Metal-Dependent SOD Mimics

Superoxide dismutase is an important antioxidant to control the free radical reactions related to superoxide generated in biological system. However, its large molecule and short lifespan in vivo limit its clinical use. Extensive studies have been carried out to find the suitable SOD-mimics to substitute it. In order to acts as SOD mimics, a compound should be non-toxic, stable, easy to reach its targets and retain high SOD activity in vivo. Many compounds have shown SOD-like activity in vitro, however, no metal-dependent SOD mimics can really replace it. W. Sun Metal-Dependent SOD Mimics 3 Introduction Superoxide is a major factor in radiation damage, inflammation, tumor promotion, and re-perfusion injury [1]. Fortunately, we have evolved an effective defense system against the toxicity of O2, such as superoxide dismutase and catalase. In 1969, McCord and Fridovich first reported that the erythrocyte protein functioned as superoxide dismutase enzyme [2]. Generally there are three groups of SOD; each has different metal in the active site. According to the metals, they are termed as CuZnSOD, MnSOD and FeSOD. Acting as one of the most important antioxidants, all SODs catalyze the dismutation of O2 as: 2 2 2 2 O O H 2H O + → + + − • SOD can greatly accelerate this process. Under physiological pH, the rate constant for uncatalyzed dismutation is 5 x 10 Ms; for CuZnSOD, k = 1.6 x 10 Ms; for MnSOD, k= 1.8 x 10Ms [3]. However, the therapeutic use of SOD is limited by the facts that SOD cannot penetrate across cell membranes and it can be rapidly cleared by the kidney [4]. So, it is important to find SOD mimics, which have the activity of SOD and at the same time, are stable, non-toxic and capable of crossing cell membrane. There are two kinds of SOD mimics: metal-dependent and metalindependent mimics. This paper will focus on the metaldependent SOD mimics, their assays, chemical characters and usage. Catalyst Mechanism of SOD and Its Mimics SOD catalyze the dismutation of superoxide through a so called “Ping-Pong mechanism”: First the metal cation (M) in SOD is reduced to M (n-1)+ and reoxidized back to M: 2 1) (n k1 2 n O M O M + →  + + • + (1) 2 2 n k2 2 1) (n M O M O H + →  + + • + (2) The net reaction is: 2 2 2 kcat 2 O O H 2H 2O + →  + + − • (3) W. Sun Metal-Dependent SOD Mimics 4 2 1 2 1 cat k k k k 2 k + = For a typical experiment system, where O2 is generated by the oxidation of xanthine, the flux of O2 production is about 1.0 μM/min and 10 μM of SOD has a protective effect. Required Qualifications of SOD Mimics SOD mimics are those compounds that can function as SOD to catalyze the dismutation of superoxide. To act as a qualified SOD mimic in vivo conditions, a compound need to meet following qualities [5]: 1. The compound should not be toxic at the concentration needed for its SOD activity; 2. The compound should have a relatively long metabolic halflife for it to carry out its SOD activity; 3. The compound should be able to penetrate into the cells so to reach the target region; 4. The compound should retain its high SOD activity in vivo. Assay of SOD Activity To directly assay the SOD activity, we need to initially generate high concentration of O2 and follow its decay by measuring the absorbance change at 250 nm in the absence and in the presence of a tested compound. The most often used methods are “indirect assay”. In an indirect assay, O2 is generated with a constant flux (often by a mixture of xanthine oxidase and xanthine). Then O2 reacts with detector molecules such as Fe (III)cytochrome c [6]: Cyt c (Fe(III)) + O2→ O2 + cyt c Fe(II) (k= 2 x 10 Ms, pH =7.8, 25°C) (4) Fe(II)cytochrome c has an absorbance at 550 nm. SOD competes with Fe(III)cytochrome c for O2. Since the ratio of the rate constant for O2 dismutation for the catalysis by SOD and that for reduction of Fe(III)cytochrome c is 2 x 10 Ms/2 x 10Ms, one part of SOD will W. Sun Metal-Dependent SOD Mimics 5 compete with 10,000 parts of cytochrome c and inhibit the change of absorbance. One unit of SOD activity is defined as the amount of SOD that inhibits the cytochrome c reduction by 50%. However, this assay can be interfered with by various factors, such as oxidization of cytochrome c by peroxynitrite, cytochrome oxidase and hydrogen peroxide [3]. Cytochrome c can be replaced by other molecules, such as nitroblue tetrazolium (NBT), adrenaline, and lluciferin. Copper-containing complex CuZnSOD contains two protein subunits; each of its active site contains one copper and one zinc ion. The zinc ion acts to stabilize the enzyme, while the copper ion acts as the functional metal. Since copper acts as the active center in CuZnSOD, many copper complexes have been synthesized and tested. Research has shown that many complexes have SOD-like activities, such as Cu(II)(3,5-diisopropylsalicylic acid)2 (CuDIPS) [7], Cu(II) histidine complexes [8], Cu(II) complexes of macrocyclic polyamine derivatives [9], Bis(2,9-dimethyl-1,10-phenaanthroline)Cu(I)nitrate (Cu(I)(DMP)2) [10] and Cu(II)-oligopeptide [11]. Table 1 [12] SOD-Like Activity, Percent Reactivity, and Rate of Superoxide Dismutation for some Copper complexes and CuZnSOD Complex Concentration (μM) % reactivity Rate (x10 Ms) CuZnSOD 0.02 100 1.3 Cu (II) (DIP)2 2.9 0.70 1-2 Cu (II) (salicylate)2 4.6 0.65 1.6 Since the dismutation reaction involves the redox cycle of Cu(II) and Cu(I), it is reasonable to expect that the redox potential of Cu(II)complex/Cu(I) complex can influence the SOD-like activity, while the ligand of the complex determines the redox potential [9]. It was shown that although His-Phe-Cu(II) complexes had a relatively high SOD activity (k = 3.57 x10 Ms), Phe-His-Cu(II) displayed no SOD activity (k = 9.9 x10 Ms) [13]. After comparing a W. Sun Metal-Dependent SOD Mimics 6 series of Cu(II)-macrocyclic polyamine derivatives, Kimura [9] proposed that the activity depended on ring size, type and subsistent on the macrocycles. By modifying their chemical structure, we can improve their biological activity. Although in vitro studies show promising results of these copper complexes, in vivo experiments are often disappointing. The first reason is that many complexes undergo dissociation in vivo, yielding copper ions that subsequently combine with serum components and lose their SOD-like activity. The second reason relates to the reoxidation of the reduced compound by O2. Unlike SOD (k-1 = 0.44 Ms), most copper compounds have a k-1 at the range of 10-10 Ms [14]. At physiological situation, the concentration of O2 is about 10 M. If k-1 [O2]> k2 [O2], the rate constant of metal compounds to catalyze O2 dismutation would be [14]: ) ] [O ] O [ k k (k k 2k k 2 2 1 2 1 2 1 real − • − + + = So, these compounds can mimic SOD only when k-1 < 10 Ms. Otherwise, they can only act as scavengers. Iron-containing compounds FeSOD is found in bacteria, algae and higher plants. During the catalytic cycle, the iron “oscillates” between Fe(III) and Fe(II) state. Some useful iron complexes with SOD-like activity include: Fe-ehylenediaminetetraacetic acid (Fe-EDTA), Fe(III)-tetrakis (4-N-methylpyridyl) porphine (Fe(III)TMPyP), Fe(II)-tetrakis-N,N,N’,N’(2-pyridylmethyl)ethylenediamine (FeTPEN) and Fe(III)-tris [N-(2-pyridylmethyl-2-aminoethyl] amine (Fe-TPAA). At pH 10.1, Fe(III)-TMPyP can catalyze dismutation with a rate constant of 3 x 10 Ms [15]. Nagano [16] reported that in the xanthine oxidase-cytochrome c assay, 0.8 μM Fe-TPEN and 7.5 μM FeTPAA were equivalent to 1 unit of SOD activity. Paraquat can be reduced to monocation radical (PQ), which then reacts rapidly with molecular oxygen to yield O2. Unlike copper-containing W. Sun Metal-Dependent SOD Mimics 7 complexes, which cannot suppress paraquat toxicity, both Fe-TPEN and Fe-TPAA can block the toxic effect of paraquat on E. coli growth and survival [16]. Some other TPAA-analogues liganding with Fe or Cu also show high SOD-like activity. It is suggested that the pyridine rings of TPAA might provide the metal an environment similar to that of native SOD [17]. However, the activity in vitro does not always correspond to those in vivo. In vitro, the SOD-like activity of Fe-TPEN is 100 times higher than that of Fe-TPAA; in vivo, Fe-TPAA has a much higher level of protection (10 fold) than Fe-TPEN [16]. As a potential Fenton catalyst, in the presence of H2O2, Fe(II)-TPEN reacted with H2O2 to generate hydroxyl radical and Fe(III)TPEN; Fe (III)-TPEN then undergoes reducing by GSH or ascorbate [18] Fe(II)-TPEN + H2O2 → Fe(III)-TPEN + OH+ OH (5) So, the in vivo use of Fe(II)-TPEN as a SOD mimic is severely impaired by its capacity to generate OH. Manganese-Containing Compound MnSOD is mainly located in the mitochondria. The “resting” enzyme contains Mn(III) at its active site. At pH 7.0, the rate constant of MnSOD catalyzed O2 dismutation is similar to that of CuZnSOD, but it decreases at alkaline conditions. The ligands for manganese-containing SOD mimics usually have a macrocycle structure, such as TMPyP, EDTA and NTA. Like its Fe-containing counterpart, Mn(III)-TMPyP also effectively catalyzes the dismutation of O2 and acts against paraquat [19]. Desferrioxamine-Mn(III) (DF-Mn) is another low-molecular-weight SOD mimic that protects cells from oxidative damage. In addition to facilitating the removal of O2, DF-Mn can also oxidize reduced metals, such as DNA-bound Fe(II) or Cu(I), so DF-Mn can protect cells through O2–independent mechanism [20]. W. Sun Metal-Dependent SOD Mimics 8 Unfortunately, in vivo, the resultant Mn( II) complex can be reduced at a rate constant of 4 x 10 Ms [21], which means in vivo, Mn-porphyrins only act as a NADPH/GSH: O2 oxidoreductase rather than O2 dismutase. Pharmaceutical Usage of SOD Mimics Superoxide is deleterious in vivo. The direct targets of O2 include many enzymes such as ribonucleotide reductase, which is responsible for DNA synthesis and 6-phosphogluconate dehydratase. In addition, O2 can generate more reactive species, including OH, NO2. Acting as SOD mimics, many metal complexes have shown promising clinical effects. Oberley [22] proposed that copper complexes were effective in antiinflammation, anti-ucler and anti-diabetic. Pretreatment with CuDIPs can inhibit TPA-induced carcinogenesis in mouse skin [23]. A Mn(II) complex with bis (cyclohexylpyridine)–substituted macrocyclic ligand has been proved to be stable in vivo and can increase the survival from ischemia-reperfusion injury [24]. Summary Compared with SOD, metal-dependent SOD mimics have several advantages. First, they are easy and cheap to synthesize; second, as low-molecular-weight-molecules, they can across the cell membrane easily; third, they are easily modified in the lab. However, they also have several shortcomings. The use of copper complexes in vivo is often severely limited by inactivation on processes due to chelating agents normally found in living cells. Although manganese and iron complexes are less influenced by chelating agents; some other limiting factors have to be taken into account. So, there still a long way for us to find the suitable nontoxic, stable and effective SOD mimics. W. Sun Metal-Dependent SOD Mimics 9 Reference 1. 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