Diverse functions of cationic Mn(III) N-substituted pyridylporphyrins, recognized as SOD mimics

Abstract

Oxidative stress, a redox imbalance between the endogenous reactive species and antioxidant systems, is common to numerous pathological conditions such as cancer, central nervous system injuries, radiation injury, diabetes etc. Therefore, compounds able to reduce oxidative stress have been actively sought for over 3 decades. Superoxide is the major species involved in oxidative stress either in its own right or through its progeny, such as ONOO⁻, H₂O₂, •OH, CO₃•⁻, and •NO₂. Hence, the very first compounds developed in the late 1970-ies were the superoxide dismutase (SOD) mimics. Thus far the most potent mimics have been the cationic meso Mn(III) N-substituted pyridylporphyrins and N,N'-disubstituted imidazolylporphyrins (MnPs), some of them with k(cat)(O₂·⁻) similar to the k(cat) of SOD enzymes. Most frequently studied are ortho isomers MnTE-2-PyP⁵⁺, MnTnHex-2-PyP⁵⁺, and MnTDE-2-ImP⁵⁺. The ability to disproportionate O₂·⁻ parallels their ability to remove the other major oxidizing species, peroxynitrite, ONOO⁻. The same structural feature that gives rise to the high k(cat)(O₂·⁻) and k(red)(ONOO⁻), allows MnPs to strongly impact the activation of the redox-sensitive transcription factors, HIF-1α, NF-κB, AP-1, and SP-1, and therefore modify the excessive inflammatory and immune responses. Coupling with cellular reductants and other redox-active endogenous proteins seems to be involved in the actions of Mn porphyrins. While hydrophilic analogues, such as MnTE-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺ are potent in numerous animal models of diseases, the lipophilic analogues, such as MnTnHex-2-PyP⁵⁺, were developed to cross blood brain barrier and target central nervous system and critical cellular compartments, mitochondria. The modification of its structure, aimed to preserve the SOD-like potency and lipophilicity, and diminish the toxicity, has presently been pursued. The pulmonary radioprotection by MnTnHex-2-PyP⁵⁺ was the first efficacy study performed successfully with non-human primates. The Phase I toxicity clinical trials were done on amyotrophic lateral sclerosis patients with N,N'-diethylimidazolium analogue, MnTDE-2-ImP⁵⁺ (AEOL10150). Its aggressive development as a wide spectrum radioprotector by Aeolus Pharmaceuticals has been supported by USA Federal government. The latest generation of compounds, bearing oxygens in pyridyl substituents is presently under aggressive development for cancer and CNS injuries at Duke University and is supported by Duke Translational Research Institute, The Wallace H. Coulter Translational Partners Grant Program, Preston Robert Tisch Brain Tumor Center at Duke, and National Institute of Allergy and Infectious Diseases. Metal center of cationic MnPs easily accepts and donates electrons as exemplified in the catalysis of O₂·⁻ dismutation. Thus such compounds may be equally good anti- and pro-oxidants; in either case the beneficial therapeutic effects may be observed. Moreover, while the in vivo effects may appear antioxidative, the mechanism of action of MnPs that produced such effects may be pro-oxidative; the most obvious example being the inhibition of NF-κB. The experimental data therefore teach us that we need to distinguish between the mechanism/s of action/s of MnPs and the effects we observe. A number of factors impact the type of action of MnPs leading to favorable therapeutic effects: levels of reactive species and oxygen, levels of endogenous antioxidants (enzymes and low-molecular compounds), levels of MnPs, their site of accumulation, and the mutual encounters of all of those species. The complexity of in vivo redox systems and the complex redox chemistry of MnPs challenge and motivate us to further our understanding of the physiology of the normal and diseased cell with ultimate goal to successfully treat human diseases.

Figure 1

The most frequently studied redox-active Mn porphyrins for treating oxidative stress disorders, MnTE-2-PyP⁵⁺, MnTnHex-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺.

Figure 2

Redox-active compounds which are able to diminish oxidative stress injuries. Shown are modified porphyrins, corroles with one less meso position; the methyl analogue of MnTE-2-PyP⁵⁺ (Figure 1) [18], and the anionic corrole with sulfonato groups on pyrrolic positions whose Ga complex is explored as anticancer drugs and Mn and Fe complexes for treating ROS-related injuries [18]. The non-porphyrin-based SOD mimics [,–31], metal oxides and metal nanoparticles are presented too. Mn²⁺ in its own right, when complexed in vivo to low-molecular weight ligands such as lactate or oxo/hydroxo/acetate ligands, exerts high SOD-like activity [18]. Cerium oxide nanoparticles [32,33], osmium tetroxide, OsO₄ [34] and platinum nanoparticles [35] have shown SOD-like efficacy in vivo. Natural compounds, primary those bearing phenol functionalities reportedly bear potential to reduce oxidative stress also, as exemplified here with genistein [36], curcumin [37] and celastrol [38]. Among them curcumin has a marginal SOD-like activity [18].

Figure 3

The dismutation of O₂^·− catalyzed by superoxide dismutases. The enzyme-catalyzed dismutation is ~3 oders of magnitude faster than O₂^·− self-dismutation, and occurs at potential that is midway between the potential for O₂^·− oxidation to oxygen, and its proton-dependent reduction to H₂O₂. Under physiological conditions peroxide is removed in a subsequent step by peroxide-removing enzymes such as glutathione peroxidases and catalases.

Figure 4

Historical overview of the optimization of the MnP structure for suppressing oxidative stress. Among structures shown, the most potent SOD mimic is the meta isomer of the Mn(II) β-octabrominated meso-tetrakis(N-methylpyridyl porphyrin, MnBr₈TM-3-PyP⁴⁺; its log k_cat ≥ 8.85 is nearly identical to the one of SOD enzymes. The k_cat of SODs obtained by different researchers ranged from 8.84 to 9.30 (Table 1). Yet, with highly positive E_1/2 of +468 mV vs NHE, Mn in MnBr₈TM-3-PyP⁴⁺ is in +2 oxidation state. The metal complex is thus insufficiently stable and readily falls apart. While not of practical importance as a therapeutic, its developmenmt justifies the use of porphyrin as an excellent ligand for optimizing SOD mimics.

Figure 5

Historical overview of the development of MnPs from the conception, synthesis, testing in prokaryotic E. coli, eukaryotic S. cerevisiae and whole organisms (rats, mice and rabbits), over non human primates to humans. Few in vivo studies are referred here only. Other studies are listed in Table 2. For the details on dosing and outcome of the studies please see refs 18,29–31.

Figure 6

The lipophilicity gain upon reduction of ortho and meta isomers of Mn(III) N-alkylpyridylporphyrins as a function of a number of carbon atoms, nC. The gain is larger with ortho than with meta isomers, and is peaking with butyl compounds [81].

Figure 7

(A) The structures of meta isomers, where hexyl and ethyl compounds are derivatized with methoxy groups: MnTMOHex-3-PyP⁵⁺ and MnTMOE-3-PyP⁵⁺. The modification was meant to decrease the toxicity of MnTnHex-3-PyP⁵⁺. Ethyl analogue was originally intended for comparison, but appeared of unexpectedly high efficacy in vivo. This is explained as a result of favorably balanced several structural features; (B) The lipophilicities of metal-free porphyrins and their ligands expressed in terms of chromatographic retention factor, R_f. Lipophilicity of Mn complexes is lower than of their metal-free ligands due to the higher solvation of the metal site. The effect is more drastic with porphyrins bearing shorter substituents. Longer-chained analogues, alkyl and methoxyalkyl are more lipophilic than their shorter-chained analogues. Introduction of methoxy group reduces lipophilicity of longer alkyl analogue, H₂TnHex-3-PyP⁴⁺ and its Mn complex; (C) The effect of the MnPs on the growth of SOD-deficient E. coli in minimal five amino acids-medium. The growth was measured as absorbance at 600 nm. The data are the average of 5 different studies where each compound was tested in triplicates. The data at 5 and 10 μM MnPs were provided; for the rest see ref . The growth of JI132 and AB1152 alone is shown also. At 5 μM MnTMOHex-3-PyP⁵⁺ is similarly potent as MnTMOE-3-PyP⁵⁺, and more efficacious than MnTE-2-PyP⁵⁺. The alkyl analogue, MnTnHex-3-PyP⁵⁺ is already very toxic at 5 μM. At 10 μM MnTMOHex-3-PyP⁵⁺ becomes toxic. MnTMOE-3-PyP⁵⁺ is the most efficacious of all compounds tested. Adapted from [53].

Figure 8

Introduction of oxygens at different positions within alkyl chains dramatically affects the lipophilicity of MnPs, while maintaining the same redox-based abilities as exemplified with high k_cat. Shown are the data related to the compounds that bear oxygens at the very end of the chains, MnTMOHex-2-PyP⁵⁺, and relatively close to the porphyrin core, MnTnBuOE-2-PyP⁵⁺, as well as their alkyl analogues of comparable chain length, MnTnHex-2-PyP⁵⁺ and MnTnHep-2-PyP⁵⁺ [18].

Figure 9

The reactivity of MnPs towards reactive species and transcription factors thus far studied [18,30]. The log values of rate constants listed relate predominantly to ortho isomers. The rate constant of MnPs with HClO was estimated based on the reported data on the reaction of MnTM-4-PyP⁵⁺ and MnTBAP³⁻ with HClO [89]. Data on the reaction of reduced Mn porphyrin, Mn^IITE-2-PyP⁴⁺ with ^·NO is also listed. The highest log k_cat(O₂^·−) ≥ 8.85 refers to Mn^IIBr₈TM-3-PyP⁴⁺. Based on our present knowledge, the ability of MnPs to catalyze peroxide removal is weak. Based on the reactivity to other species listed, it is highly likely that potent SOD mimics would scavenge ^·NO₂ radical also.

Figure 10

The production of cytotoxic H₂O₂ when MnP was exposed to ascorbate in aerobic environment. Together, MnP and ascorbate can produce superoxide which can then undergo self-dismutation, or enzyme-catalyzed dismutation to H₂O₂. In the presence of Fe, the Fenton chemistry can lead to the production of hydroxyl radical ^·OH. Reduction of Fe³⁺ to Fe^{2+ ·−}. The abbreviations are: A, dehydroascorbic acid; HA⁻, monodeprotonated ascorbate; can happen with either ascorbate or O₂ HA^·, ascorbyl radical; A^·− deprotonated ascorbyl radical.

Figure 11

Cytotoxicity of sodium ascorbate at 3.3 mM without or with 30 μM MnTnHex-2-PyP⁵⁺. 5 mM mannitol only weakly suppressed the cytotoxicity when MnP was combined with sodium ascorbate, while the viability of Caco-2 cells increased with the addition of 1500 U/mL catalase. This indicates that the cells were killed predominantly by hydrogen peroxide (WST-1-based cytotoxicity assay). The assay is based on the reduction of tetrazolium salt WST-1 to a water-soluble colored formazan by metabolically active cell. 10,000 viable cells were plated in each well of a 96-well plate in a complete medium. After 24 h in 5% CO₂ at 37 ^oC, the medium was replaced with 100 μl of a fresh complete medium. A positive control containing the same number of cells in complete medium without any drug was used. A negative control without cells was used as blank. The plates were incubated for 96 h. At the end of the incubation, the cells were washed twice with PBS. Then 100 μl of Opti-Media (Invitrogen, USA) with 10 μl of proliferation reagent WST-1 was added to each well and the plates were incubated at 37 ^oC for another 2 h. The absorbance was measured in a microplate reader (BMG) at 450/595 nm and the percent viability was calculated from the following equation: (A_sample−A_blank)/(A_control−A_blank) × 100. Adopted from [68].

Figure 12

(A) The growth of the wild type parental E. coli AB1157, and SOD-deficient JI132 E. coli with 5 and 10 μM MnP −/+ ascorbate (1 mM) in minimal, five amino acids medium at 14 h; (B) The growth of the wild type AB1157 and SOD-deficient E. coli JI132 with 5 and 10 μM MnP/ascorbate (1 mM) in M9CA medium at 18 h, and in minimal 5 amino acids medium at 24 h, respectively. P=parental, or SOD⁻ = SOD-deficient E. coli, Asc=ascorbate, E3=MnTE-3-PyP⁵⁺. (*) represents statistical significance. The data are adopted from [44].

Figure 13

(A) Effect of catalase on MnTE-3-PyP⁵⁺/ascorbate toxicity on the growth of E. coli at 9 hours in M9CA nutrient rich medium. The parental AB1157 E. coli (P) grew with or without 1 μM MnP and 1 mM ascorbate. Catalase (C) was added to the growth medium at 1,000 units/mL five minutes before the addition of MnP and ascorbate; (B) The effect of MnP/ascorbate on the upregulation of catalases and peroxidases. The parental (GC4468) cells were incubated for 2 hours in the presence of 1 μM MnTE-3-PyP⁵⁺ and 1 mM ascorbate. E. coli responds to the increased peroxide levels as a consequence of MnTE-3-PyP⁵⁺/ascorbate-based peroxide formation by upregulating catalases and peroxidases (E3+Asc bar vs nontreated bar). P=parental, C=catalase, Asc=ascorbate, E3=MnTE-3-PyP⁵⁺; (*) represents statistical significance. The data are adopted from [44].