds have been extensively evaluated in a variety of in vitro models of redox stress. For example, MitoVit E was shown to protect isolated rat liver mitochondria against oxidative damage induced by tert-butylhydroperoxide [28] and MitoQ was shown to protect Jurkat cells from hydrogen peroxide-induced apoptosis [23]. Importantly, Adlam et al. recently reported that pre-treating rats for 14 days with oral MitoQ resulted in preservation of myocardial function when hearts were studied ex vivo, using a Langendorff constant pressure system, and subjected to transient coronary ischemia and reperfusion [29]. In another recent study, Esplugues and colleagues showed that pre-treating rats with oral MitoQ for 14 days blunted the development of tolerance to the vasodilating properties of nitroglycerin when aortic rings were studied ex vivo [30]. In contrast to these positive studies, Covey et al. recently reported that directly infusing MitoVit E into the brain (using an osmotic pump) failed to provide protection against striatal injury in a neonatal rat model of cerebral hypoxia and ischemia [31]. The authors of this study interpreted their findings as showing that mitochondrial oxidative damage does not contribute significantly to the death of striatal neurons after perinatal hypoxia– ischemic injury, but an equally plausible interpretation is that MitoVitE was ineffective (for some reason) as a scavenger of mitochondria-derived oxidants in this in vivo model system. As pointed out recently by Szeto [17], drugs like MitoQ and MitoVit E may have only limited utility because their selective uptake by mitochondria depends on the electrical polarization of the organelles. During sepsis and shock as well as in some more chronic diseases, mitochondrial function is impaired, leading (at least in some cases) to mitochondrial depolarization [32–34]. Furthermore, the uptake of triphenylphosphonium-conjugated ROS scavengers is reduced at concentrations greater than 50 mM [28], possibly because mitochondria depolarize when cations accumulate in the matrix. Another problem with at least one of the compounds in this class, MitoQ, is that it can undergo autoxidation in mitochondria, leading to O2 generation and promoting H2O2 efflux from mitochondria [17]. In addition to compounds like MitoQ and MitoVit E, another novel class of small cell-permeable peptide anti-oxidants that target mitochondria was recently described [35]. The essential structural motif of these peptides consists of alternating aromatic residues and basic amino acids [35]. Tyrosine- or dimethyltyrosine-containing analogs are capable of scavenging H2O2, OH, and ONOO and can inhibit lipid peroxidation in vitro. These peptides contain an amino acid sequence that allows them to freely penetrate cells despite carrying a 3+ net charge at physiologic pH [36], and they are highly concentrated in mitochondria [37,35]. Membrane depolarization only slightly inhibits mitochondrial uptake [35]. Administration to rats of one of these compounds (20 ,60 -dimethyltyrosine-DArg-Phe-Lys-NH2) has been shown to ameliorate myocardial
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dysfunction after transient occlusion of the left anterior descending coronary artery followed by reperfusion [38]. One of these novel peptide-based mitochondria-targeted ROS scavengers, SS-31 (D-Arg-Dmt-Lys-Phe-NH2, where Dmt stands for 20 ,6-dimethyltyrosine), is capable of reducing infarct volume when administered to mice immediately after reperfusion following transient acute cerebral ischemia [39]. SS-31 also has been shown to protect cultured N2a murine neuroblastoma neuron-like cells against death induced by exposure to H2O2 in vitro [40]. Furthermore, daily intraperitoneal injections of SS-31 (5 mg/kg), starting at 30 days of age, improve the survival and motor performance of G93A transgenic mice, an animal model of the degenerative neurological disease, amyotrophic lateral sclerosis [40]. Yet another approach has been to use manganese-centered SOD mimics. For example, Asayama et al. covalently coupled a mitochondria-targeting peptide leader sequence, Met-LeuSer-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Gly-Cys, to a water soluble manganese-centered porphyrin SOD mimic, 5-(4aminophenyl)-10,15,20-tris(N-methyl-4-pyridyl)porphine [41]. When certain Mn-centered porphyrin-based SOD mimics are employed, it may not be necessary to deliberately include structural features, which are designed to improve mitochondrial localization. Thus, when mice are injected intraperitoneally with 10 mg/kg of the ortho isomer of Mn(III) mesotetrakis(N-ethylpyridinium-2-yl)porphyrin, which is a potent water soluble SOD mimic (and peroxynitrite scavenger), pharmacologically relevant concentrations of the compound can be detected in rat heart mitochondria [42].
4. Hemigramicidin-dragged mitochondriatargeted ROS scavengers prevent oxidant-induced apoptosis of cultured cells Another distinct and novel class of mitochondria-targeted electron and ROS scavengers was recently described by the authors of the present report. These molecules consist of a ‘‘payload’’ – the portion of the molecule with electron – and ROS-scavenging activities – and a ‘‘targeting moiety’’ – the portion of the molecule, which promotes selective accumulation within mitochondria. For the payload portion, these molecules utilize stable nitroxide radicals, such as 4-hydroxy2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL; Fig. 1), which have been extensively investigated as cytoprotectors in a number of experimental models of oxidative stress [43–46]. By accepting one-electron, nitroxide radicals are converted to the corresponding hydroxylamine. Hydroxylamines act as effective ROS scavengers and in the process are converted back into nitroxides. In other words, these compounds undergo redox recycling [47]. Furthermore, nitroxide radicals exert SOD mimetic activity [48,49], which contributes to the prevention of the reaction of O2 with nitric oxide, thereby inhibiting formation of the highly toxic species, ONOO. Thus, nitroxides combine several important anti-oxidant and electron-scavenging properties in a single functional moiety. Nevertheless, it must be pointed out that stable nitroxide radicals like TEMPOL can have some deleterious effects as well. Specifically, TEMPOL-like compounds can be reduced by anti-oxidants and by ubiquinol in the respiratory chain [50,51], thereby
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Fig. 1 – Chemical structures of XJB-5-131 and XJB-5-208.
consuming cellular defenses against redox stress and interfering with mitochondrial respiration. Maximizing the beneficial effects of TEMPOL-like compounds while minimizing their deleterious effects probably depends, at least to some extent, on optimizing the concentration of the drug at the site(s) of action. An important aspect of the design of these molecules was the use of a targeting sequence that delivered the scavenging agent preferentially to the mitochondrial membrane. In accordance with this concept, we synthesized a series of compounds that consist of 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-NH2–TEMPO) conjugated to fragments of the membrane-active cyclopeptide antibiotic, gramicidin S [52]. The gramicidin segments were used to target the 4-NH2–TEMPO payload to mitochondria because antibiotics of this group have a high affinity for bacterial membranes [53] and because of the close relationship between bacteria and mitochondria [54]. We selected the Leu-D-Phe-Pro-Val-Orn fragment of gramicidin S as the targeting sequence, because it encompasses the betaturn motif that directs most of the polar functionality of the peptide strand into the core, and acylated the amino functions
of Leu and Orn in order to reduce gramicidin S-related cytotoxicity [55]. We sought to determine whether various conjugates of 4NH2–TEMPO and derivatives of gramicidin S (‘‘hemigramicidin– TEMPO conjugates’’) are concentrated within mitochondria [56]. Because of the presence of an unpaired electron, nitroxides, such as 4-NH2–TEMPO, display a distinctive and characteristic triplet signal (with hyperfine splitting constants of 16.6 G) when analyzed by electron paramagnetic resonance (EPR) spectroscopy. Accordingly, we employed EPR spectroscopy to monitor the cellular delivery and metabolic fate of hemigramicidin– TEMPO conjugates, such as XJB-5-131 (Fig. 1). Mouse embryonic cells (MECs) were incubated with 10 mM XJB-5-131 and cytosolic and mitochondrial fractions were prepared (Fig. 2A). The cells elicted the characteristic nitroxide EPR signal as did the mitochondrial preparation from the cells. In contrast, the cytosolic fraction did not elicit the EPR signature of nitroxide radicals, indicating that [1] XJB-5-131 is incorporated into MECs and [2] the compound localizes within the mitochondria of these cells. Similar results were observed with another conjugate, XJB-5-125 (data not shown). In contrast, 4-NH2–
Fig. 2 – EPR-based analysis of the integration and reduction of hemigramicidin–TEMPO conjugates in MECs. Cells (10 million/ mL) were incubated with 10 mM 4-NH2–TEMPO or 10 mM XJB-5-131 (labeled 5a) for 15 min. Recovered nitroxide radicals in whole cells, mitochondria, or cytosol fractions were resuspended in PBS in the presence or absence of 2 mM K3Fe(CN)6 Panel A depicts representative EPR spectra of XJB-5-131 (5a) in different fractions of MECs in the presence of K3Fe(CN)6. Note that distinctive EPR signals were detectable from cells and mitochondria but not from the cytosol. 4-NH2–TEMPO, the non-GSconjugated parental compound, did not significantly partition into cells. Panel B depicts the presence of nitroxides (n = 3); * p < 0.01 vs. K3Fe(CN)6; #p < 0.01 vs. XJB-5-131 (labeled 5a) under the same conditions. Reprinted from Reference [56] with permission.
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TEMPO did not effectively partition into either cells or mitochondria. Incubation of MECs in the presence of XJB-5131 resulted not only in its uptake into the cells but also in its one-electron reduction, as evidenced by a significant increase in the magnitude of the EPR signal intensity upon addition of a one-electron oxidant, ferricyanide (Fig. 2B). Using flow cytometry to monitor the oxidation of dihydroethidium to the fluorescent compound, ethidium, we carried out experiments to determine whether XJB-5-131 and XJB-5-125 are capable of preventing intracellular generation of O2 following incubation MECs with actinomycin D (ActD). Both XJB-5-131 and XJB-5-125 (but not 4-NH2–TEMPO) completely inhibited ActD-induced O2 production in MECs. Additionally, XJB-5-131 dramatically inhibited the induction of apoptosis (as assessed using three different assays) when MECs were incubated with ActD. Importantly, 4-NH2–TEMPO failed to inhibit ActD-induced apoptosis. We also tested another conjugate of 4-NH2–TEMPO, which is highly lipophilic (like XJB-5-131) but lacks the gramicidin S-based mitochondrial targeting motif. This conjugate also failed to block ActDinduced apoptosis. In more recent studies designed to better characterize the structure–activity relationships for hemigramicidin–TEMPO conjugates, ActD-induced apoptosis was again used as the primary measure of activity [57]. In these studies, we determined that a high degree of hydrophobicity and effective mitochondrial integration are necessary but not sufficient conditions for high anti-apoptotic/anti-oxidant activity for these nitroxide-based compounds. The presence of a betaturn/beta-sheet secondary structure is essential for optimal activity. Based on Monte-Carlo simulations in model lipid membranes, conservation of the D-Phe-Pro reverse turn in hemi-gramicidin analogs is important to ensure positioning of the nitroxide moiety at the mitochondrial membrane interface in order to maximize the protective effects of these compounds (at least in a reductionist in vitro assay system using cultured MECs).
5. XJB-5-131 and XJ B-5-208 ameliorate intestinal mucosal barrier dysfunction following hemorrhagic shock in rats Recognizing the inherent limitations of any cell-based screening assay, we sought to develop an in vivo assay system that would avoid the necessity to administer the compounds systemically to experimental animals, but instead would permit assessments of the test compounds in a local milieu. Because hemorrhagic shock in rats leads to marked derangements in intestinal mucosal barrier function [58,59], we used ileal mucosal permeability to fluorescein-labeled dextran (4 kDa average molecular mass) as a read-out. For the assay, the ileum was divided (like links of sausage) into a series of well-vascularized compartments. The lumen of each compartment was filled with the same volume (0.3 ml) of test solution. In every case, two of the compartments were filled with vehicle alone and thus served as internal controls to account for animal-to-animal variations in the severity of shock or the response of the mucosa to it. Using this assay system, we evaluated eight compounds: TEMPOL, one dipeptidic TEMPO analog, three hemigramici-
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din–TEMPO conjugates, and three hemigramicidin compounds lacking the TEMPO payload. As expected, the mucosal permeability of intestinal segments from hemorrhaged animals was significantly greater than the permeability of segments from normal rats. TEMPOL was previously shown to ameliorate organ damage [60,46] when used to treat rats subjected to hemorrhagic shock. Accordingly, we used intraluminal TEMPOL as a ‘‘positive control’’ for the gut mucosal protection assay. As anticipated, TEMPOL concentrations 1 mM in the gut lumen ameliorated hemorrhagic shock-induced ileal mucosal hyperpermeability [61]. Two of the TEMPO conjugates, namely XJB-5-208 and XJB-5-131, also significantly ameliorated hemorrhagic shock-induced ileal mucosal hyperpermeability. The lowest effective concentration for XJB-5-208 and XJB-5-131 was 1 mM; i.e., both of these compounds were 1000-fold more potent than TEMPOL. Two other compounds carrying the TEMPO payload, XJB-5-125 and XJB-5-197, failed to provide protection against gut barrier dysfunction induced by hemorrhage. XJB-5-133 has the same (hemigramicidin-based) mitochondrial-targeting moiety as XJB-5-131 but lacks the TEMPO payload. It is noteworthy, therefore, that XJB-5-133 did not afford protection from the development of ileal mucosal hyperpermeability. Ineffective as well were the two other hemigramicidin-based compounds that also lacked the TEMPO payload. Of the compounds screened, XJB-5-131 appeared to be the most effective, reducing hemorrhagic shock-induced mucosal hyperpermeability to approximately 60% of the control value.
6. XJB-5-131 ameliorates peroxidation of mitochondrial phospholipids and activation of Caspases 3/7 in intestinal mucosal samples from hemorrhaged rats Cardiolipin (CL) is an anionic phospholipid found exclusively in the inner mitochondrial membrane of eukaryotic cells [62]. Under normal conditions, the pro-apoptotic protein, cytochrome c, is anchored to the mitochondrial inner membrane by its specific and stoichiometric association with CL [63]. The acyl chains of CL are unsaturated and, therefore, susceptible to peroxidation by ROS. When ROS are generated within mitochondria in excessive quantities, cytochrome c bound to CL can function as an oxidase and induce extensive peroxidation of CL in the inner mitochondrial membrane [64,65]. Peroxidation of CL has two important consequences. First, peroxidized CL fails to bind cytochrome c tightly [66], leading to release of this protein into the intermembrane space. Second, peroxidation of CL is important for opening of the mitochondrial permeability transition pore [67,68]. Opening of the mitochondrial permeability transition pore promotes mitochondrial swelling and release of cytochrome c into the cytosol. Thus, peroxidation of CL promotes the release of cytochrome c and, on this basis, apoptosis [62]. In view of the preceding, we carried out another series of in vivo experiments. Again, isolated segments of intestine were prepared and filled with either vehicle or a 10 mM solution of XJB-5-131, the most active of the hemigramicidin–TEMPO conjugates examined in the previous series of experiments. The rats were subjected to 2 h of hemorrhagic shock, and at
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the end of this period, samples of ileal mucosa from the gut sacs filled with vehicle or XJB-5-131 solution were obtained. Samples of ileal mucosa from completely normal rats were also obtained for purposes of comparison. The samples were assayed for caspase 3/7 activity (an index of the extent of mucosal cell aptoptosis) as well as peroxidation of CL and several other phospholipids. When mucosal samples from normal animals were compared to those from vehicle-treated segments from shocked rats, it was apparent that hemorrhage was associated with significant peroxidation of key phospholipids, including phosphatidylcholine (PC; Fig. 3A), phosphatidylethanolamine (PE; Fig. 3B), phosphatidylserine (PS; Fig. 3C) and CL (Fig. 3D) [61]. Treatment with XJB-5-131 significantly ameliorated hemorrhagic shock-induced peroxidation of CL, the only one of the four phospholipids found exclusively in mitochondria. In contrast, treatment with XJB-5-131 had only a small effect on PE peroxidation and no effect on peroxidation of PC and PS. These data indicate that HS is associated with substantial oxidative stress even in the absence of resuscitation (reperfusion). Furthermore, these data support the view that XJB-5-131 is an effective ROS scavenger, which localizes predominantly in mitochondria where it is capable of protecting CL from peroxidation. Relative to the activity measured in samples from normal animals, the activity of caspases 3 and 7 was markedly increased in vehicle-treated mucosal samples from hemorrhaged rats. However, when the ileal segments were filled with XJB-5-131 solution instead of its vehicle, the level of caspases 3
and 7 activity after hemorrhagic shock was significantly decreased. Consistent with previously reported findings [69,70], our observations support the view that hemorrhagic shock is associated with activation of pro-apoptotic pathways in gut mucosal cells. Moreover, our data support the view that this process is significantly ameliorated following (local) treatment with XJB-5-131.
7. Treatment with XJB-5-131 prolongs survival of rats subjected to lethal hemorrhagic shock Whereas the previous experiments tested the activity of a solution of XJB-5-131 applied directly to the intestinal mucosa, the next series of experiments tested the therapeutic efficacy of this compound when administered systemically by intravenous infusion. These studies were designed to determine whether systemic administration of this compound could prolong the survival of rats subjected to massive blood loss, even in the absence of standard resuscitation with blood and crystalloid solution. Sixteen rats were used. The rats were hemorrhaged over 60 min, according to a strict protocol, which resulted in the removal of almost 60% of the estimated blood volume. Three rats died during the hemorrhage phase of the protocol (i.e., during the first 60 min) and were excluded from the analyses of data. Thirteen survived for at least 60 min and received the full dose of either XJB-5-131 solution or vehicle [61]. Six of seven animals in the vehicle-treated (control) group died
Fig. 3 – (A–D) Effect of intraluminal XJB-5-131 on hemorrhagic shock-induced peroxidation of phospholipids in intestinal mucosa. Tissue samples were harvested after 2 h of shock and assayed for oxidation products in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and cardiolipin (CL). Samples were obtained from intestinal segments of normal rats not subjected to hemorrhagic shock (control; n = 3), intestinal segments filled with vehicle from rats subjected to hemorrhagic shock (n = 5), and intestinal segments filled with 10 mM XJB-5-131 from rats subjected to hemorrhagic shock (n = 5). Data are means W S.E.; # indicates p < 0.05 vs. control; * indicates p < 0.05 vs. HS + vehicle. Reprinted from Reference [61] with permission.
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pharmacologically relevant concentrations of ROS scavengers within mitochondria. The field has matured to the point that investigators are now moving rapidly from purely in vitro studies to in vivo proof-of-principle experiments, and the early results suggest that mitochondria-targeted anti-oxidants could prove to be valuable therapeutic agents for a variety of acute and chronic medical conditions. At the present time, little is known about the toxicology of agents in this general class, and targeting ROS scavengers to mitochondria might prove to have deleterious effects. This cautionary note notwithstanding, the next several years should provide valuable new insights into the therapeutic possibilities for this fascinating new pharmacological approach.
Acknowledgements Fig. 4 – Effects of intravenous treatment with XJB-5-131 on survival of rats subjected to volume-controlled hemorrhagic shock, Rats were treated with 2.8ml/kg of vehicle or the same volume of XJB-5-131 solution during the final 20 min of the bleeding protocol. The total dose of XJB-5-131 infused was 2 mmol/kg. The difference in survival time between the two groups was statistically significant ( p < 0.01). Reprinted from Reference [61] with permission.
within 1 h after the end of the bleeding protocol and all were dead within 125 min (Fig. 4). The rats treated with XJB-5-131 survived significantly longer ( p < 0.01). Three of six survived for longer than 3 h after completion of the hemorrhage protocol and one rat survived for the whole 6 h post-bleeding observation period. These findings suggest that treatment with XJB-5131 might be able to prolong the period of time that patients can survive after losing large quantities of blood due to traumatic injuries or other catastrophes (e.g., rupture of an abdominal aortic aneurysm). By extending the treatment window before irreversible shock develops, treatment in the field with XJB-5131 might extend survival time enough to allow transport of more badly injured patients to locations where definitive care, including control of bleeding and resuscitation with blood products and asanguinous fluids, can be provided. Our findings using a rodent model of hemorrhagic shock also open up the possibility that drugs like XJB-5-131 might be beneficial in other conditions associated with marked tissue hypoperfusion, such as stroke and myocardial infarction. Although previous studies have shown that treatment with TEMPOL is beneficial in rodent models of HS [60,46], a relatively large dose (175 mmol/kg bolus + 175 mmol/kg per h) of the compound was required. In contrast, treatment with a dose of XJB-5-131 (about 2 mmol/kg) that was more than 80-fold lower was clearly beneficial. The greater potency of XJB-5-131 as compared to TEMPOL presumably reflects the tendency of the former compound to localize in mitochondrial membranes.
8.
Summary
During the past decade or so, medicinal chemists and biochemists have developed a variety of strategies for achieving
Defense Advanced Research Projects Administration (DARPA contract W81XWH-05-2-0026) as well as U.S. Public Health Service National Institutes of Health (GM067082). Figs. 1, 3 and 4 were used with permissions from Lippincott Williams & Wilkins: Ann Surg 2007 Feb; 245(2):305–14.
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