Emerging small molecule inhibitors of Bach1 as therapeutic agents: Rationale, recent advances, and future perspectives.

The transcription factor Nrf2 is the master regulator of cellular stress response, facilitating the expression of cytoprotective genes, including those responsible for drug detoxification, immunomodulation, and iron metabolism. FDA-approved Nrf2 activators, Tecfidera and Skyclarys for patients with multiple sclerosis and Friedreich's ataxia, respectively, are non-specific alkylating agents exerting side effects. Nrf2 is under feedback regulation through its target gene, transcriptional repressor Bach1. Specifically, in Parkinson's disease and other neurodegenerative diseases with Bach1 dysregulation, excessive Bach1 accumulation interferes with Nrf2 activation. Bach1 is a heme sensor protein, which, upon heme binding, is targeted for proteasomal degradation, relieving the repression of Nrf2 target genes. Ideally, a combination of Nrf2 stabilization and Bach1 inhibition is necessary to achieve the full therapeutic benefits of Nrf2 activation. Here, we discuss recent advances and future perspectives in developing small molecule inhibitors of Bach1, highlighting the significance of the Bach1/Nrf2 signaling pathway as a promising neurotherapeutic strategy.

Degradation Kinetics of Disulfide Cross-Linked Microgels: Real-Time Monitoring by Confocal Microscopy.

Although biodegradable microgels represent a useful drug delivery system, questions remain regarding the kinetics of gel degradation and subsequent drug release. Spherical microgels (~Ø10-300 µm) were synthesized using an inverse suspension polymerization method. Specifically, acrylamide and acrylonitrile monomers were thermally co-polymerized with N,N'-bis(acryloyl)cystamine as a cross-linker with disulfide bridges. The kinetics and mechanism of degradation of these cross-linked, degradable, fluorescently labeled microgels (PAAm-AN-BAC-FA) were quantitatively studied under confocal microscopy at various concentrations of glutathione (reducing agent) ranging from 0.06 to 91.8 mM. It was found that polymer network degradation via the cleavage of disulfide bonds was accompanied by two overlapping processes: diffusion-driven swelling and dissolution-driven erosion. A slow increase in microgel size (swelling) resulted from partial de-cross-linking in the bulk of the microgel, whereas a faster decrease in fluorescence intensity (erosion) resulted from the complete cleavage of disulfide bonds and the release of uncleaved polymeric chains from the microgel immediate surface into the solution. Swelling and erosion exhibited distinct kinetics and characteristic times. Importantly, the dependence of kinetics on glutathione concentration for both swelling and erosion suggests that degradation would occur faster in cancer cells (higher concentration of reductants) than in normal cells (lower concentration of reductants), such that drug release profiles would be correspondingly different. A greater comprehension of microgel degradation kinetics would help in (i) predicting the drug release profiles for novel multifunctional drug delivery systems and (ii) using redox-sensitive degradable hydrogel particles to determine the concentrations of reducing agents either in vitro or in vivo.

Carotenoids from Starfish Patiria pectinifera: Therapeutic Activity in Models of Inflammatory Diseases.

The carotenoids mixture (MC) isolated from the starfish Patiria. pectinifera contains more than 50% astaxanthin, 4-6% each zeaxanthine and lutein, and less pharmacologically active components such as free fatty acids and their glycerides. Astaxanthin, the major component of MC, belongs to the xanthophyll class of carotenoids, and is well known for its antioxidant properties. In this work, in vitro and in vivo studies on the biological activity of MC were carried out. The complex was shown to exhibit anti-inflammatory, anti-allergic and cancer-preventive activity, without any toxicity at a dose of 500 mg/kg. MC effectively improves the clinical picture of the disease progressing, as well as normalizing the cytokine profile and the antioxidant defense system in the in vivo animal models of inflammatory diseases, namely: skin carcinogenesis, allergic contact dermatitis (ACD) and systemic inflammation (SI). In the skin carcinogenesis induced by 7,12-dimethylbenzanthracene, the incidence of papillomas was decreased 1.5 times; 1% MC ointment form in allergic contact dermatitis showed an 80% reduced severity of pathomorphological skin manifestations. Obtained results show that MC from starfish P. pectinifera is an effective remedy for the treatment and prevention of inflammatory processes.

Synthesis and Anti-Inflammatory Activity of 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-Derived NRF2 Activators.

This is the first study investigating the nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity of compounds containing a new scaffold, tetrahydrobenzo[b]thiophene. Eighteen compounds were synthesised and confirmed their NRF2 activation through NQO1 enzymatic activity and mRNA expression of NQO1 and HO-1 in Hepa-1c1c7 cells. The compounds disrupted the interaction between Kelch-like ECH-associated protein 1 (KEAP1) and NRF2 via interfering with the KEAP1's Kelch domain. The compounds exhibited anti-inflammatory activity in Escherichia coli Lipopolysaccharide (LPSEc )-stimulated RAW 264.7 cells. The anti-inflammatory activity of the compounds was associated with their ability to activate NRF2. The compounds reversed the elevated levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IFN-γ) and inflammatory mediators (PGE2, COX-2, and NF-κB). The compounds were metabolically stable in human, rat, and mouse liver microsomes and showed optimum half-life (T1/2 ) and intrinsic clearance (Clint ). The binding mode of the compounds and physicochemical properties were predicted via in silico studies.

Harnessing the Therapeutic Potential of the Nrf2/Bach1 Signaling Pathway in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative movement disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although a complex interplay of multiple environmental and genetic factors has been implicated, the etiology of neuronal death in PD remains unresolved. Various mechanisms of neuronal degeneration in PD have been proposed, including oxidative stress, mitochondrial dysfunction, neuroinflammation, α-synuclein proteostasis, disruption of calcium homeostasis, and other cell death pathways. While many drugs individually targeting these pathways have shown promise in preclinical PD models, this promise has not yet translated into neuroprotective therapies in human PD. This has consequently spurred efforts to identify alternative targets with multipronged therapeutic approaches. A promising therapeutic target that could modulate multiple etiological pathways involves drug-induced activation of a coordinated genetic program regulated by the transcription factor, nuclear factor E2-related factor 2 (Nrf2). Nrf2 regulates the transcription of over 250 genes, creating a multifaceted network that integrates cellular activities by expressing cytoprotective genes, promoting the resolution of inflammation, restoring redox and protein homeostasis, stimulating energy metabolism, and facilitating repair. However, FDA-approved electrophilic Nrf2 activators cause irreversible alkylation of cysteine residues in various cellular proteins resulting in side effects. We propose that the transcriptional repressor of BTB and CNC homology 1 (Bach1), which antagonizes Nrf2, could serve as a promising complementary target for the activation of both Nrf2-dependent and Nrf2-independent neuroprotective pathways. This review presents the current knowledge on the Nrf2/Bach1 signaling pathway, its role in various cellular processes, and the benefits of simultaneously inhibiting Bach1 and stabilizing Nrf2 using non-electrophilic small molecules as a novel therapeutic approach for PD.

Structure-Activity Relationships and Transcriptomic Analysis of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors.

To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and "branched-tail" oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors' IC50 values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure-activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 µM concentrations matching the effect of 30 µM roxadustat and 500 µM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.

Probable Mechanisms of Doxorubicin Antitumor Activity Enhancement by Ginsenoside Rh2.

Ginsenoside Rh2 increases the efficacy of doxorubicin (DOX) treatment in murine models of solid and ascites Ehrlich's adenocarcinoma. In a solid tumor model (treatment commencing 7 days after inoculation), DOX + Rh2 co-treatment was significantly more efficacious than DOX alone. If treatment was started 24 h after inoculation, the inhibition of tumor growth of a solid tumor for the DOX + Rh2 co-treatment group was complete. Furthermore, survival in the ascites model was dramatically higher for the DOX + Rh2 co-treatment group than for DOX alone. Mechanisms underlying the combined DOX and Rh2 effects were studied in primary Ehrlich's adenocarcinoma-derived cells and healthy mice's splenocytes. Despite the previously established Rh2 pro-oxidant activity, DOX + Rh2 co-treatment revealed no increase in ROS compared to DOX treatment alone. However, DOX + Rh2 treatment was more effective in suppressing Ehrlich adenocarcinoma cell adhesion than either treatment alone. We hypothesize that the benefits of DOX + Rh2 combination treatment are due to the suppression of tumor cell attachment/invasion that might be effective in preventing metastatic spread of tumor cells. Ginsenoside Rh2 was found to be a modest activator in a Neh2-luc reporter assay, suggesting that Rh2 can activate the Nrf2-driven antioxidant program. Rh2-induced direct activation of Nrf2 might provide additional benefits by minimizing DOX toxicity towards non-cancerous cells.

Bach1 derepression is neuroprotective in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of nigrostriatal dopaminergic neurons. Mounting evidence suggests that Nrf2 is a promising target for neuroprotective interventions in PD. However, electrophilic chemical properties of the canonical Nrf2-based drugs cause irreversible alkylation of cysteine residues on cellular proteins resulting in side effects. Bach1 is a known transcriptional repressor of the Nrf2 pathway. We report that Bach1 levels are up-regulated in PD postmortem brains and preclinical models. Bach1 knockout (KO) mice were protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity and associated oxidative damage and neuroinflammation. Functional genomic analysis demonstrated that the neuroprotective effects in Bach1 KO mice was due to up-regulation of Bach1-targeted pathways that are associated with both Nrf2-dependent antioxidant response element (ARE) and Nrf2-independent non-ARE genes. Using a proprietary translational technology platform, a drug library screen identified a substituted benzimidazole as a Bach1 inhibitor that was validated as a nonelectrophile. Oral administration of the Bach1 inhibitor attenuated MPTP neurotoxicity in pre- and posttreatment paradigms. Bach1 inhibitor-induced neuroprotection was associated with the up-regulation of Bach1-targeted pathways in concurrence with the results from Bach1 KO mice. Our results suggest that genetic deletion as well as pharmacologic inhibition of Bach1 by a nonelectrophilic inhibitor is a promising therapeutic approach for PD.

HIF1α stabilization in hypoxia is not oxidant-initiated.

Hypoxic adaptation mediated by HIF transcription factors requires mitochondria, which have been implicated in regulating HIF1α stability in hypoxia by distinct models that involve consuming oxygen or alternatively converting oxygen into the second messenger peroxide. Here, we use a ratiometric, peroxide reporter, HyPer to evaluate the role of peroxide in regulating HIF1α stability. We show that antioxidant enzymes are neither homeostatically induced nor are peroxide levels increased in hypoxia. Additionally, forced expression of diverse antioxidant enzymes, all of which diminish peroxide, had disparate effects on HIF1α protein stability. Moreover, decrease in lipid peroxides by glutathione peroxidase-4 or superoxide by mitochondrial SOD, failed to influence HIF1α protein stability. These data show that mitochondrial, cytosolic or lipid ROS were not necessary for HIF1α stability, and favor a model where mitochondria contribute to hypoxic adaptation as oxygen consumers.

Non-canonical Keap1-independent activation of Nrf2 in astrocytes by mild oxidative stress.

The transcription factor Nrf2 is a stress-responsive master regulator of antioxidant, detoxification and proteostasis genes. In astrocytes, Nrf2-dependent gene expression drives cell-autonomous cytoprotection and also non-cell-autonomous protection of nearby neurons, and can ameliorate pathology in several acute and chronic neurological disorders associated with oxidative stress. However, the value of astrocytic Nrf2 as a therapeutic target depends in part on whether Nrf2 activation by disease-associated oxidative stress occludes the effect of any Nrf2-activating drug. Nrf2 activation classically involves the inhibition of interactions between Nrf2's Neh2 domain and Keap1, which directs Nrf2 degradation. Keap1 inhibition is mediated by the modification of cysteine residues on Keap1, and can be triggered by electrophilic small molecules such as tBHQ. Here we show that astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling. Keap1 deficiency elevates basal Nrf2 target gene expression in astrocytes and occludes the effects of tBHQ, oxidative stress still induced strong Nrf2-dependent gene expression in Keap1-deficient astrocytes. Moreover, while tBHQ prevented protein degradation mediated via Nrf2's Neh2 domain, oxidative stress did not, consistent with a Keap1-independent mechanism. Moreover the effects of oxidative stress and tBHQ on Nrf2 target gene expression are additive, not occlusive. Mechanistically, oxidative stress enhances the transactivation potential of Nrf2's Neh5 domain in a manner dependent on its Cys-191 residue. Thus, astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling, meaning that further Nrf2 activation by Keap1-inhibiting drugs may be a viable therapeutic strategy.

Identification of a potent Nrf2 displacement activator among aspirin-containing prodrugs.

Aspirin is a desired leaving group in prodrugs aimed at treatment of neurodegeneration and other conditions. A library of aspirin derivatives of various scaffolds potentially activating Nrf2 has been tested in Neh2-luc reporter assay which screens for direct Nrf2 protein stabilizers working via disruption of Nrf2-Keap1 interaction. Most aspirin prodrugs had a pro-alkylating or pro-oxidant motif in the structure and, therefore, were toxic at high concentrations. However, among the active compounds, we identified a molecule resembling a well-known Nrf2 displacement activator, bis-1,4-(4-methoxybenzenesulfonamidyl) naphthalene (NMBSA). The direct comparison of the newly identified compound with NMBSA and its improved analog in the reporter assay showed no quenching with N-acetyl cysteine, thus pointing to Nrf2 stabilization mechanism without cysteine alkylation. The potency of the newly identified compound in the reporter assay was much stronger than NMBSA, despite its inhibitory action in the commercial fluorescence polarization assay was observed only in the millimolar range. Molecular docking predicted that mono-deacetylation of the novel prodrug should generate a potent displacement activator. The time-course of reporter activation with the novel prodrug had a pronounced lag-period pointing to a plausible intracellular transformation leading to an active product. Treatment of the novel prodrug with blood plasma or cell lysate demonstrated stepwise deacetylation as judge by liquid chromatography-mass spectrometry (LC-MS). Hence, the esterase-catalyzed hydrolysis of the prodrug liberates only acetyl groups from aspirin moiety and generates a potent Nrf2 activator. The discovered mechanism of prodrug activation makes the newly identified compound a promising lead for future optimization studies.

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

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.

Neuroprotective Effect of HIF Prolyl Hydroxylase Inhibition in an In Vitro Hypoxia Model.

A novel potent analog of the branched tail oxyquinoline group of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, neuradapt, has been studied in two treatment regimes in an in vitro hypoxia model on murine primary hippocampal cultures. Neuradapt activates the expression of HIF1 and HIF2 target genes and shows no toxicity up to 20 µM, which is more than an order of magnitude higher than its biologically active concentration. Cell viability, functional activity, and network connectivity between the elements of neuronal networks have been studied using a pairwise correlation analysis of the intracellular calcium fluctuations in the individual cells. An immediate treatment with 1 µÐœ and 15 µÐœ neuradapt right at the onset of hypoxia not only protects from the death, but also maintains the spontaneous calcium activity in nervous cells at the level of the intact cultures. A similar neuroprotective effect in the post-treatment scenario is observed for 15 µÐœ, but not for 1 µÐœ neuradapt. Network connectivity is better preserved with immediate treatment using 1 µÐœ neuradapt than with 15 µÐœ, which is still beneficial. Post-treatment with neuradapt did not restore the network connectivity despite the observation that neuradapt significantly increased cell viability at 1 µÐœ and functional activity at 15 µÐœ. The preservation of cell viability and functional activity makes neuradapt promising for further studies in a post-treatment scenario, since it can be combined with other drugs and treatments restoring the network connectivity of functionally competent cells.

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

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.

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

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.

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

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 (Aß) deposition, tau hyperphosphorylation and oxidative stress. Benfotiamine (BFT) rescued cognitive deficits and reduced Aß 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.

In vitro and in silico liver models: Current trends, challenges and opportunities.

Most common drug development failures originate from either bioavailability problems, or unexpected toxic effects. The culprit is often the liver, which is responsible for biotransformation of a majority of xenobiotics. Liver may be modeled using "liver on a chip" devices, which may include established cell lines, primary human cells, and stem cell-derived hepatocyte-like cells. The choice of biological material along with its processing and maintenance greatly influence both the device performance and the resultant toxicity predictions. Impediments to the development of "liver on a chip" technology include the problems with standardization of cells, limitations imposed by culturing and the necessity to develop more complicated fluidic contours. Fortunately, recent breakthroughs in the development of cell-based reporters, including ones with fluorescent label, permits monitoring of the behavior of the cells embed into the "liver on a chip" devices. Finally, a set of computational approaches has been developed to model both particular toxic response and the homeostasis of human liver as a whole; these approaches pave a way to enhance the in silico stage of assessment for a potential toxicity.

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

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.

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

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.

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.

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.