Simple Biological Systems for Assessing the Activity of Superoxide Dismutase Mimics

Abstract

Significance: Half a century of research provided unambiguous proof that superoxide and species derived from it—reactive oxygen species (ROS)—play a central role in many diseases and degenerative processes. This stimulated the search for pharmaceutical agents that are capable of preventing oxidative damage, and methods of assessing their therapeutic potential. Recent Advances: The limitations of superoxide dismutase (SOD) as a therapeutic tool directed attention to small molecules, SOD mimics, that are capable of catalytically scavenging superoxide. Several groups of compounds, based on either metal complexes, including metalloporphyrins, metallocorroles, Mn(II) cyclic polyamines, and Mn(III) salen derivatives, or non-metal based compounds, such as fullerenes, nitrones, and nitroxides, have been developed and studied in vitro and in vivo. Very few entered clinical trials. Critical Issues and Future Directions: Development of SOD mimics requires in-depth understanding of their mechanisms of biological action. Elucidation of both molecular features, essential for efficient ROS-scavenging in vivo, and factors limiting the potential side effects requires biologically relevant and, at the same time, relatively simple testing systems. This review discuses the advantages and limitations of genetically engineered SOD-deficient unicellular organisms, Escherichia coli and Saccharomyces cerevisiae as tools for investigating the efficacy and mechanisms of biological actions of SOD mimics. These simple systems allow the scrutiny of the minimal requirements for a functional SOD mimic: the association of a high catalytic activity for superoxide dismutation, low toxicity, and an efficient cellular uptake/biodistribution. Antioxid. Redox Signal. 20, 2416–2436.

Introduction

Excessive production of reactive species derived from oxygen is implicated in various pathological processes. Promising results with superoxide dismutase (SOD) preparations (9,47,81) stimulated an intense search for pharmacological agents that combine highly efficient detoxification of superoxide radical (O₂^•−) with minimal side effects. Advancement in understanding of the mechanisms of the SOD-catalyzed O₂^•− dismutation, and the fact that certain transition metal complexes are capable of substituting for the SODs, directed the efforts toward creation of functional, artificial SOD catalysts—SOD mimics. In a relatively short period, a variety of SOD mimics, often based on low-molecular-weight transition-metal complexes, were produced. Clinical trials, however, are limited mainly due to a lack of information about bioavailability, absorption, pharmacokinetics, toxicity, and biotransformation of such antioxidants. For many of them, it is not known whether they retain in vivo the activities assigned based on in vitro assays. The need to study in vivo the mechanisms of action of SOD mimics, and to predict which compounds might have potential medical applications, posed a strong demand for appropriate biological testing systems. Since all aerobic organisms inevitably generate superoxide, finding a suitable biosystem may appear easier than it actually is. Numerous articles report beneficial effects of SOD mimics in animal models of diseases and pathological conditions having oxidative stress as a common mechanism [for recent reviews, see (11,16,86,120)]. However, higher eukaryotic organism-based models are too complex, and biological activity of any studied agent depends on complicated pharmacokinetics, tissue gradient distribution, and others. In addition, the compound can be modified or degraded, obscuring the benefits that could arise from the superoxide dismuting ability of the potential SOD mimic itself. Since most SOD mimics display a variety of activities and do not selectively react with O₂^•−, reported beneficial effects can be attributed to actions other than superoxide scavenging (54). Similar shortcomings apply to cell culture systems in which O₂^•−, along with other reactive oxygen species (ROS), are generated by the addition of chemical agents (84,101), enzymatically (xanthine plus xanthine oxidase) (84), by radiation exposure (110); by activation of macrophages (33), and so on. The lack of a clear understanding of both the nature of the ROS responsible for damage and the mechanisms of action of the tested compounds can be listed among the reasons for failures in clinical trials (8,23,38,44,52,76,109). The use of so-called “SOD mimics” with low superoxide-scavenging activity casts doubt on mechanistic studies in which such compounds are applied “to prove” O₂^•− involvement.

At least some of the limitations of the more complicated systems listed earlier can be eliminated by simple, unicellular prokaryotic and eukaryotic organisms in which a high steady-state intracellular O₂^•− concentration can be maintained by aerobic metabolism, and where the effects of superoxide can be relatively easily quantified. The advantages of such microbial systems for studying the consequences of oxidative stress, elegantly summarized in a recent review (62), apply equally to the study of SOD mimics. Among such advantages are the selection of medium components to manipulate metabolic pathways, ability to grow under anaerobic as well as aerobic conditions, sufficient knowledge about pathways that are sensitive to O₂^•−, and, most importantly, genetic manipulations producing mutants lacking specific ROS-scavenging enzymes.

Irrespective of their relative simplicity, even such unicellular organisms possess subcellular structures and metabolic activities that can noticeably modulate the bioefficacy and the mode of action of the tested SOD mimics. Similar to the multicellular eukaryotes, these simple organisms are well protected by SOD enzymes, which catalyze O₂^•− dismutation with a diffusion limited rate and, thus, overwhelm the action of any externally added SOD scavenger. This problem was solved by the use of mutants lacking SOD enzymes. The first such organism to be isolated was the unicellular eukaryote, Saccharomyces cerevisiae (34) followed by a prokaryote, Escherichia coli (35).

The SOD-Deficient Mutants

The SOD-deficient E. coli

The facultative anaerobe E. coli has three SOD isozymes. MnSOD and FeSOD are cytoplasmic and are encoded by the sodA and sodB genes, respectively (62,119). The third isozyme, a CuZnSOD, encoded by sodC, is periplasmic and is induced when bacteria enter a non-growing state, called stationary phase (25,32). In 1986, Carlioz and Touati (35) reported the construction of a sodA sodB mutant lacking the cytosolic MnSOD and FeSOD. The mutant is indistinguishable from its SOD-replete parent if grown in the absence of oxygen, but it aerobically exhibits defects directly resulting from the lack of cytoplasmic SODs. The defects include slow aerobic growth even in media containing all the required nutrients (1,35) and an inability to grow on non-fermentable carbon sources or in the absence of sulfur-containing (31), branched-chain (35), and aromatic (30) amino acids. Absence of cytoplasmic SODs was also associated with leaky membranes (64), sensitivity to mild heat shock (28), hypersensitivity to hydrogen peroxide (H₂O₂) and redox-cycling agents (35), and a high rate of spontaneous mutagenesis (29,49). All these defects can be eliminated by the expression of active SOD (87,118), which implies that a compound acting as an artificial SOD enzyme should have a similar effect. In addition, the auxotrophy (inability to synthesize a particular organic compound required for growth) for branched-chain, sulfur-containing, and aromatic amino acids can be suppressed by spontaneous mutations producing pseudorevertants that are capable of growing in aerobic minimal medium without expressing cytoplasmic SODs (63,64) or by supplementation of the growth medium with manganese salts (1).

The SOD-deficient S. cerevisiae

The unicellular eukaryote, S. cerevisiae, is commonly used as a single-cell model for higher eukaryotic organisms. As in the majority of eukaryotes, the most abundant SOD in S. cerevisiae is a cytoplasmic CuZnSOD (SOD1). It is also found in the nucleus and mitochondrial intermembrane space. In addition, eukaryotes express an MnSOD (SOD2) in the mitochondrial matrix. Similar to human MnSOD, and in contrast to bacterial MnSODs, which are dimers, S. cerevisiae MnSOD is a tetramer (94). Yeast with a mutated mitochondrial MnSOD gene (sod2Δ) are sensitive to oxygen (124). Under aerobic conditions, such mutants cannot grow on non-fermentable carbon sources, but do not show defects when grown on glucose. Strains with mutations in the cytoplasmic CuZnSOD gene (sod1Δ) are much more affected and show multiple defects. Similar to sodA sodB E. coli, they exhibit poor growth in normoxic conditions, increased mutation rate, decreased stationary phase survival (75), and amino-acid auxotrophies for lysine and methionine (34,60,129). Similarities with sodA sodB E. coli also include suppression of all growth defects by supplementation of the growth medium with manganese (106) or by growth under anaerobic conditions. The defects of SOD1 mutants can be genetically reversed by mutations classified in two main groups. One group comprises mutations influencing transition metal ions homeostasis and includes the BSD1 and BSD2 (bypass SOD defects) genes (69). BSD1 is identical to the PMR1 gene, coding for a microsomal Ca²⁺ ATP-ase, which is eventually involved in the transport of other cations, including manganese. BSD1 mutants accumulate elevated levels of intracellular manganese and copper (69), and it is known that Mn²⁺ can form unstable complexes that are capable of catalyzing O₂^•− dismutation (7,10). The BSD2 gene codes for a protein that is important in copper ion transport and accumulation (74). Overexpression of another gene, ATX1, which is important in copper ion accumulation, also suppresses oxygen sensitivity of SOD-deficient yeast (70), which implies that Cu-containing complexes might play a protective role (77).

A second group consists of various mutations that suppress amino-acid auxotrophies without preventing hypersensitivity to oxidants (114).

The SOD-deficient E. coli and S. cerevisiae as useful tools for screening of SOD mimics

E. coli and S. cerevisiae strains containing deletions in the SOD genes could be considered very useful for the initial screening of potential SOD mimics. As mentioned earlier, the lack of SODs in such organisms entails growth deficiencies, which can be relieved by active SOD or by a molecule that is capable of functionally substituting for it. Since SOD-deficient mutants aerobically grow slowly and cannot grow if certain amino acids are absent in the growth medium, most often, the activity of SOD mimics is assessed by their effect on the growth of the mutants. Growth is easy to follow turbidimetrically, and the rates of growth of the SOD-deficient mutants with and without the tested mimic are compared with those of the SOD-proficient parent.

This section describes growth media and general procedures for storage, inoculation, and growth of E. coli and S. cerevisiae.

Escherichia coli

E. coli frozen stocks are prepared by snap-freezing aliquoted overnight Luria–Bertani (LB) cultures (described below) preincubated for 30 min at room temperature with glycerol at a final concentration of 30%. In procedures used by different laboratories, frozen stock cultures are initially grown in LB medium. All media for E. coli growth are adapted from Sambrook et al. (105). LB medium contains 10 g of bactotryptone, 5 g of yeast extract, 10 g of sodium chloride (NaCl), and 2 g of glucose per liter. M9CA medium consists of M9 salts (components are listed next), 0.2% casamino acids, 0.2% glucose, 3 mg pantothenate, and 5 mg of thiamine per liter.

Among the restricted media, minimal medium is most often used. It consists of M9 salts supplemented with 3 mg of pantothenate, 5 mg of thiamine, and 2 g of glucose per liter. Filter-sterilized L-histidine, L-leucine, L-threonine, L-arginine, and L-proline (to 0.5 mM each) are added to satisfy the genetic auxotrophies of strains derived from AB1157. This medium is referred to as a five amino acid (5AA) medium. M9 salts are prepared by autoclaving 0.6 g of disodium phosphate, 0.3 g of monopotassium phosphate, 0.05 g of NaCl, and 0.1 g of ammonium chloride per liter in distilled water. After cooling, separately autoclaved solutions of magnesium sulfate and calcium chloride are added to a final concentration of 1.0 mM.

Liquid cultures

According to the simplest procedure, parental and SOD-deficient E. coli frozen stocks are initially inoculated in liquid LB medium, supplemented with appropriate antibiotics for the SOD-deficient mutants.

The LB culture is grown on a shaking water bath (200 rpm) under aerobic atmosphere for 24 h at 37°C. If compounds are to be tested in M9CA medium, the LB overnight culture is diluted into the medium usually to A_600nm=0.005 (∼5×10⁶ cells/ml) [A₆₀₀ of 1.0=∼1×10⁹ cells/ml (85)]. If tests are carried out in a minimal/5AA medium, the cells are washed thrice with M9 salts to avoid transferring nutrients from LB to the minimal medium. The cells are then resuspended to A₆₀₀ ∼0.005 in 10–15 ml of minimal (or 5AA medium, depending on the genetic background of strains) and grown aerobically in 50 ml microbiological flasks.

As mentioned earlier, sodA sodB cultures tend to accumulate pseudorevertants that grow in aerobic minimal medium irrespective of the lack of cytoplasmic SODs (63). One way to suppress the growth of pseudorevertants is to grow the initial cultures under an anaerobic atmosphere (85% nitrogen, 10% hydrogen, and 5% carbon dioxide) in LB medium supplemented with 0.2% glucose (59). The overnight LB cultures are then diluted and grown overnight in anaerobic minimal medium. For testing SOD mimics, the anaerobic minimal cultures are diluted in minimal medium, and growth is followed under aerobic atmosphere.

Solid cultures

Another strategy for minimizing the overgrowth of suppressor mutants is to streak the frozen stocks on LB agar plates supplemented with 0.2% sucrose (64). In a procedure adopted by Munroe and coworkers (85), the agar plates are incubated in air for 24 h at 37°C. To ensure that the response is not specific for a particular clone, four independent colonies for each strain are each inoculated into 3.0 ml LB sucrose and grown overnight in air at 37°C and 220 rpm. The overnight 3.0 ml cultures are then inoculated in 15 ml LB-sucrose to A₆₀₀=0.01 (parental) or 30 ml LB-sucrose to A₆₀₀=0.02 (sodA sodB) in 50 ml culture flasks. After incubation for 2–3 h at 37°C and 220 rpm, the cells are washed thrice with minimal medium and resuspended in the same medium to the initial volume. Experimental cultures are prepared by diluting the cell suspension 1/10 with minimal or 5AA medium.

Growth is usually monitored turbidimetrically at 600 nm or if metalloporphyrins are tested, at 700 nm, in order to minimize the contribution of the absorbance of the compounds (50).

Until recently, test cultures were standardly grown in 50 ml flasks on a shaker at 200 rpm and 37°C, which limited the number of compounds that could be tested simultaneously. The use of 96-well microtiter plates, instead, has allowed simultaneous comparisons of a large number of compounds at a wide range of concentrations, but requiring minimal amounts of the tested compounds (85). The microtiter plates (100 μl of cell suspension per well) are usually shaken at 220 rpm in a 37°C incubator. Growth is measured by recording turbidity at 600 nm using a microtiter plate reader.

Saccharomyces cerevisiae

Using CuZnSOD-deficient S. cerevisiae as a biosensor for antioxidants was first proposed by Zyracka et al. (131). Three possible tests were listed, based on the estimation of lifespan, abolition of superoxide-induced auxotrophy, and growth in hypertonic medium. As with E. coli, a turbidimetric growth-based assay appears to be the easiest and the most often applied.

Two types of media are used for initial growth and for testing compounds: (i) YPD, a standard complete medium, contains 1% yeast extract and 2% peptone. Glucose (dextrose) is separately autoclaved and added to a final concentration of 2%. (ii) Synthetic (defined) dextrose medium (SD) is made of 1.8 g yeast nitrogen base (without amino acids and ammonium sulfate), 5.0 g ammonium sulfate, and 0.7 g monosodium phosphate. The pH is adjusted to 6.0 with 10 M sodium hydroxide. The SD medium is supplemented with glucose to 2%, and appropriate amino acids (SDC medium) (125).

For a routine pregrowth, the strains lacking CuZnSOD activity are cultured in microaerophilic conditions (5% oxygen) using CampyPaks (BBL) for plates or, for liquid cultures, 5 ml of medium in a 16×100 mm culture tube. For flask cultures, the low aeration is achieved by decreasing the surface-to-volume ratio and/or reducing the shaking rate to 100 rpm. High aeration for experimental samples is achieved by using a flask volume/medium volume ratio of 5:1 (typically 50 ml of medium in a 250-ml flask) and shaking at 200 rpm (41).

In the procedure described by Munroe et al. (85), 20% glycerol freezer stocks were streaked onto YPD agar plates and were grown under low oxygen at 30°C for 3 days. Pre-cultures were prepared by inoculating single colonies in 5 ml of SD medium in 16 ml culture tubes and growing them overnight at 30°C and 220 rpm. Experimental cultures were then inoculated at A₆₀₀=0.05 (∼5×10⁵ cells/ml; A₆₀₀=1.0 corresponds to ∼1×10⁷ cells/ml) in 10 ml liquid medium in 50 ml flasks and grown in air at 30°C and 220 rpm. Usually two to four independent colonies are used for each experiment.

In general, slight variations in the procedures used by different laboratories practically do not affect experimental outcomes, and if all precautions to avoid artifacts are taken into consideration, reported results about the efficacy of the tested redox-active compounds are consistent.

The Physico-Chemical Properties and the Biological Effects of SOD Mimics

The requirements for a good SOD mimic

A list of requirements for a compound to mimic SOD in vivo can be found in a publication by Czapski and Goldstein (40). Theoretically, an SOD mimic should exert protective effects by scavenging O₂^•− before it reacts with [4Fe-4S] clusters (71) or with other cellular targets (55). The efficiency of this process would depend on the concentration of the SOD mimic, its specificity (39), and the rate constant for reaction with O₂^•−. Keeping in mind that the SOD mimic should outcompete biological superoxide targets and should complement endogenous SOD enzymes, it becomes clear that the SOD mimic should react with O₂^•− with a rate constant comparable to that of the SOD enzymes and should be present at comparable concentrations. This is not easily achievable, because SOD-catalyzed dismutation occurs at a rate of >10⁹ M⁻¹ s⁻¹ (56) and intracellular SODs reach a concentration of 10–20 μM (62).

Alternatively, if catalytic activity is lower, the SOD mimic should accumulate at proportionally higher concentrations at the site of action. This, in turn, implies that in addition to catalytic activity, information about uptake and subcellular distribution of SOD mimics is essential for proper evaluation of their action. Caution should be exercised in interpreting data when compounds with low reactivity toward O₂^•− exert noticeable protection. Unless the tested compound preferentially accumulates at the sites of O₂^•− production, its effects are most probably due to actions other than O₂^•− scavenging.

Most of our current knowledge about the mechanisms of action and potential pitfalls in applying SOD mimics in vivo has been obtained using unicellular SOD-deficient organisms as a model system.

The mechanism of action of Mn porphyrins in SOD-deficient E. coli: preliminary insights

Faulkner et al. (50) were the first to use sodA sodB E. coli for determining the biological activity of several Mn porphyrins (MnPs), including MnTM-4-PyP⁵⁺ and MnTBAP³⁻ [Mn(III) meso-tetrakis(4-benzoic acid) porphyrin, also known as MnTCPP³⁻ and AEOL10201)] (Figs. 1 and 3). These researchers made the important observation that intracellular MnTM-4-PyP⁵⁺ looks greenish, suggesting that it exists in its reduced form, as Mn(II)TM-4-PyP⁴⁺ (50). This observation helped explain the in vivo mechanism of catalytic O₂^•− removal by MnP-based SOD mimics. Reduction of MnTM-4-PyP⁵⁺ by cellular reductants (some reaching millimolar intracellular concentrations) rather than by O₂^•−, avoids the rate-limiting first step of MnP-catalyzed O₂^•− dismutation [equation (1)]. Under such conditions, an MnP would act as superoxide reductase rather than SOD (50).

FIG. 1.

Chemical structures, superoxide dismutase activity (catalytic rate constant for O₂^•− dismutation, log k_cat), and Mn^III/Mn^II reduction potential (E_1/2 in mV vs. normal hydrogen electrode) of various cationic and anionic Mn porphyrins.

FIG. 3.

Chemical structure, Mn^III/Mn^II reduction potential (E_1/2 in mV vs. normal hydrogen electrode), and SOD activity (catalytic rate constant for O₂^•− dismutation, log k_cat) of ortho, meta, and para isomers of MnTMPyP⁵⁺.

The importance of the growth medium

Since SOD deficiency slows the multiplication of microorganisms even in media containing all needed nutrients (35), the simplest way to test a potential SOD mimic would be to determine whether it restores the growth rate of the mutants to that of the SOD-replete parents. Evaluating potential SOD mimic in complete medium, however, poses a risk of erroneously taking for an SOD mimic a compound that stimulates the growth by an action other than catalytic scavenging of O₂^•−. In complete (M9CA) medium, MnTBAP³⁻ accelerated the aerobic growth of the sodA sodB E. coli (50). MnTBAP³⁻, however, has neither the thermodynamic nor kinetic properties needed to act as an SOD mimic (98) and does not help the aerobic growth of SOD-deficient E. coli in medium lacking aromatic, sulfur-containing, and branched-chain amino acids, such as 5AA medium (Fig. 2) (13). The lack of superoxide scavenging activity of MnTBAP³⁻ has been confirmed when tested on sod1Δ S. cerevisiae (85). These results point to the importance of using restricted medium for growth of sodA sodB E. coli when evaluating the potential of an SOD mimic.

FIG. 2.

Cell density (based on A₆₀₀) of SOD-deficient Escherichia coli (JI132) after 20 h of growth. Cultures were grown in the presence of 20 or 200 μM MnTE-2-PyP⁵⁺ and MnTBAP^3– in five amino acid medium. The A₆₀₀ of parental (SOD-proficient, AB1157) E. coli strain was set at 100%. Adapted from (13). SOD, superoxide dismutase.

Ortho, meta, and para Mn(III) N-alkylpyridylporphyrins

The importance of appropriate growth medium for assessing SOD mimics was further supported when the effects of ortho, meta, and para isomers of MnTMPyP⁵⁺ [MnTM-2(or 3, or 4)-PyP⁵⁺] were compared in cultures grown in different media (12) (Figs. 3 and 4). In M9CA medium, all three isomers stimulated the aerobic growth of the sodA sodB strain. Growth in 5AA medium, however, revealed clear differences among the isomers; the ortho isomer was the most efficient in stimulating the growth of the SOD-deficient mutant, the meta isomer was similar, while the para isomer did not show any beneficial effect (Fig. 4) (12). Among the reasons for the lack of beneficial effect of the para isomer in vivo is the intercalation of its more planar molecule (relative to the ortho and meta isomers) in DNA, leading to toxicity and loss of SOD activity (50).

FIG. 4.

Aerobic growth of SOD-deficient E. coli (JI132) in five amino acid medium in the absence or presence of 25 μM ortho MnTM-2-PyP⁵⁺, meta MnTM-3-PyP⁵⁺, and para MnTM-4-PyP⁵⁺ isomers. Growth was monitored turbidimetrically at 700 nm. The parental strain AB1157 was used as a control. Adapted from (12).

Does catalytic activity predict the biological activity?

Since high catalytic activity [k_cat(O₂^•−)] of a potential SOD mimic is considered the main factor contributing to its biological efficacy, efforts were directed toward bringing the k_cat(O₂^•−) values of MnPs closer to the k_cat(O₂^•−) values of the SOD enzymes. Testing the effect of such compounds on sodA sodB E. coli, however, revealed that a high k_cat(O₂^•−) does not necessarily translate into a high biological efficacy. To increase the catalytic activity of MnP-based SOD mimics, the pyrrolic moiety was derivatized with electron-withdrawing groups (e.g., Br and Cl), which stabilized Mn in the +2 oxidation state, instead of the +3 state usually found in the non-derivatized analogs. The electron-deficient ligand produced by such modification cannot support the higher Mn +3 oxidation state. Thus, no oxidation occurs on metallation of the ligand with manganese(II) chloride (MnCl₂), and Mn complex bearing Mn in the +2 oxidation state was isolated. With β-octabrominated MnBr₈TM-4-PyP⁴⁺ [log k_cat≥8.67, E_1/2=+480 mV vs. normal hydrogen electrode (NHE)] (14) and MnBr₈TM-3-PyP⁴⁺ (log k_cat≥8.85, E_1/2=+468 mV vs. NHE) (42), catalytic activity and reduction potentials comparable to those of the SOD enzymes (log k_cat=8.84–9.30, E_1/2+300 mV vs. NHE) were achieved (42). When tested on sodA sodB E. coli, however, MnBr₈TM-3-PyP⁴⁺ demonstrated a concentration-dependent protection with maximum growth rate (∼50% of that of the parent) reached at 1.0 μM (Fig. 5) (42). Higher concentrations were toxic. The para isomer was toxic even at 1 μM, and the maximum effect achieved at 0.5 μM was only ∼30% growth compared with the parental strain (Fig. 5). The reason for such low efficacy in vivo can be found in the very low stability constant (K) of these perbrominated MnPs (log K=8 for MnBr₈TM-4-PyP⁴⁺). Consequently, they rapidly decompose, presumably generating toxic products. These results highlight the fact that the high catalytic activity for O₂^•− dismutation is not a sufficient predictor of biological efficacy. Stability of the compound, its cellular uptake, subcellular distribution, and biological transformations are factors that could override the impact of k_cat.

FIG. 5.

Effect of β-brominated Mn(II) N-alkylpyridylporphyrins and their metal-free ligands on the growth of SOD-deficient E. coli in five amino acid medium. Cell density at the 13th hour of growth is presented. Adapted from (42).

The accumulation of MnPs by E. coli and S. cerevisiae

In general, the main features that determine the uptake and subcellular distribution of an externally added compound are (i) charge, (ii) shape and size, and (iii) lipophilicity of the molecule.

Charge

Comparison between anionic [MnT(2,6-Cl₂-3-SO₃-P)P³⁻, MnTSPP³⁻, and MnTBAP³⁻] and cationic [MnTrM-2-PyP⁴⁺, MnTE-2-PyP⁵⁺, MnT(TFTrMA)P⁵⁺, and MnT(TrMA)P⁵⁺] MnPs (Fig. 1) revealed that positive charge is essential for biological activity (19). Cellular/mitochondrial uptake of positively charged molecules is driven by the membrane potential, which thermodynamically favors their accumulation to levels exceeding those of the surrounding medium. The importance of positive charges is illustrated by the fact that none of the anionic compounds could substitute for the missing cytoplasmic SODs in E. coli. Among the cationic MnPs tested, MnTM-2-PyP⁵⁺ (Fig. 3) and MnTE-2-PyP⁵⁺ (Fig. 1), with an E_1/2 close to the potential of the SOD enzyme of +300 mV, accelerate the aerobic growth of the sodA sodB mutant to a rate typical for the SOD-containing parent (Fig. 6). These two cationic MnPs with a log k_cat(O₂^•−) ∼7.8 remain the most active and the least toxic, and are among the most studied MnPs in animal models of oxidative stress (21,22,43,108). They are commonly used as a positive control in SOD-deficient E. coli and S. cerevisiae studies. Anionic MnPs of low metal-centered reduction potentials (insufficiently redox-active) such as MnTBAP³⁻ (log k_cat=3.16 and E_1/2=−194 mV vs. NHE), which repel the anionic O₂^•−, cannot act as SOD mimics and do not protect the SOD-deficient E. coli (Fig. 2). Therefore, the therapeutic efficacy of MnTBAP³⁻ is likely based on a mechanism other than O₂^•− scavenging (13).

FIG. 6.

Effect of 25 μM ortho (2) and meta (3) isomeric Mn(III) N-alkylpyridyl porphyrins (alkyls being methyl to butyl) on the aerobic growth of SOD-deficient E. coli (JI132) in five amino acid medium. Growth was monitored turbidimetrically at 700 nm. Cell density at 18 h is presented. Adapted from ref. (66).

Shape and size of the molecule

The three-dimensional structure of the molecule exerts a strong influence on the biological efficacy of the SOD mimics. For example, the k_cat(O₂^•−) of the meta isomers (alkyl side chain varying from methyl to octyl) is an order of magnitude lower than k_cat(O₂^•−) of the corresponding ortho isomers, and their redox potentials differ significantly. E_1/2 of the meta MnP compounds falls in the region of 52–74 mV versus NHE, while for the ortho analogs, it is in the range from 220 to 367 mV versus NHE, depending on the nature of the alkyl side chains (11,122). Nevertheless, both ortho and meta analogs efficiently substituted for SOD when tested on sodA sodB E. coli (Fig. 6). In contrast, Mn(III) N,N′-disubstituted imidazolium analogs (MnTDE-2-ImP⁵⁺, MnTM,MOE-2-ImP⁵⁺, and MnTDMOE-2-ImP⁵⁺, Fig. 7) despite having lipophilicity (R_f) comparable to and a k_cat similar to or even higher than that of their N-substituted pyridyl analogs, MnTM-2-PyP⁵⁺ and MnTE-2-PyP⁵⁺, were biologically much less efficient (Fig. 8). Such low bioefficacy could be attributed to the bulkiness of the molecule due to the N,N′-imidazolyl substituents located above and below the porphyrin plane (19), which hinders the diffusion across the membranes and prevents a sufficient cellular uptake.

FIG. 7.

Structures, Mn^III/Mn^II reduction potential (E_1/2 in mV vs. normal hydrogen electrode), and SOD-like activity (catalytic rate constant for O₂^•− dismutation, log k_cat) of Mn(III) N,N′-disubstituted imidazolium porphyrins: MnTDE-2-ImP⁵⁺, MnTM,MOE-2-ImP⁵⁺, and MnTDMOE-2-ImP⁵⁺. The imidazolium compounds have two imidazolyl substituents, one placed below and the other placed above the porphyrin ring, which makes them bulkier than N-substituted pyridylporphyrins (19).

FIG. 8.

Growth of SOD-deficient E. coli in five-amino-acid medium in the presence of 25 μM Mn(III) N,N′-disubstituted imidazolium porphyrins: MnTDE-2-ImP⁵⁺, MnTM,MOE-2-ImP⁵⁺, and MnTDMOE-2-ImP⁵⁺. Their N-substituted pyridyl analog, MnTE-2-PyP⁵⁺, was also tested for comparison. Adapted from (88).

Lipophilicity

Investigations of the relationship between MnP lipophilicity, influenced by the length of the aliphatic chains attached to the meso pyridyl nitrogen atoms at the porphyrin ring, and biological activity, have demonstrated that more lipophilic SOD mimics (MnTnHex-2-PyP⁵⁺ and MnTnOct-2-PyP⁵⁺) are efficacious at lower concentrations (0.1–3.0 μM); whereas less lipophilic compounds, MnTM-2-PyP⁵⁺, MnTE-2-PyP⁵⁺, and MnTnPr-2-PyP⁵⁺, are highly efficient only at concentrations above 10 μM (Fig. 9) (88). Since all tested SOD mimics have similar log k_cat(O₂^•−) values, the higher biological activity of the more lipophilic compounds could be attributed to more efficient cellular uptake resulting from facilitated diffusion across membranes.

FIG. 9.

Aerobic growth of SOD-deficient E. coli (JI132) in the presence of 25 μM Mn(III) meso-tetrakis(N-alkylpyridinium-2-yl)porphyrins, where the alkyl group is methyl (MnTM-2-PyP⁵⁺), ethyl (MnTE-2-PyP⁵⁺), n-propyl (MnTnPr-2-PyP⁵⁺), n-hexyl (MnTnHex-2-PyP⁵⁺), or n-octyl (MnTnOct-2-PyP⁵⁺) in restricted (5AA) medium. Data from 14 h of growth are shown. MnTnHex-2-PyP⁵⁺ and MnTnOct-2-PyP⁵⁺ were toxic at a 25 μM concentration but supported the growth of E. coli when supplied at a concentration as low as 0.3 μM. Adapted from (88).

Figure 10A shows that those MnPs which have high catalytic rate constants for O₂^•− dismutation (>10⁷ M⁻¹·s⁻¹) and protect the SOD-deficient mutants against O₂^•−, accumulate in cells to concentrations that are comparable to or exceed those of the native SOD. The figure also shows that cellular accumulation of the most lipophilic Mn-hexyl derivatives is ∼20–30-fold higher than the accumulation of the hydrophilic Mn-methyl derivatives, which explains why amphiphilic MnPs are efficient at concentrations lower than 1.0 μM.

FIG. 10.

Relationship between lipophilicity and cellular uptake of MnPs. (A) Accumulation of ortho and meta isomeric Mn(III) meso-tetrakis(N-alkylpyridyl)porphyrins in the cytosolic fractions of wild-type E. coli (AB1157) after 1 h of incubation with 5 μM Mn porphyrin (MnP) in M9CA medium. Bars represent mean±SE. (B) Lipophilicity of ortho and meta isomeric MnPs defined by chromatographic retention factor, R_f. The R_f depends on the number of carbon atoms in alkylpyridyl chains of MnPs and correlates well with partition coefficient between n-octanol and water, P_ow (66,67). (C) Effect of ascorbate on MnP uptake. Wild-type E. coli (AB1157) was incubated for 1 h at 37°C in M9CA medium containing 5 μM Mn(III) meso-tetrakis(N-alkylpyridyl)porphyrins with or without 1 mM sodium ascorbate. Bars represent mean±SE. SE, standard error.

These findings helped formulate the key requirements that should be met by the compounds expected to act as SOD mimics in vivo: high k_cat(O₂^•−), high stability, and efficient cellular uptake.

Investigations of the relationship between MnPs lipophilicity and their cellular uptake and distribution, carried out with E. coli and S. cerevisiae, revealed the general rules that are applicable to higher organisms: (i) Longer aliphatic chain analogs with lipophilic properties accumulate in cytosol and membranes to higher levels than more hydrophilic molecules. Meta isomers, which are more flexible and more lipophilic (Fig. 10B), cross cell membranes easier than their respective ortho analogs, and are found in the cytosol at higher concentrations (Fig. 10A); and (ii) in vivo, a lower k_cat for O₂^•− dismutation can be compensated for by better cellular uptake of the SOD mimic, resulting in higher efficacy (Fig. 6) (66). Compensation for low catalytic activity by higher uptake, however, is limited by potential toxicity of the SOD mimics.

Cellular uptake of MnPs could be significantly influenced by the components of the growth medium. Reducing compounds, such as ascorbate, could significantly enhance the uptake of MnPs (Fig. 10C) (111). Reduction of Mn^IIIP to Mn^IIP by ascorbate leads to the loss of a single charge at the metal center, which decreases the overall charge of the molecule from +5 to +4 and increases the lipophilicity of the metalloporphyrin. The importance of the metal center reduction is illustrated by the fact that the uptake of meta isomeric MnP, MnTM-3-PyP⁵⁺ was not affected by ascorbate. MnTM-3-PyP⁵⁺, with a reduction potential of only 52 mV is much less reducible than the ortho analog, MnTM-2-PyP⁵⁺ with E_1/2 +220 mV versus NHE. While the ortho isomer exists as Mn^IITM-2-PyP⁴⁺ in biological milieu, the meta isomer remains unchanged, as a pentacationic species. Based on such observations, one could predict that in higher organisms, cellular/subcellular accumulation and distribution of SOD mimics may be strongly affected by endogenous reductants, some of which reach millimolar intracellular levels.

Studies on the subcellular localization of SOD mimics revealed similarities among different classes of organisms. A positive correlation between the lipophilicity and accumulation in mitochondria relative to the cytosol was observed in yeast cells incubated with a series of MnP analogs. For the least lipophilic compound, MnTM-2-PyP⁵⁺, the mitochondria to cytosol ratio was ∼1.5; while for the much more lipophilic MnTnHex-2-PyP⁵⁺, the ratio was >10, that is, the concentration was 10-fold higher in the mitochondria than in the cytosol. Compounds of intermediate lipophilicity, MnTE-2-PyP⁵⁺ and MnTnBu-2-PyP⁵⁺, showed intermediate distribution, with ratios of around 2 and 4, respectively (Li et al., unpublished observation) (112). A similar distribution was found when MnPs were administered to mice. The mitochondria-to-cytosol ratio was 3.6 for MnTnHex-2-PyP⁵⁺ and 1.6 for MnTE-2-PyP⁵⁺ (126). The reason for such similarities lies in the identical forces and principles that govern cellular transport. Uptake of cationic metalloporphyrins by E. coli and mitochondria of eukaryotic cells is driven by the electrochemical proton gradient across membranes and is facilitated by the amphiphilic properties of the molecule. Depending on the alkyl chain length, MnPs accumulate more in membranes or in cytosol; the longer alkyl-chain analogs tend to accumulate more in membranes than in cytosol (66).

The toxicity of porphyrins

Toxicity is the main factor restricting the use of metalloporphyrin-based SOD mimics. Studies with various classes of organisms have shown that bacteria are much more sensitive to the toxicity of metalloporphyrins than other organisms, including the yeast S. cerevisiae.

Light-independent porphyrin toxicity to bacteria is well known, and has been proposed as a treatment modality against antibiotic-resistant strains (113). Several possible mechanisms of porphyrin toxicity have been suggested: interference with redox reactions, generation of ROS, distortion of the membrane lipid bilayer, and insertion of a non-functional heme-like porphyrin in heme-containing proteins, thus blocking their functions (113).

E. coli responds to oxidative stress by inducing specific regulons, which, in turn, prompt the expression of hundreds of genes [reviewed in details in (37,62)]. Activation of this response can serve as an indication of toxicity due to the reversed action of the tested SOD mimics, that is, pro-oxidative instead of antioxidative. For example, increased production of ROS explains the toxicity of some MnPs when they are combined with natural reductants. Such MnPs can be easily reduced by ascorbate, tetrahydrobiopterin, or glutathione (18,21,130) and can be subsequently reoxidized by either O₂^•− or dioxygen, generating H₂O₂ as an ultimate product. Reductants, usually present in all cells, can sustain the redox cycling, generating toxic levels of H₂O₂ (Fig. 11A) (15). E. coli reacts to the combination of MnP with ascorbate by inducing members of the oxyR regulon, including catalase and peroxidases (Fig. 11C, D), which is evidence for H₂O₂ production. The importance of this cellular response is illustrated by the fact that an oxyR-deficient mutant, unable to induce protection against H₂O₂, was highly sensitive to the combination of MnP and ascorbate; in the absence of ascorbate, the MnP did not cause any deleterious effects (Fig. 11B). The toxicity of the Mn(III) salen EUK-8 has also been attributed to the increased production of O₂^•− and H₂O₂ (78).

FIG. 11.

Ascorbate-dependent toxicity of MnTE-(2 and 3)-PyP. (A) Suppression of E. coli growth by MnTE-2-PyP plus ascorbate (M9CA medium) (B). Growth of oxyR-deficient E. coli mutant in the presence of MnTE-2-PyP plus ascorbate (C, D); Induction of catalase and peroxidase. Adapted from ref. (15).

The E. coli-based discovery of MnP-ascorbate cytotoxicity (92) brought up the idea that the combination can be used for anticancer treatment (48,95,117,130). The therapeutic potential of pharmacological doses of ascorbate alone has already been demonstrated in several preclinical and clinical trials (46,61,83,127) and is based on the increased production of reactive species due to ascorbate oxidation, catalyzed by endogenous metalloproteins (36). MnPs, however, are advantageous, as their redox properties can be optimized in order to achieve the maximal rate of ascorbate oxidation. Consequently, a maximal production of H₂O₂ and other reactive species can be achieved, causing a more efficient tumor destruction. Promising results of such combinational catalytic therapy have been reported (48,95,130). In vitro experiments have shown that some cancer cell lines are more sensitive to either cationic MnPs or Fe porphyrins (FePs) alone and to the MnP-ascorbate combination than are normal cells (121). Among the reasons is preferential accumulation of porphyrins in neoplastic cells (51,72).

Toxicity of amphiphilic SOD mimics could be explained by the membrane damage due to the detergent-like action. Such action, however, requires a high SOD mimic/lipid ratio (107) that cannot be achieved at the concentrations usually used. Further, no signs of membrane damage were detected when amphiphilic metalloporphyrins were tested at concentrations of approximately 50 μM (Benov et al., unpublished observation). Since such compounds accumulate in cells to much higher concentrations than their hydrophilic analogs, their higher toxicity most probably results from blocking proteins/enzymes that require heme for their function (113).

Some toxicity of metalloporphyrin-based SOD mimics could be related to the light-dependent generation of ROS, mainly singlet oxygen, as demetallation of these compounds in the cytoplasm yields a photosensitizing metal-free porphyrin ligand in situ. Such compounds show much lower toxicity in the dark than when cultures are illuminated (96,121). Of note, MnPs and FePs are not photosensitizers per se, whereas their corresponding free ligands or the Zn analogues (ZnPs) have been explored as potent photosensitizers for photodynamic therapy (2–4,24,26,27).

Depending on the nature of the metalloporphyrin, its concentration, and the environment, including growth media and cellular reductants, different mechanisms can eventually combine, leading to cell damage.

The strategies for decreasing the MnP toxicity

As mentioned earlier, the bioefficacy of amphiphilic MnP-based SOD mimics is limited by their higher toxicity. Further efforts have been directed toward decreasing the toxicity of SOD mimics while preserving the high SOD activity and lipophilicity. High catalytic activity was maintained by preserving the ortho cationic pyridyl nitrogens that dominate the thermodynamics and kinetics of metalloporphyrins-catalyzed O₂^•− dismutation. Previous investigations have shown that replacement of a CH₂ group in the butyl chain of MnTnBu-2-PyP⁵⁺ with an oxygen atom diminishes its toxicity, but at the expense of decreased lipophilicity and, consequently, decreased cellular accumulation (20). Applying such a strategy to MnTnHex-2-PyP⁵⁺ led to the synthesis of its methoxy analog, MnTMOHex-2-PyP⁵⁺. Its lipophilicity was, indeed, much lower than that of MnTnHex-2-PyP⁵⁺. Since meta isomers are more lipophilic than their ortho analogs (Fig. 10B), meta isomers of hexyl and ethyl species were synthesized and examined using sodA sodB E. coli (123). Both MnTMOHex-3-PyP⁵⁺ and MnTMOE-3-PyP⁵⁺ appeared more efficacious than MnTnHex-3-PyP⁵⁺ and MnTE-3-PyP⁵⁺ in supporting the aerobic growth of SOD-deficient E. coli (Fig. 12).

FIG. 12.

Comparison of the efficacy/toxicity profiles of methoxy-derivatized cationic Mn(III) N-substituted pyridyl porphyrins (MnTMOE-3-PyP⁵⁺ and MnTMOHex-3-PyP⁵⁺) with their respective alkyl analogs (ortho MnTE-2-PyP⁵⁺, MnTnHex-2-PyP⁵⁺ and meta MnTE-3-PyP⁵⁺, MnTnHex-3-PyP⁵⁺). Growth of SOD-deficient E. coli strain (JI132) in five amino acid medium was followed turbidimetrically at 600 nm (123). Cell density at 16th hour of growth is shown. The meta ethyl has similar R_f as meta methoxyethyl, and the meta hexyl has R_f similar to meta methoxyhexyl (inset) (123).

In an attempt to preserve the lipophilicity of alkoxyalkylated porphyrins, the oxygen atoms were pushed deeper into the cavity encircled by the N-alkylpyridyl substituents. The reduced oxygen exposure to solvent prevented its solvation, and, thus, preserved the lipophilicity of the oxygenated derivatives. Such an optimized molecule, Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP⁵⁺) was as lipophilic as its non-derivatized seven-carbon chain alkyl analog, MnTnHep-2-PyP⁵⁺ and stimulated the aerobic growth of SOD-deficient S. cerevisiae (Fig. 13). At concentrations 5–30 μM, MnTnBuOE-2-PyP⁵⁺ improved the aerobic growth of the sod1Δ yeast strain, whereas MnTnHep-2-PyP⁵⁺ was toxic even at 5 μM (Fig. 13). The applicability of these results to higher organisms was tested in a mouse toxicity study, where MnTnBuOE-2-PyP⁵⁺ was much less toxic than either MnTnHex-2-PyP⁵⁺ or MnTnHep-2-PyP⁵⁺ (93,123) [reviewed in detail in (120)].

FIG. 13.

Growth of wild-type (EG 103) and SOD-deficient (EG118, sod1Δ) Saccharomyces cerevisiae strains in the presence of MnTnHep-2-PyP⁵⁺ and MnTnBuOE-2-PyP⁵⁺ (1, 5, 10, or 30 μM). Cultures were grown in YPD medium, and growth was monitored turbidimetrically at 600 nm. Adapted from ref. (93).

The SOD mimics and E. coli growth curves

The growth pattern of most microorganisms in a closed habitat can be divided in three distinct phases: lag phase, during which the organisms adapt to the new environment and do not grow; exponential phase, where cell number increases in geometric progression; and stationary phase, where growth practically stops due to the lack of nutrients, accumulation of inhibitory products, or limit of space (90). Important information about the mechanism of action of tested compounds can be obtained by analyzing the kinetics of microbial growth. The significance of analyzing growth curves has been illustrated using the FeP-based SOD mimics. Para cationic Fe(III) N-methylpyridylporphyrin, FeTM-4-PyP⁵⁺ reported in the late 1970s was the first metalloporphyrin to possess a high catalytic rate constant for O₂^•− dismutation [log k_cat(O₂^•−)=7.20)] (89). Based on the ortho-driven design of N-alkylpyridylporphyrins, FePs FP-15, WW-85, and INO-4885 have been synthesized. Beneficial effects of such FePs have been reported in various pathological conditions, including spinal cord injury, burn and smoke inhalation injury, septic shock, diabetes, and so on (57,68,79,80,91,115,116). The effect was attributed to the catalytic decomposition of peroxynitrite by FePs. Since Fe(III) N-alkylpyridylporphyrins have SOD activity that is very similar to their Mn analogs, as indicated by their log k_cat(O₂^•−) values (19,122), the in vivo FeP-based protection could have ensued from both peroxynitrite decomposition and superoxide dismutation (17,120). Initially, the Fe(III) N-alkylpyridylporphyrins were tested on the sodA sodB E. coli at a concentration optimal for the MnP analogs, 25 μM. Under such conditions, neither cationic nor anionic FePs (FeTM-2-PyP⁵⁺, Fe^IIITE-2-PyP⁵⁺, Fe^IIIT(TMA)P⁵⁺, Fe^IIITCPP³⁻, Fe^IIIT(TFTMA)P⁵⁺, and Fe^IIITSPP³⁻) afforded protection; moreover, they acted as bacteriostatics to both SOD-deficient and SOD-proficient E. coli under aerobic and anaerobic conditions (19) (Fig. 14). At much lower concentrations, 0.01–1.0 μM, FePs stimulated the growth of the sodA sodB E. coli (Fig. 15). With FePs, however, the sodA sodB growth pattern was different than that of the SOD-containing and MnP-stimulated SOD-deficient cultures. If a compound substitutes for the SOD enzymes, then the SOD-deficient mutants should follow a growth pattern similar to that of the SOD-containing strains. As mentioned, both sodA sodB E. coli and sod1Δ S. cerevisiae can accumulate mutations that suppress the aerobic defects, including the auxotrophies for amino acids (63,69). After prolonged incubation in restricted media, the SOD-deficient strains acquire the ability to overcome the auxotrophies and to restore growth without producing functional SODs. This is exhibited as a growth curve with a long lag followed by slow exponential growth. Some candidates for SOD mimics are able to act at this late stage and accelerate the growth rate of the sodA sodB cultures, without shortening the length of the lag phase. The growth stimulatory effect of FePs shown in Figure 15 could not be attributed to their SOD activity for two reasons: (i) Since FePs and MnPs have similar values of k_cat(O₂^•−), the effect of FePs should have occurred at a concentration range at which analogous MnPs are efficacious. (ii) The growth curve of sodA sodB+FeP should be similar to the growth curve of parental E. coli. The data (122) indicate that at such low concentrations, FePs act in an identical way as simple Fe salts, thus supporting metabolism by providing Fe as an Fe source.

FIG. 14.

Comparison between MnPs and FePs. (A) Growth of wild-type E. coli AB1157 in casamino acids (M9CA) medium in the presence of 25 μM of ortho (H₂O)MnTM-2-PyP⁵⁺, or ortho, meta, and para (OH)FeTMPyP⁴⁺ under anaerobic conditions. The other axial ligand in MnPs and FePs is water molecule. Identical data were obtained with the JI132 SOD-deficient strain (not shown); anaerobically with and without MnTM-2-PyP⁵⁺ the SOD-deficient strain grows as well as wild type. (B) Structures of ortho, meta, and para (OH)FeTMPyP⁴⁺ and ortho (OH)FeTE-2-PyP⁵⁺. (C) Effect of (H₂O)MnTM-2-PyP⁵⁺, (H₂O)MnTE-2-PyP⁵⁺, (OH)FeTM-2-PyP⁵⁺, and (OH)FeTE-2-PyP⁵⁺ at 25 μM on the aerobic growth of SOD-deficient E. coli JI132 in 5AA medium. A_700nm after 56 h of growth is shown. Adapted from ref. (19).

FIG. 15.

Growth of SOD-deficient JI132 and wild-type AB1157 E. coli in five-amino-acid medium in the presence of FePs and MnPs. FeP has essentially the same log k_cat(O₂^•−) as MnP (17). Adapted from (122).

The sodA sodB E. coli versus the sod1Δ S. cerevisiae

A good strategy for screening the potential of SOD mimics is to compare their effects on sodA sodB E. coli and on sod1Δ S. cerevisiae. This strategy was used to screen the representatives from different classes of SOD mimics (85). Even though some of the tested compounds reportedly ameliorated oxidative stress in animal model systems, investigations of their ability to substitute for the missing cytoplasmic SODs produced unexpected results; only the Mn N-alkylpyridylporphyrin complexes MnTM-2-PyP⁵⁺, MnTE-2-PyP⁵⁺, and MnTM-4-PyP⁵⁺, (the last one at low concentrations of 3 μM), stimulated the aerobic growth of SOD-deficient E. coli (85). MnTM-2-PyP⁵⁺ and MnTE-2-PyP⁵⁺ were also capable of supporting the aerobic growth of sod1Δ S. cerevisiae.

The two unicellular organisms responded differently to the same SOD mimic when other classes of compounds were studied. Thus, some compounds found to be efficient in rescuing the aerobic growth of the sodA sodB E. coli proved inefficient when tested in the sod1Δ yeast system. It has been hypothesized that such differences could be due either to the mislocalization or to the lack of sufficient concentration buildup of active species of these compounds in compartments where the radicals are present (85). For example, none of the Mn salen derivatives tested (Eukarion, EUK-134 and EUK-8) were efficient in sod1Δ yeast, but to a certain extent, improved the growth of sodA sodB E. coli (85). Since in Mn salen, chelated metal is weakly held (65), the growth stimulation could be due to Mn ions that dissociate from the ligands. It has been suggested that the protective action of Mn chelates could result from the facilitated import of Mn, which forms intracellular Mn complexes possessing SOD-like activity (1,6,7,10) and replaces the Fe in Fe-containing enzymes (5). The facilitated Mn transport may explain the beneficial effects of those SOD mimics that have low metal/ligand stability such as Mn salen and MnBr₈TSPP³⁻. Although MnBr₈TSPP³⁻ is a poor SOD mimic, as indicated by its low log k_cat(O₂^•−), its ability to protect SOD-deficient E. coli mirrored that of the potent mimic MnTM-2-PyP⁵⁺ (Fig. 16). It has been reported that in contrast to the stable MnP SOD mimics, which are found intact in the E. coli cytosol, cells loaded with MnBr₈TSPP³⁻ contained metal-free ligand (96). The following scenario was suggested to accommodate the spectroscopic and E. coli growth data: The Mn(III) complex is taken up by the cell and is reduced to its Mn(II) analog, which is then demetallated in situ, yielding Mn²⁺ and metal-free ligand. The nature of the resulting Mn²⁺ species inside the cell remains unknown, whereas the free ligand, exported out of the cell, is found in the medium. No demetallation was observed in a cell-free medium. Therefore, MnBr₈TSPP³⁻ was protecting the SOD-deficient cells not by scavenging superoxide but by facilitating the Mn import. A study using Criptococcus neoformans suggests that Mn salen (EUK-8) is also acting as a Mn carrier (58). A similar effect of Mn was reported for sod1Δ S. cerevisiae (106), which implies that growth stimulation by the unstable Mn-based compounds might simply reflect their ability to transport manganese.

FIG. 16.

Effect of anionic β-brominated 4-sulfonatophenylporphyrin and its 4-carboxylatophenyl analog on the aerobic growth of SOD-deficient E. coli (JI132) in 5AA medium (data at 24th hour time point is presented). MnTM-2-PyP⁵⁺ was used as a positive control. Results are presented as a percentage of the growth of the parental strain. Adapted from (96).

The purity of the tested compounds

Various commercial SOD mimics contain impurities, which can affect the outcomes of biological trials. Impurities are probably the main reason behind the contradictory reports about the bioefficacy of MnTBAP³⁻ (98,99). Impure preparations of MnTE-2-PyP⁵⁺ obtained from CalBiochem (containing ∼25% tetraethylated MnTE-2-PyP⁵⁺; the remaining ∼75% comprised tri-, di-, monoethylated, and non-ethylated derivatives) (99) may be a reason for the lower-than-anticipated efficacy of this SOD mimic, when tested on SOD-deficient E. coli and S. cerevisiae (85). Chemicals used in the preparation of SOD mimics, traces of which might remain in the final product, should also be tested for their biological activity. For example, neither tetra-n-butylammonium chloride nor ammonium hexafluorophosphate, used in the preparation of MnP-based SOD mimics caused any adverse effects when tested on E. coli (100) (Fig. 17). It is also important to quantify the amount of residual metal (Mn, Fe, etc.) present in the preparations (97), and to assess the biological effect of respective metal salts at matching concentrations.

FIG. 17.

Testing the effect of chemicals used for preparation of SOD mimics. Growth of SOD-deficient (JI132) and wild-type (AB1157) E. coli in the presence of: (A) TBACl in M9CA medium (Benov et al., unpublished), (B) NH₄PF₆ (100), in five amino acid medium. Both compounds are used in the isolation and purification of cationic MnPs and, thus, may be present as impurities in final preparation. The compounds have been tested at the following concentrations: (i) TBACl, 20 μM; (ii) NH₄PF₆, 60 μM; (iii) MnTM-2-PyP⁵⁺, 20 μM; and (iv) MnTE-2-PyP⁵⁺, 1 μM. TBACl, tetra-n-butylammonium chloride; NH₄PF₆, ammonium hexafluorophosphate.

The role of SOD-deficient E. coli and S. cerevisiae in distinguishing SOD mimics from non-SOD mimics

Various metal- or non-metal-based compounds (Fig. 18) were reportedly capable of suppressing oxidative stress in animal models: Mn(III) salen derivatives, such as EUK-8 (45,103); Mn(II) cyclic polyamine/azacrown ethers, such as M40403 (102); nitrones, such as NXY-059 (53); nitroxides, such as tempol (17,110); and MnTBAP³⁻ (73,128). When tested on SOD-deficient E. coli or S. cerevisiae, none of them acted as SOD mimics (Figs. 19 and 20). Such results are not unexpected, because the log k_cat(O₂^•−) of some of those compounds is lower than the rate constant of spontaneous O₂^•− dismutation (5×10⁵ M⁻¹·s⁻¹), while the others have a high log k_cat(O₂^•−) (M40403, EUK-8), but are unstable and eventually decompose and lose the redox-active metal. In a comprehensive study, Munroe and coworkers reported that EUK-8 and EUK-134 at concentrations of approximately 100 μM improved the growth of the sodA sod B E. coli, but the effect was similar to that of Mn(II)EDTA²⁻ or MnCl₂ (85). None of the two Mn salen compounds improved the growth of the sod1Δ S. cerevisiae (85). No positive results were reported for M40403 (85). Since all these compounds are redox active, they most probably act by mechanisms other than catalysis of O₂^•− dismutation (17,82,104).

FIG. 18.

Structures of various classes of compounds that have been tested as SOD mimics. Summarized in (17,82).

FIG. 19.

Growth of E. coli and S. cerevisiae in the presence of different redox-active compounds and SOD mimics. Wild-type AB1157 and SOD-deficient JI132 E. coli (A) were grown in five amino acid medium. Wild-type EG103 and SOD1-deficient EG118 S. cerevisiae strains (B) were grown in YPD medium. Growth was monitored turbidimetrically at 600 nm. Adapted from (13) (Benov et al., unpublished). Similar results were reported by Munroe et al. (85).

FIG. 20.

Effect of nitrone, NXY-059 on the growth of SOD-deficient E. coli (JI132) in five amino acid medium (Benov et al., unpublished). NXY-059 has been widely tested in animal models of oxidative stress with fair success, but failed in the clinical trials on stroke patients (44,109).

Concluding Remarks

Results obtained so far support the benefit of using SOD-deficient unicellular organisms for predicting the therapeutic potential of artificial SOD substitutes and for studying the mechanisms of action of redox-active compounds. The absence of superoxide-scavenging enzymes in these organisms offers an unambiguous background for testing in vivo the superoxide-scavenging capacity of SOD mimics. Such simple systems allow growth conditions and growth media to be manipulated, which is crucial for studying the mechanisms of action and, to some extent, the biological transformations of newly synthesized compounds. The relative simplicity of E. coli and S. cerevisiae is essential in the exploration of factors that affect accumulation and subcellular distribution of various types of compounds. Information on the impact of stability, charge, lipophilicity, size, shape, and bulkiness of molecules on the uptake and subcellular distribution of redox-active compounds could be obtained using these unicellular organisms. No less important, knowledge obtained with E. coli and S. cerevisiae could be successfully translated to higher organisms. Application of SOD-deficient E. coli and S. cerevisiae models proved that compounds with low catalytic rate constants (k_cat) for O₂^•− dismutation and unsuitable reduction potentials (E_1/2) cannot act in vivo as SOD mimics, and indicate that the beneficial effects observed in various animal model systems most probably result from activities other than the removal of O₂^•−. High catalytic constant, however, is not a sufficient predictor of an SOD mimic's bioefficacy. Stability, size, shape, charges, lipophilicity, and other intrinsic properties of the molecule, which affect cellular uptake, subcellular distribution, and biological transformations, are factors that can outweigh the impact of k_cat and should be tested on relevant biological systems before a compound is identified as an SOD mimic. It is important to keep in mind that irrespective of their apparent simplicity, the E. coli and S. cerevisiae microorganisms have complex physiology and metabolism, and interpretation of their responses to externally added compounds is not straightforward. Various factors, including composition of the growth media, aeration, physiological state of the initial inoculum, time of incubation, and even illumination, could affect cellular response and should be taken into consideration when results are analyzed. It becomes clear that a compound should not be recognized as an SOD mimic based on the results obtained only in one model system, and that a thorough investigation of biological actions other than superoxide scavenging should be carried out.

Abbreviations Used

5AA

five amino acid, minimal, restricted medium

E_1/2

half-wave reduction potential

EG118 and EG103

SOD-deficient and SOD-proficient Saccharomyces cerevisiae strains, respectively

EUK-134 and EUK-8

Mn salen derivatives

FeP

Fe porphyrin

FeTM-2-PyP⁵⁺

Fe(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin

FeTM-3-PyP⁵⁺

Fe(III) meso-tetrakis(N-methylpyridinium-3-yl)porphyrin

FeTM-4-PyP⁵⁺

Fe(III) meso-tetrakis(N-methylpyridinium-4-yl)porphyrin

FeTE-2-PyP⁵⁺

Fe(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin

FP-15

Fe (III) meso-tetrakis(N-(1-(2-(2(-2-methoxyethoxy)ethoxy)ethyl)pyridinium-2-yl) porphyrin

H₂O₂

hydrogen peroxide

INO-4885

Fe(III) meso-tetrakis[N-(4-carboxylatobenzyl)pyridinium-2-yl]porphyrin

JI132 and AB1157

SOD-deficient and SOD-proficient Escherichia coli strains, respectively

M40403

cyclic polyamine

M9CA

casamino acid complete medium Mn(III) meso-tetrakis(N-alkylpyridinium-2-yl)porphyrins, alkyl being methyl (M, MnTM-2-PyP⁵⁺⁺, AEOL10112), ethyl (E, MnTE-2-PyP⁵⁺⁺, AEOL10113, BMX-010), n-propyl (nPr, MnTnPr-2-PyP⁵⁺), n-butyl (nBu, MnTnBu-2-PyP⁵⁺), n-hexyl (nHex, MnTnHex-2-PyP⁵⁺), n-heptyl (nHep, MnTnHep-2-PyP⁵⁺), n-octyl (nOct, MnTnOct-2-PyP⁵⁺) Mn(III) meso-tetrakis(N-alkylpyridinium-3-yl)porphyrins, alkyl being methyl (M, MnTM-3-PyP⁵⁺), ethyl (E, MnTE-3-PyP⁵⁺), n-propyl (nPr, MnTnPr-3-PyP⁵⁺), n-butyl (nBu, MnTnBu-3-PyP⁵⁺), n-hexyl (nHex, MnTnHex-3-PyP⁵⁺)

MnBr₈TBAP³⁻

Mn(III) β-octabromo-meso-tetrakis(4-carboxylatophenyl)porphyrin, also known as

MnBr₈TM-3(or 4)-PyP⁴⁺

Mn(III) β-octabromo-meso-tetrakis(N-methylpyridinium-3(or 4)-yl))porphyrin

MnBr₈TSPP³⁻

Mn(III) β-octabromo-meso-tetrakis(4-sulfonatophenyl)porphyrin

MnP

Mn porphyrin (charges of MnPs are omitted in figures but not in figure legends)

MnT(2,6-Cl₂-3-SO₃-P)P³⁻

Mn(III) meso-tetrakis(2,6-dichloro-3-sulfonatophenyl)porphyrin

MnT(TFTeMa)P⁵⁺

Mn(III) meso-tetrakis(2,3,5,6 tetrafluoro-N,N,N-trimethylanilinium-4-yl)porphyrin

MnT(TrMA)P⁵⁺

Mn(III) meso-tetrakis(2,3,5,6 tetrafluoro- N,N,N-trimethylanilinium-4-yl)porphyrin

MnTBAP³⁻

Mn(III) meso-tetrakis(4-carboxyphenyl)porphyrin, also known as MnTCPP³⁻ and AEOL10201

MnTDE-2-ImP⁵⁺

Mn(III) meso-tetrakis(N,N′-diethylimidazolium-2-yl)porphyrin, AEOL10150

MnTDMOE-2-ImP⁵⁺

Mn(III) meso-tetrakis[N,N′-di(2-methoxyethyl)imidazolium-2-yl]porphyrin

MnTM-4-PyP⁵⁺

Mn(III) meso-tetrakis(N-methylpyridinium-4-yl)porphyrin

MnTM,MOE-2-ImP⁵⁺

Mn(III) meso-tetrakis[(N-methyl-N′-methoxethyl)imidazolium-2-yl]porphyrin

MnTMOE-3-PyP⁵⁺

Mn(III) meso-tetrakis(N-(2′-methoxyethyl)pyridinium-3-yl)porphyrin

MnTMOHex-3-PyP⁵⁺

Mn(III) meso-tetrakis(N-(6′-methoxyhexyl)pyridinium-3-yl)porphyrin

MnTnBuOE-2-PyP⁵⁺

Mn(III) meso-tetrakis(N-(n-butoxyethyl)pyridinium-2-yl)porphyrin, also BMX-001

MnTSPP³⁻

Mn(III) meso-tetrakis(4-sulfonatophenyl)porphyrin

NaCl

sodium chloride

NH₄PF₆

ammonium hexafluorophosphate

NHE

normal hydrogen electrode

NIH

National Institutes of Health

nitrone

NXY-059, disulfonated PBN, phenyl-tert-butylnitrone

O₂^•−

superoxide

ONOO⁻

peroxynitrite

ROS

reactive oxygen species

SE

standard error

SOD

superoxide dismutase

TBACl

tetra-n-butylammonium chloride

tempol

4-OH-2,2,6,6,-tetramethylpiperidine-1-oxyl

WW-85

Fe(III) meso-tetrakis(N-carboxylatobenzylpyridyl)porphyrin

Acknowledgments

The authors appreciate financial support from Duke University's Critical and Translational Science Awards grant 1 UL 1 RR024128-01 from National Center for Research Resources/National Institutes of Health (A.T.), NIH U19AI067798 (A.T.), and Batinic-Haberle's general research funds (A.T.). L.B. acknowledges financial support from Kuwait University, grants MB 01/09 and MB 03/07, and Research Unit grant SRUL02/13. The authors are thankful to Irwin Fridovich and Ines Batinic-Haberle for critically reading the article. They are also grateful to the anonymous reviewers for their constructive criticism and valuable comments.