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.

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.

Structure-Functional Examination of Novel Ribonucleoside Hydrolase C (RihC) from Limosilactobacillus reuteri LR1.

Ribonucleoside hydrolase C (RihC, EC 3.2.2.1, 3.2.2.2, 3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases Rih and catalyzes the cleavage of ribonucleosides to nitrogenous bases and ribose. RihC is one of the enzymes that are synthesized by lactobacilli in response to the presence of Klebsiella. To characterize this protein from Limosilactobacillus reuteri LR1, we cloned and expressed it. The activity of the enzyme was studied towards a wide range of substrates, including ribonucleosides, deoxyribonucleosides as well as an arabinoside. It was shown that the enzyme is active only with ribonucleosides and arabinoside, with the best substrate being uridine. The thermal stability of this enzyme was studied, and its crystal structure was obtained, which demonstrated the tetrameric architecture of the enzyme and allowed to shed light on a correlation between its structure and enzymatic activity. Comprehensive comparisons of all known RihC structures, both existing crystal structures and computed model structures from various species, were made, allowing for the identification of structural motifs important for enzyme functioning.

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.

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.

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.

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

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.

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.

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.

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.

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

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 in silico 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.

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

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.

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

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.

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

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.