RESUMEN
Catalases (CAT) are antioxidant metalloenzymes necessary for life in oxygen-metabolizing cells to regulate H2O2 concentration by accelerating its dismutation. Many physiopathological situations are associated with oxidative stress resulting from H2O2 overproduction, during which antioxidant defenses are overwhelmed. We have used a combinatorial approach associated with an activity-based screening to discover a first peptidyl di-copper complex mimicking CAT. The complex was studied in detail and characterized for its CAT activity both in solutions and in cells using different analytical methods. The complex exhibited CAT activity in solutions and, more interestingly, on HyPer HeLa cells that possess a genetically encoded ratiometric fluorescent sensors of H2O2. These results highlight the efficiency of a combinatorial approach for the discovery of peptidyl complexes that exhibit catalytic activity.
Asunto(s)
Antioxidantes/metabolismo , Catalasa/metabolismo , Cobre/metabolismo , Metaloproteínas/metabolismo , Péptidos/metabolismo , Antioxidantes/química , Catalasa/química , Cobre/química , Células HeLa , Humanos , Peróxido de Hidrógeno/metabolismo , Metaloproteínas/química , Péptidos/química , Células Tumorales CultivadasRESUMEN
Oxidative stress is known to play a major role in the pathogenesis of inflammatory bowel diseases (IBDs), and, in particular, superoxide dismutase (SODs) defenses were shown to be weakened in patients suffering from IBDs. SOD mimics, also called SOD mimetics, as low-molecular-weight complexes reproducing the activity of SOD, constitute promising antioxidant catalytic metallodrugs in the context of IBDs. A Mn(II) complex SOD mimic (Mn1) based on an open-chain diaminoethane ligand exerting antioxidant and anti-inflammatory effects on an intestinal epithelial cellular model was shown to experience metal exchanges between the manganese center and metal ions present in the biological environment (such as Zn(II)) to some degrees. As the resulting complexes (mainly Zn(II)) were shown to be inactive, improving the kinetic inertness of Mn(II) complexes based on open-chain ligands is key to improve their bioactivity in a cellular context. We report here the study of three new Mn(II) complexes resulting from Mn1 functionalization with a cyclohexyl and/or a propyl group meant to limit, respectively, (a) metal exchanges and (b) deprotonation of an amine from the 1,2-diaminoethane central scaffold. The new manganese-based SOD mimics display a higher intrinsic SOD activity and also improved kinetic inertness in metal ion exchange processes (with Zn(II), Cu(II), Ni(II), and Co(II)). They were shown to provide anti-inflammatory and antioxidant effects in cells at lower doses than Mn1 (down to 10 µM). This improvement was due to their higher inertness against metal-assisted dissociation and not to different cellular overall accumulations. Based on its higher inertness, the SOD mimic containing both the propyl and the cyclohexyl moieties was suitable for intracellular detection and quantification by mass spectrometry, quantification, that was achieved by using a 13C-labeled Co-based analog of the SOD mimics as an external heavy standard.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Manganeso , Antioxidantes/farmacología , Células Epiteliales , Humanos , Ligandos , Manganeso/farmacología , Metales , Superóxido DismutasaRESUMEN
Oxidative stress that results from an imbalance between the concentrations of reactive species (RS) and antioxidant defenses is associated with many pathologies. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase are among the key enzymes that maintain the low nanomolar physiological concentrations of superoxide and hydrogen peroxide. The increase in the levels of these species and their progeny could have deleterious effects. In this context, chemists have developed SOD and CAT mimics to supplement them when cells are overwhelmed with oxidative stress. However, the beneficial activity of such molecules in cells depends not only on their intrinsic catalytic activities but also on their stability in biological context, their cell penetration and their cellular localization. We have employed cellular assays to characterize several compounds that possess SOD and CAT activities and have been frequently used in cellular and animal models. We used cellular assays that address SOD and CAT activities of the compounds. Finally, we determined the effect of compounds on the suppression of the inflammation in HT29-MD2 cells challenged by lipopolysaccharide. When the assay requires penetration inside cells, the SOD mimics Mn(III) meso-tetrakis(N-(2'-n-butoxyethyl)pyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP5+) and Mn(II) dichloro[(4aR,13aR,17aR,21aR)-1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21,21a-eicosahydro-11,7-nitrilo-7Hdibenzo[b,h] [1,4, 7,10] tetraazacycloheptadecine-κN5,κN13,κN18,κN21,κN22] (Imisopasem manganese, M40403, CG4419) were found efficacious at 10 µM, while Mn(II) chloro N-(phenolato)-N,N'-bis[2-(N-methyl-imidazolyl)methyl]-ethane-1,2-diamine (Mn1) requires an incubation at 100 µM. This study thus demonstrates that MnTnBuOE-2-PyP5+, M40403 and Mn1 were efficacious in suppressing inflammatory response in HT29-MD2 cells and such action appears to be related to their ability to enter the cells and modulate reactive oxygen species (ROS) levels.
Asunto(s)
Catalasa/metabolismo , Manganeso/metabolismo , Compuestos Organometálicos/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Antioxidantes/metabolismo , Línea Celular , Glutatión Peroxidasa/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Metaloporfirinas/metabolismo , Imitación Molecular , Oxidación-Reducción , Estrés Oxidativo , Porfirinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
A combinatorial approach using a one-bead-one-compound method and a screening based on a SOD-activity assay was set up for the discovery of an efficient peptidyl copper complex. The complex exhibited good stability constants, suitable redox potentials and excellent intrinsic activity. This complex was further assayed in cells for its antioxidant properties and showed beneficial effects when cells were subjected to oxidative stress.
Asunto(s)
Materiales Biocompatibles/metabolismo , Cobre/química , Péptidos/química , Secuencia de Aminoácidos , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Colon/citología , Colon/efectos de los fármacos , Colon/metabolismo , Cobre/metabolismo , Células HT29 , Humanos , Interleucina-8/metabolismo , Lipopolisacáridos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Péptidos/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
A superoxide dismutase mimic (Mn1) was functionalized with three positively charged-peptides: RRRRRRRRR (Mn1-R9), RRWWWRRWRR (Mn1-RW9) or Fx-r-Fx-K (Mn1-MPP). Characterization of the physico-chemical properties of the complexes show that they share similar binding affinity for Mn2+, apparent reduction potential and intrinsic superoxide dismutase activity. However, their accumulation in cells is different (Mn1-R9 < Mn1-MPP < Mn1-RW9 < Mn1), as well as their subcellular distribution. In addition, the three functionalized-complexes display a better anti-inflammatory activity than Mn1 when assayed at 10 µM. This improvement is due to a combination of an anti-inflammatory effect of the peptidyl moiety itself, and of the SOD mimic for Mn1-RW9 and Mn1-MPP. In contrast, the enhanced anti-inflammatory activity of Mn1-R9 is solely due to the SOD mimic.