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Effect of some kynurenines under succinate deshidrogenase activity and ATP production: in
vitro experimental and computational assays.
Introduction Mitochondria is the primary site of reactive oxygen species (ROS) production and is uniquely vulnerable to oxidative damage that may play a critical role in controlling neuronal excitability. Mitochondrial membrane-bound Succinate dehydrogenase or Complex II is present in all aerobic cells, is linked to the respiratory chain and a member of the tricarboxylic acid (TCA); its activity is modulated by several activators and inhibitors; but it also differs from other dehydrogenases due to its unique redox properties. Alterations in mitochondrial function includes ionic imbalance, Ca2+ overload and ATP depletion (Brookes et al; 2004) . Growing evidence indicates that some metabolites derived from the kynurenine pathway (KP), the major route of L-tryptophan catabolism, are involved in the neurotoxicity associated with several brain disorders in which mitochondrial respiratory parameters are altered. Actually, inhibitors of mitochondrial respiratory chain complexes have been used experimentally to model neurochemical, histological, and clinical features of neurodegerative disorders (Cooper and Schapira, 1997; Borlongan et al., 1997; Betarbet et al., 2000; Beal, 2001; Brouillet et al., 2005). Mitochondrial respiratory parameters, including those on the activities of the respiratory chain complexes I–IV have been demonstrated to be altered by some kynurenines (Baran et al., 2001, 2003). Quinolinic acid (QUIN), a metabolite of the kynurenine pathway, inhibits complex II in rat brain homogenates (Baran et al., 2003). Additionally, it has been reported that altered levels of other metabolites of the KP, such as kynurenic acid (KYNA), anthranilic acid and 3-hidoxykynurenine (3HK), together with a simultaneous mitochondrial dysfunction, are common features present in neurodegenerative disorders. Here, we first calculate the electrostatic contribution of the SDH-metabolites interaction to the free energy bound using theoretical-computational tools. Later on, we investigated the in vitro effect of KYNA, 3HK and QUIN on succinate dehydrogenase (SDH) activity and ATP production in isolated brain mitochondria.
Materials and methods • Computational modeling The electrosatic contribution to the binding free energy in the Succinate dehydrogenase flavoprotein subunit (SDH)-inhibitors interaction, was estimated through of the solvation energy contribution under the continuous solvent approximation using the numerical solution to the nonlinear Poisson–Boltzmann equation implemented in Adaptive Poisson–Boltzmann Solver (APBS), the Coulombic energy contribution using APBS external program Coulomb and non polar solvation energy was estimated by the change in solvent accessible surface area (ASA) upon binding with the program Visual Molecular Dynamics (VMD). Then the previously generated conformers for QUIN, 3HK and KYNA were scanned against the SDH surface using 100,000 poses of probe for each inhibitor in order to include virtually all orientations for binding each of those compounds. The best complexes were then submitted to an energy minimization process in order to improve the interactions at the binding sites. • Mitochondrial isolation Mitochondria were isolated as described by Mirandola et al. (2010) with minor modifications. Tissue was dissected and extensively washed with ice-cold isolation buffer containing 225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 0.05% bovine serum albumin and 10 mM HEPES pH 7.2. After, tissue was homogenized and the homogenate was centrifuged for 5 min at 2,000 x g; the resulting supernatant was recentrifuged for 10 min at 12,000 x g. The pellet was resuspended in isolation buffer with 0.2% digitonin and recentrifuged for 10 min at 12 x 000 g. The supernatant was discarded and the final dark pellet gently washed and resuspended in ‘isolation buffer’ devoid of EGTA, at an approximate protein concentration of 3-4 mg/mL. • Protein Determination Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard. • Complex II (Succinate dehydrogenase) activity This was evaluated through a spectrophotometric assay previously described (Mao et al., 2007) with slight modifications. The reaction medium consisted in 20 mM succinate, 2 μg/ml of antimycin A, 2 μg/ml rotenone, 2 mM KCN and 50 μM DCPIP in 10 mM potassium phosphate, pH 7.4. Thirty mg of mitochondrial protein was added and the reaction was started by addition of 50 μM DB. The reduction of DCPIP was monitored at 600 nm for 3 min. Calculations for activity were obtained using the extinction coefficient of 19.1 mM-1cm-1 for DCPIP.
• ATP production
The rate of ATP synthesis was measured using an enzyme-linked assay. The reduction of NADP+ was followed at 340 nm in a multimodal microplate reader (Biotek) during 5 min. Succinate was used as a respiratory substrate, and the enzyme-linked assay was 163 μg/mL hexokinase, 2 U/mL glucose-6-phosphate dehydroge- nase, 20 mM glucose, and 1.4 mM NADP+. The reaction was started with 200 μM ADP. ATP production from sources other than electron transport chain was determined by performing the assay in the presence of oligomycin. The last was subtracted from the total activity.
Results • Overlapping of kynurenines and 3-NP on SDH-binding site A) B) Figure 1.0. 3D representation of the interaction of kynurenines (QUIN, KYNA, 3HK) and 3NP on SDH-binding site. A) SDH-binding site and kynurenines on 3D SDH protein of chicken. B) SDH-binding site and kynurenines in a homologous 3D protein structure of SDH of rat. Prostethetic group FAD is also represented, where blue ends represent Nitrogen, red represents Oxygen, and gray carbons.
• Schematic representation of the electrosatic contribution to the binding free energy in the succinate
dehydrogenase flavoprotein subunit (SDH)-inhibitors interaction. Figure 2.0 Schematic representation of the electrosatic contribution of the aminoacid residues of each kynurenine (2-4) and 3NP (1) to the binding free energy (ΔGb) in the interaction with SDH-binding site. Hydrogen bonds are represented with dotted lines, FAD is also represented. A conserved Arg is observed in the interaction of each kynurenine with the SDH-binding site.
• Table 1.0 Theoretical calculus of SDH-inhibitor binding.
• SDH activity and ATP production in isolated mitochondrias.
Cortex
Cerebellum
3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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50
75
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125
150 10 min 2 h
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3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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100
10 min 2 h
* *
* * * **
% o
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3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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50
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100
125
10 min 2 h
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3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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150 10 min 2 h
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3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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10 min 2 h
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3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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125 10 min 2 h
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3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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120 10 min 2 h
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Table 2.0. Summary of the in vitro inhibition effect on SDH activity of each kynurenine and 3NP in liver and distinct brain regions at different times of incubation (10 min and 2h) . This table shows differences between each brain region and liver, and also at different times of 37ºC incubation.
Hipoccampus
Striatum
Liver
• Table 2.0 Comparative inhibition of SDH activity by kynurenines vs 3NP in liver and distinct brain regions.
Conclusions Kynurenines exhibited a high simulated binding affinity for SDH. The results showed that the electrostatic energy for each complex could be ordered as follows: 3-NP (ΔGele= -513 kJ/mol) > QUIN (ΔGele= -41 kJ/mol) > KYNA (ΔGele= -37 kJ/mol) > 3HK (ΔGele= -36 kJ/mol). 3NP and kynurenines interact in the SDH-binding site with the arginine 297 residue (R290 of the sequence numbering of SDH-QUIN by docking), whose positive charge is important for the binding affinity of negatively charged inhibitors.
Our data show a differential effect of kynurenines on SDH activity, depending on each brain region. We observed a more marked effect in SDH activity of kynurenines in all brain regions after 2 h of incubation, an effect not seen in liver in which only 3-NP, our positive control, exhibited a notably effect since the first 10 min of incubation.
The effect exerted by each kynurenine and 3-NP on SDH activity was not directly correlated with the reduction of ATP levels. For instance, while at 10 min of incubation SDH activity in cortex and cerebellum was not altered when compared to controls, ATP depletion is observed and remains the same after 2 h when the effect of kynurenines on SDH activity exerted its major effect. The striatum showed a major susceptibility to kynurenines and 3-NP, since reduction of SDH activity is seen even from the first 10 min and continues up to 2 h after incubation. ATP levels are drastically diminished
References
Baran, H., et al. 2001. Pharmacology 62:119–123. Baran, H. et al.2003.Life Sci. 72:1103–1115. Cooper JM, Schapira AH. 1997. J Bioenerg Biomembr 29:175–183. Beal, M. 2004. J. Bioenerg. Biomembr. 36:381–386. Betarbet R et al. 2000. Nat Neurosci 3:1301–1306. Borlongan CV, et al. 1997. Neurosci Biobehav Rev 21:289–293. Brookes PS, et al. 2004. Am J Physiol Cell Physiol. 287:C817–33. Brouillet E, et al. 2005. J Neurochem 95:1521–1540. Lowry O. H., et al.1951. J. Biol. Chem. 193, 265–275 Mirandola SR, et al. 2008 . J Inherit Metab Dis 31:44–54. Rosenthal R; et al. 1987. J Cereb Blood Flow Metab 7:752–758. Singer TP. 1974. Methods Biochem Anal 22:123–175.
T. BLANCO AYALA1,4, I. SERRATOS ÁLVAREZ 2, M. OROZCO IBARRA1, J. REYES OCAMPO1, C. MILLÁN PACHECO2, N. PASTOR3, P. GARCÍA GUTIÉRREZ2, P. DÍAZ1, J. PEDRAZA CHAVERRÍ4, A. SANTAMARÍA1,V. PÉREZ DE LA CRUZ1,4; 1Inst. Nacional de Neurología y Neurocirugía, Ciudad de México, D.F., México; 2Univ. Autónoma Metropolitana, México,D.F., México; 3Univ. Autónoma del Estado de Morelos, Cuernavaca, Morelos, México; 4Univ. Nacional Autónoma de México, Mexico City, Mexico
1) SDH-3NP 2) SDH-QUIN
3) SDH-KYNA 4) SDH-3HK
3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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3-NP QUIN KYNA 3-HK 3-NP QUIN KYNA 3-HK0
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Table 1.0 Values of the binding constant and the binding free energy of each kynurenine and 3NP with SDH. A) Values calculated for SDH-binding site interaction of chicken B) Values for SDH-binding site interaction of rat.
A) B)
a.a)
b.a)
c.a.)
d.a.)
a.b)
b.b)
c.b.)
d.b.)
e.a.) e.b.)
Figures a-e. In vitro effect of 3-nitropropionic acid , quinolinic acid (QUIN), kynurenic acid (KYNA) and 3-hydroxykynurenine (3HK)) in cerebral cortex (a.a, a.b.), cerebellum (b.a.,b.b.), hipoccampus (c.a.,c.b.), striatum (d.a,d.b.) and liver (e.a.,e.b.) from Wistar male rats on SDH activity and ATP production. The results are expressed as percentage compared to controls.
