Publications by authors named "Dmitry M Hushpulian"

11 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

Breast cancer organoid model allowed to reveal potentially beneficial combinations of 3,3'-diindolylmethane and chemotherapy drugs.

Biochimie 2020 Dec 22;179:217-227. Epub 2020 Oct 22.

Faculty of Biology and Biotechnologies, Higher School of Economics, Moscow, 101000, Russia; P. A. Hertsen Moscow Oncology Research Center, Branch of the National Medical Research Radiological Center, Ministry of Health of the Russian Federation, Moscow, 125284, Russia. Electronic address:

Epigenetic alterations represent promising therapeutic targets in cancer treatment. Recently it was revealed that small molecules have the potential to act as microRNA silencers. Capacity to bind the discrete stem-looped structure of pre-miR-21 and prevent its maturation opens opportunities to utilize such compounds for the prevention of initiation, progression, and chemoresistance of cancer. Molecular simulations performed earlier identified 3,3'-diindolylmethane (DIM) as a potent microRNA-21 antagonist. However, data on DIM and microRNA-21 interplay is controversial, which may be caused by the limitations of the cell lines.
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http://dx.doi.org/10.1016/j.biochi.2020.10.007DOI Listing
December 2020

"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

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

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

Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease.

J Neurosci 2016 06;36(23):6332-51

Departments of Pharmacology and Toxicology, Neurology,

Unlabelled: A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic.

Significance Statement: Almost two centuries since its first description by James Parkinson, Parkinson's disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.
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http://dx.doi.org/10.1523/JNEUROSCI.0426-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899530PMC
June 2016

Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode.

Anal Chem 2015 Nov 13;87(21):10807-14. Epub 2015 Oct 13.

Department of Physical Chemistry, University of Sevilla , Profesor García González 1, 41012, Sevilla, Spain.

Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 μM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 μM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions.
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http://dx.doi.org/10.1021/acs.analchem.5b01710DOI Listing
November 2015

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