Publications by authors named "Irina G Gazaryan"

21 Publications

  • Page 1 of 1

Challenges and Limitations of Targeting the Keap1-Nrf2 Pathway for Neurotherapeutics: Bach1 De-Repression to the Rescue.

Front Aging Neurosci 2021 8;13:673205. Epub 2021 Apr 8.

Darby Children's Research Institute, Medical University of South Carolina, Charleston, SC, United States.

The Keap1-Nrf2 signaling axis is a validated and promising target for cellular defense and survival pathways. This minireview discusses the potential off-target effects and their impact on future drug development originating from Keap1-targeting small molecules that function as displacement activators of the redox-sensitive transcription factor Nrf2. We argue that small-molecule displacement activators, similarly to electrophiles, will release both Nrf2 and other Keap1 client proteins from the ubiquitin ligase complex. This non-specificity is likely unavoidable and may result in off-target effects during Nrf2 activation by targeting Keap1. The small molecule displacement activators may also target Kelch domains in proteins other than Keap1, causing additional off-target effects unless designed to ensure specificity for the Kelch domain only in Keap1. A potentially promising and alternative therapeutic approach to overcome this non-specificity emerging from targeting Keap1 is to inhibit the Nrf2 repressor Bach1 for constitutive activation of the Nrf2 pathway and bypass the Keap1-Nrf2 complex.
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http://dx.doi.org/10.3389/fnagi.2021.673205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060438PMC
April 2021

HIF Prolyl Hydroxylase Inhibitors for COVID-19 Treatment: Pros and Cons.

Front Pharmacol 2020 29;11:621054. Epub 2021 Jan 29.

P. A. Hertsen Moscow Oncology Research Center, Branch of the National Medical Research Radiological Center, Ministry of Health of the Russian Federation, Moscow, Russia.

The review analyzes the potential advantages and problems associated with using HIF prolyl hydroxylase inhibitors as a treatment for COVID-19. HIF prolyl hydroxylase inhibitors are known to boost endogenous erythropoietin (Epo) and activate erythropoiesis by stabilizing and activating the hypoxia inducible factor (HIF). Recombinant Epo treatment has anti-inflammatory and healing properties, and thus, very likely, will be beneficial for moderate to severe cases of COVID-19. However, HIF PHD inhibition may have a significantly broader effect, in addition to stimulating the endogenous Epo production. The analysis of HIF target genes reveals that some HIF-targets, such as furin, could play a negative role with respect to viral entry. On the other hand, HIF prolyl hydroxylase inhibitors counteract ferroptosis, the process recently implicated in vessel damage during the later stages of COVID-19. Therefore, HIF prolyl hydroxylase inhibitors may serve as a promising treatment of COVID-19 complications, but they are unlikely to aid in the prevention of the initial stages of infection.
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http://dx.doi.org/10.3389/fphar.2020.621054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878396PMC
January 2021

"Branched Tail" Oxyquinoline Inhibitors of HIF Prolyl Hydroxylase: Early Evaluation of Toxicity and Metabolism Using Liver-on-a-chip.

Drug Metab Lett 2019 ;13(1):45-52

Dmitry Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology, Healthcare Ministry of Russia, 117997 Moscow, Russian Federation.

Background: "Branched tail" oxyquinolines, and adaptaquin in particular, are potent HIF prolyl hydroxylase inhibitors showing promising results in in vivo hemorrhagic stroke models. The further improvement of the potency resulted in identification of a number of adaptaquin analogs. Early evaluation of toxicity and metabolism is desired right at the step of lead selection.

Objective: The aim of the study is to characterize the toxicity and metabolism of adaptaquin and its new improved analogs.

Method: Liver-on-a-chip technology with differentiated HepaRG cells followed by LC-MS detection of the studied compounds and metabolites of the P450 substrate-inhibitor panel for CYP2B6, CYP2C9, CYP2C19, and CYP3A4.

Results: The optimized adaptaquin analogs show no toxicity up to a 100-fold increased range over EC50. The drugs are metabolized by CYP3A4 and CYP2B6 as shown with the use of the cytochrome P450 substrate-inhibitor panel designed and optimized for preclinical evaluation of drugs' in vitro biotransformation on a 3D human histotypical cell model using "liver-on-a-chip" technology. Activation of CYP2B6 with the drugs tested has been observed. A scheme for adaptaquin oxidative conversion is proposed.

Conclusion: The optimized adaptaquin analogs are suitable for further preclinical trials. Activation of CYP2B6 with adaptaquin and its variants points to a potential increase in Tylenol toxicity if administered together.
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http://dx.doi.org/10.2174/1872312813666181129100950DOI Listing
January 2020

Benfotiamine treatment activates the Nrf2/ARE pathway and is neuroprotective in a transgenic mouse model of tauopathy.

Hum Mol Genet 2018 08;27(16):2874-2892

Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY 10065, USA.

Impaired glucose metabolism, decreased levels of thiamine and its phosphate esters, and reduced activity of thiamine-dependent enzymes, such as pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and transketolase occur in Alzheimer's disease (AD). Thiamine deficiency exacerbates amyloid beta (Aβ) deposition, tau hyperphosphorylation and oxidative stress. Benfotiamine (BFT) rescued cognitive deficits and reduced Aβ burden in amyloid precursor protein (APP)/PS1 mice. In this study, we examined whether BFT confers neuroprotection against tau phosphorylation and the generation of neurofibrillary tangles (NFTs) in the P301S mouse model of tauopathy. Chronic dietary treatment with BFT increased lifespan, improved behavior, reduced glycated tau, decreased NFTs and prevented death of motor neurons. BFT administration significantly ameliorated mitochondrial dysfunction and attenuated oxidative damage and inflammation. We found that BFT and its metabolites (but not thiamine) trigger the expression of Nrf2/antioxidant response element (ARE)-dependent genes in mouse brain as well as in wild-type but not Nrf2-deficient fibroblasts. Active metabolites were more potent in activating the Nrf2 target genes than the parent molecule BFT. Docking studies showed that BFT and its metabolites (but not thiamine) bind to Keap1 with high affinity. These findings demonstrate that BFT activates the Nrf2/ARE pathway and is a promising therapeutic agent for the treatment of diseases with tau pathology, such as AD, frontotemporal dementia and progressive supranuclear palsy.
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http://dx.doi.org/10.1093/hmg/ddy201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077804PMC
August 2018

Activation of Nrf2 and Hypoxic Adaptive Response Contribute to Neuroprotection Elicited by Phenylhydroxamic Acid Selective HDAC6 Inhibitors.

ACS Chem Neurosci 2018 05 17;9(5):894-900. Epub 2018 Jan 17.

D. Rogachev Federal Scientific and Clinical Centre of Pediatric Hematology, Oncology and Immunology , Samora Mashela 1 , Moscow 117997 , Russian Federation.

Activation of HIF-1α and Nrf2 is a primary component of cellular response to oxidative stress, and activation of HIF-1α and Nrf2 provides neuroprotection in models of neurodegenerative disorders, including ischemic stroke, Alzheimer's and Parkinson's diseases. Screening a library of CNS-targeted drugs using novel reporters for HIF-1α and Nrf2 elevation in neuronal cells revealed histone deacetylase (HDAC) inhibitors as potential activators of these pathways. We report the identification of phenylhydroxamates as single agents exhibiting tripartite inhibition of HDAC6, inhibition of HIF-1 prolyl hydroxylase (PHD), and activation of Nrf2. Two superior tripartite agents, ING-6 and ING-66, showed neuroprotection against various cellular insults, associated with stabilization of both Nrf2 and HIF-1, and expression of their respective target genes in vitro and in vivo. Discovery of the innate ability of phenylhydroxamate HDAC inhibitors to activate Nrf2 and HIF provides a novel route to multifunctional neuroprotective agents and cautions against HDAC6 selective inhibitors as chemical probes of specific HDAC isoform function.
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http://dx.doi.org/10.1021/acschemneuro.7b00435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955769PMC
May 2018

L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?

Biochimie 2018 Apr 28;147:46-54. Epub 2017 Dec 28.

Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, 119992, Russian Federation; Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences. 33, bld. 2 Leninsky Ave., Moscow 119071, Russian Federation; Innovations and High Technologies MSU Ltd, Tsymlyanskaya, 16, of 96, Moscow, 109599, Russian Federation.

L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40-100 μM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.
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http://dx.doi.org/10.1016/j.biochi.2017.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460286PMC
April 2018

The status of Nrf2-based therapeutics: current perspectives and future prospects.

Neural Regen Res 2016 Nov;11(11):1708-1711

Departments of Pharmacology, Toxicology and Neurology, Medical College of Georgia, Augusta University, Augusta, GA, USA.

This mini-review presents the authors' vision on the current status and future trends in the development of neuroprotective agents working activation of nuclear factor erythroid 2-related factor 2 (Nrf2), and in particular, disruption of Nrf2-Keap1 interaction. There are two opposite "chemical" mechanisms underlying such activation: the first one is a non-specific covalent modification of Keap1 thiols, resulting in side effects of varied severity, and the second one is the shift of the Nrf2-Kelch-like ECH associated protein-1 (Keap1) binding equilibrium in the presence of a competitive and chemically benign displacement agent. At this point, no displacement activators exhibit sufficient biological activity in comparison with common Nrf2 activators working Keap1 thiol modification. Hence, the hope in therapeutics is now linked to the FDA approved dimethylfumarate, whose derivative, monomethylfumarate, as we demonstrated recently, is much less toxic but equally biologically potent and an ideal candidate for clinical trials right now. A newly emerging player is a nuclear inhibitor of Nrf2, BTB domain and CNC homolog 1 (Bach1). The commercially developed Bach1 inhibitors are currently under investigation in our laboratory showing promising results. In our viewpoint, the perfect future drug will present the combination of a displacement activator and Bach1 inhibitor to insure safety and efficiency of Nrf2 activation.
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http://dx.doi.org/10.4103/1673-5374.194706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204211PMC
November 2016

Bioactive Flavonoids and Catechols as Hif1 and Nrf2 Protein Stabilizers - Implications for Parkinson's Disease.

Aging Dis 2016 Dec 1;7(6):745-762. Epub 2016 Dec 1.

1Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, NY 10605, USA; 5Department of Chemical Enzymology, Moscow State University, Moscow 119992, Russia; 8Department of Chemistry and Physical Sciences, Dyson College, Pace University, Pleasantville, NY 10570, USA.

Flavonoids are known to trigger the intrinsic genetic adaptive programs to hypoxic or oxidative stress via estrogen receptor engagement or upstream kinase activation. To reveal specific structural requirements for direct stabilization of the transcription factors responsible for triggering the antihypoxic and antioxidant programs, we studied flavones, isoflavones and catechols including dihydroxybenzoate, didox, levodopa, and nordihydroguaiaretic acid (NDGA), using novel luciferase-based reporters specific for the first step in HIF1 or Nrf2 protein stabilization. Distinct structural requirements for either transcription factor stabilization have been found: as expected, these requirements for activation of HIF ODD-luc reporter correlate with binding to HIF prolyl hydroxylase. By contrast, stabilization of Nrf2 requires the presence of 3,4-dihydroxy- (catechol) groups. Thus, only some but not all flavonoids are direct activators of the hypoxic and antioxidant genetic programs. NDGA from the Creosote bush resembles the best flavonoids in their ability to directly stabilize HIF1 and Nrf2 and is superior with respect to LOX inhibition thus favoring this compound over others. Given much higher bioavailability and stability of NDGA than any flavonoid, NDGA has been tested in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-animal model of Parkinson's Disease and demonstrated neuroprotective effects.
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http://dx.doi.org/10.14336/AD.2016.0505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201116PMC
December 2016

In vitro ischemia suppresses hypoxic induction of hypoxia-inducible factor-1α by inhibition of synthesis and not enhanced degradation.

J Neurosci Res 2013 Aug 4;91(8):1066-75. Epub 2013 Mar 4.

Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York, USA.

Hypoxia-inducible factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. This study investigated the mechanism by which in vitro ischemia (oxygen-glucose deprivation; OGD) affects canonical hypoxic HIF-1α stabilization. We validated the use of a reporter containing the oxygen-dependent degradation domain of HIF-1α fused to firefly luciferase (ODD-luc) to monitor quantitatively distinct biochemical events leading to hypoxic HIF-1α expression or stabilization in a human neuroblastoma cell line (SH-SY5Y). When OGD was imposed following a 2-hr hypoxic stabilization of ODD-luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolylhydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD-luc in OGD. Furthermore, OGD does not affect the half-life of ODD-luc protein following hypoxia, suggesting that OGD abrogates hypoxic HIF-1α induction by reducing HIF-1α synthesis rather than by enhancing its degradation. We observed ATP depletion under OGD vs. hypoxia and propose that ATP depletion enhances translational suppression, overcoming the selective synthesis of HIF concurrent with global decreases in protein synthesis in hypoxia. Taken together, these findings biochemically characterize a practical reporter for monitoring HIF-1α levels and support a novel model for HIF regulation in an in vitro model of human ischemia.
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http://dx.doi.org/10.1002/jnr.23204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321689PMC
August 2013

A screen for inducers of p21(waf1/cip1) identifies HIF prolyl hydroxylase inhibitors as neuroprotective agents with antitumor properties.

Neurobiol Dis 2013 Jan 27;49:13-21. Epub 2012 Aug 27.

Burke-Cornell Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605, USA; Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 525 East 68th Street, New York, NY 10065, USA. Electronic address:

Preventing neuronal death is a priority for treating neurological diseases. However, therapies that inhibit pathological neuron loss could promote tumorigenesis by preventing the physiological death of cancerous cells. To avert this, we targeted the transcriptional upregulation of p21(waf1/cip1) (p21), an endogenous tumor suppressor with neuroprotective and pro-regenerative activity. We identified potential p21 indcuers by screening a FDA-approved drug and natural product small molecule library against hippocampal HT22 cells stably expressing a luciferase reporter driven by the proximal 60bp of the p21 promoter, and tested them for neuroprotection from glutathione depletion mediated oxidative stress, and cytotoxicity to cancer cell lines (DLD-1, Neuro-2A, SH-SY5Y, NGP, CHLA15, CHP212, and SK-N-SH) in vitro. Of the p21 inducers identified, only ciclopirox, a hypoxia-inducible factor prolyl-4-hydroxylase (HIF-PHD) inhibitor, simultaneously protected neurons from glutathione depletion and decreased cancer cell proliferation at concentrations that were not basally toxic to neurons. We found that other structurally distinct HIF-PHD inhibitors (desferrioxamine, 3,4-dihydroxybenzoate, and dimethyloxalyl glycine) also protected neurons at concentrations that killed cancer cells. HIF-PHD inhibitors stabilize HIF transcription factors, mediating genetic adaptation to hypoxia. While augmenting HIF stability is believed to promote tumorigenesis, we found that chronic HIF-PHD inhibition killed cancer cells, suggesting a protumorigenic role for these enzymes. Moreover, our findings suggest that PHD inhibitors can be used to treat neurological disease without significant concern for cell-autonomous tumor promotion.
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http://dx.doi.org/10.1016/j.nbd.2012.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706502PMC
January 2013

Targeting Nrf2-mediated gene transcription by extremely potent synthetic triterpenoids attenuate dopaminergic neurotoxicity in the MPTP mouse model of Parkinson's disease.

Antioxid Redox Signal 2013 Jan 13;18(2):139-57. Epub 2012 Aug 13.

Department of Pharmacology & Toxicology, Georgia Health Sciences University, Augusta, GA 30912, USA.

Unlabelled: Although the etiology of Parkinson's disease (PD) remains unclear, ample empirical evidence suggests that oxidative stress is a major player in the development of PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that upregulates a battery of antioxidant response element (ARE)-driven antioxidative and cytoprotective genes that defend against oxidative stress.

Aims: We evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with small molecule synthetic triterpenoids (TP) attenuate MPTP-induced PD in mice.

Results: We show that synthetic TP are thus far the most potent and direct activators of the Nrf2 pathway using a novel Neh2-luciferase reporter. They upregulate several cytoprotective genes, including those involved in glutathione biosynthesis in vitro. Oral administration of TP that were structurally modified to penetrate the brain-induced messenger RNA and protein levels for a battery of Nrf2-dependent cytoprotective genes reduced MPTP-induced oxidative stress and inflammation, and ameliorated dopaminergic neurotoxicity in mice. The neuroprotective effect of these TP against MPTP neurotoxicity was dependent on Nrf2, since treatment with TP in Nrf2 knockout mice failed to block against MPTP neurotoxicity and induce Nrf2-dependent cytoprotective genes.

Innovation: Extremely potent synthetic TP that are direct activators of the Nrf2 pathway block dopaminergic neurodegeneration in the MPTP mouse model of PD.

Conclusion: Our results indicate that activation of Nrf2/antioxidant response element (ARE) signaling by synthetic TP is directly associated with their neuroprotective effects against MPTP neurotoxicity and suggest that targeting the Nrf2/ARE pathway is a promising approach for therapeutic intervention in PD.
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http://dx.doi.org/10.1089/ars.2011.4491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514006PMC
January 2013

Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.

Chem Biol 2011 Jun;18(6):752-65

Burke Medical Research Institute, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, White Plains, NY 10605, USA.

The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found--nordihydroguaiaretic acid, fisetin, and gedunin--induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.
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http://dx.doi.org/10.1016/j.chembiol.2011.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3251032PMC
June 2011

Neurotoxic lupus autoantibodies alter brain function through two distinct mechanisms.

Proc Natl Acad Sci U S A 2010 Oct 4;107(43):18569-74. Epub 2010 Oct 4.

Burke Cornell Medical Research Institute, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, White Plains, NY 10605, USA.

Damaging interactions between antibodies and brain antigenic targets may be responsible for an expanding range of neurological disorders. In the case of systemic lupus erythematosus (SLE), patients generate autoantibodies (AAbs) that frequently bind dsDNA. Although some symptoms of SLE may arise from direct reactivity to dsDNA, much of the AAb-mediated damage originates from cross-reactivity with other antigens. We have studied lupus AAbs that bind dsDNA and cross-react with the NR2A and NR2B subunits of the NMDA receptor (NMDAR). In adult mouse models, when the blood-brain barrier is compromised, these NMDAR-reactive AAbs access the brain and elicit neuronal death with ensuing cognitive dysfunction and emotional disturbance. The cellular mechanisms that underlie these deleterious effects remain incompletely understood. Here, we show that, at low concentration, the NMDAR-reactive AAbs are positive modulators of receptor function that increase the size of NMDAR-mediated excitatory postsynaptic potentials, whereas at high concentration, the AAbs promote excitotoxicity through enhanced mitochondrial permeability transition. Other synaptic receptors are completely unaffected by the AAbs. NMDAR activation is required for producing both the synaptic and the mitochondrial effects. Our study thus reveals the mechanisms by which NMDAR-reactive AAbs trigger graded cellular alterations, which are likely to be responsible for the transient and permanent neuropsychiatric symptoms observed in patients with SLE. Our study also provides a model in which local AAb concentration determines the exact nature of the cellular response.
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http://dx.doi.org/10.1073/pnas.1006980107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2972998PMC
October 2010

Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway.

Proc Natl Acad Sci U S A 2010 Oct 20;107(40):17385-90. Epub 2010 Sep 20.

Department of Neurology and Neuroscience, Weill Medical College of Cornell University, The Burke Medical Research Institute, White Plains, NY 10605, USA.

Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H(2)O(2)) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H(2)O(2) in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H(2)O(2) in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H(2)O(2) production in astrocytes cocultured with neurons, we heterologously expressed the H(2)O(2)-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H(2)O(2). Seven hours of low-level H(2)O(2) production (∼3.7 nmol·min·mg protein) in astrocytes protected neurons from oxidative stress, but higher levels (∼130 nmol·min·mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H(2)O(2), suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H(2)O(2) yet H(2)O(2)-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H(2)O(2)-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H(2)O(2) concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.
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http://dx.doi.org/10.1073/pnas.1003996107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951414PMC
October 2010

Utilization of an in vivo reporter for high throughput identification of branched small molecule regulators of hypoxic adaptation.

Chem Biol 2010 Apr;17(4):380-91

Burke Medical Research Institute, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 785 Mamaroneck Ave, White Plains, NY 10605, USA.

Small molecules inhibiting hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are the focus of drug development efforts directed toward the treatment of ischemia and metabolic imbalance. A cell-based reporter produced by fusing HIF-1 alpha oxygen degradable domain (ODD) to luciferase was shown to work as a capture assay monitoring stability of the overexpressed luciferase-labeled HIF PHD substrate under conditions more physiological than in vitro test tubes. High throughput screening identified novel catechol and oxyquinoline pharmacophores with a "branching motif" immediately adjacent to a Fe-binding motif that fits selectively into the HIF PHD active site in in silico models. In accord with their structure-activity relationship in the primary screen, the best "hits" stabilize HIF1 alpha, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.
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http://dx.doi.org/10.1016/j.chembiol.2010.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4327942PMC
April 2010

Zinc irreversibly damages major enzymes of energy production and antioxidant defense prior to mitochondrial permeability transition.

J Biol Chem 2007 Aug 12;282(33):24373-80. Epub 2007 Jun 12.

Burke Medical Research Institute, White Plains, New York 10605, USA.

Recent observations point to the role played by Zn2+ as an inducer of neuronal death. Two Zn2+ targets have been identified that result in inhibition of mitochondrial respiration: the bc1 center and, more recently, alpha-ketoglutarate dehydrogenase. Zn2+ is also a mediator of oxidative stress, leading to mitochondrial failure, release of apoptotic peptides, and neuronal death. We now present evidence, by means of direct biochemical assays, that Zn2+ is imported through the Ca2+ uniporter and directly targets major enzymes of energy production (lipoamide dehydrogenase) and antioxidant defense (thioredoxin reductase and glutathione reductase). We demonstrate the following. (a) These matrix enzymes are rapidly inhibited by application of Zn2+ to intact mitochondria. (b) Delayed treatment with membrane-impermeable chelators has no effect, indicating rapid transport of biologically relevant quantities of Zn2+ into the matrix. (c) Membrane-permeable chelators stop but do not reverse enzyme inactivation. (d) Enzyme inhibition is rapid and irreversible and precedes the major changes associated with the mitochondrial permeability transition (MPT). (e) The extent and rate of enzyme inactivation linearly correlates with the MPT onset and propagation. (f) The Ca2+ uniporter blocker, Ruthenium Red, protects enzyme activities and delays pore opening up to 2 microm Zn2+. An additional, unidentified import route functions at higher Zn2+ concentrations. (g) No enzyme inactivation is observed for Ca2+-induced MPT. These observations strongly suggest that, unlike Ca2+, exogenous Zn2+ interferes with mitochondrial NADH production and directly alters redox protection in the matrix, contributing to mitochondrial dysfunction. Inactivation of these enzymes by Zn2+ is irreversible, and thus only their de novo synthesis can restore function, which may underlie persistent loss of oxidative carbohydrate metabolism following transient ischemia.
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http://dx.doi.org/10.1074/jbc.M611376200DOI Listing
August 2007

Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase.

Biol Chem 2007 Apr;388(4):373-80

Department of Chemical Enzymology, Chemical Faculty, M.V. Lomonosov Moscow State University, Moscow 119992, Russia.

The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it 'guards' heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.
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http://dx.doi.org/10.1515/BC.2007.050DOI Listing
April 2007

Intersection between mitochondrial permeability pores and mitochondrial fusion/fission.

Neurochem Res 2007 Apr-May;32(4-5):917-29. Epub 2007 Mar 7.

Dementia Research Service, Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605, USA.

The goal of this review is to highlight recent developments in the field of mitochondrial membrane processes, which provide new insights into the relation between mitochondrial fission/fusion events and the mitochondrial permeability transition (MPT). First, we distinguish between pore opening events at the inner and outer mitochondrial membranes. Inner membrane pore opening, or iMPT, leads to membrane depolarization, release of low molecular weight compounds, cristae reorganization and matrix swelling. Outer membrane pore opening, or oMPT, allows partial release of apoptotic proteins, while complete release requires additional remodeling of inner membrane cristae. Second, we summarize recent data that supports a similar temporal and physical separation between inner and outer mitochondrial membrane fusion events. Finally, we focus on cristae remodeling, which may be the intersection between oMPT and iMPT events. Interestingly, components of fusion machinery, such as mitofusin 2 and OPA1, appear to play a role in cristae remodeling as well.
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http://dx.doi.org/10.1007/s11064-006-9252-2DOI Listing
May 2007

pH-dependent substrate preference of pig heart lipoamide dehydrogenase varies with oligomeric state: response to mitochondrial matrix acidification.

J Biol Chem 2005 Apr 14;280(16):16106-14. Epub 2005 Feb 14.

Department of Chemical Enzymology, M. V. Lomonosov Moscow State University, 119899 Moscow, Russia.

Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. Ischemia-reperfusion also leads to mitochondrial matrix acidification and dysfunction. However, the mechanism by which matrix acidification contributes to mitochondrial dysfunction, oxidative stress, and the resultant cellular injury has not been elucidated. We observe pH-dependent equilibria between monomeric, dimeric, and a previously undescribed tetrameric form of pig heart lipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme. Dynamic light scattering studies of native LADH in aqueous solution indicate that lowering pH favors a shift in average molecular mass from higher oligomeric states to monomer. Sedimentation velocity of LADH entrapped in reverse micelles reveals dimer and tetramer at both pH 5.8 and 7.5, but monomer was observed only at pH 5.8. Enzyme activity measurements in reverse Aerosol OT micelles in octane indicate that LADH dimer and tetramer possess lipoamide dehydrogenase and diaphorase activities at pH 7.5. Upon acidification to pH 5.8 only the LADH monomer is active and only the diaphorase activity is observed. These results indicate a correlation between pH-dependent changes in the LADH reaction specificity and its oligomeric state. The acidification of mitochondrial matrix that occurs during ischemia-reperfusion injury is sufficient to alter the structure and enzymatic specificity of LADH, thereby reducing mitochondrial defenses, increasing oxidative stress, and slowing the recovery of energy metabolism. Matrix acidification may also disrupt the quaternary structure of other mitochondrial protein complexes critical for cellular homeostasis and survival.
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http://dx.doi.org/10.1074/jbc.M414285200DOI Listing
April 2005

A novel approach to distinguish between enzyme mechanisms: quasi-steady-state kinetic analysis of the prostaglandin H synthase peroxidase reaction.

Biochem J 2003 Jun;372(Pt 3):713-24

International Biotechnological Center, Lomonosov Moscow State University, Leninskiye Gory, Laboratory Building B, Room 610, Moscow 119992, Russian Federation.

A method of analysis for steady-state kinetic data has been developed that allows relationships between key partial reactions in the catalytic cycle of a functioning enzyme to be determined. The novel approach is based on a concept of scalar and vector 'kinetic connectivities' between enzyme intermediates in an arbitrary enzyme mechanism. The criterion for the agreement between experimental data and a proposed kinetic model is formulated as the kinetic connectivity of intermediate forms of the enzyme. This concept has advantages over conventional approaches and is better able to describe the complex kinetic behaviour of prostaglandin H synthase (PGHS) when catalysing the oxidation of adrenaline by H(2)O(2). To interpret the experimental data for PGHS, a generalized model for multi-substrate enzyme reactions was developed with provision for irreversible enzyme inactivation. This model showed that two enzyme intermediates must undergo inactivation during the catalytic cycle. These forms are proposed to be PGHS compound I and a compound I-adrenaline complex.
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http://dx.doi.org/10.1042/BJ20030043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223445PMC
June 2003

Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase.

J Biol Chem 2002 Mar 13;277(12):10064-72. Epub 2001 Dec 13.

Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York 10605, USA.

Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the alpha-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn(2+) (K(i) approximately 0.15 microm) in both directions. Steady-state kinetic studies revealed that Zn(2+) competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn(2+) with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn(2+) accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 microm. Activation is a consequence of Zn(2+) binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn(2+) on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn(2+) can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.
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http://dx.doi.org/10.1074/jbc.M108264200DOI Listing
March 2002