This work relates to the design and synthesis of a series of novel multi-target directed ligands (MTDLs), i.e., compounds 4a-l, via a convenient one-pot three-component Hantzsch reaction. This approach targeted calcium channel antagonism, antioxidant capacity, cathepsin S inhibition, and interference with Nrf2 transcriptional activation. Of these MTDLs, 4i emerged as a promising compound, demonstrating robust antioxidant activity, the ability to activate Nrf2-ARE pathways, as well as calcium channel blockade and cathepsin S inhibition. Dihydropyridine 4i represents the first example of an MTDL that combines these biological activities.

The skin sensitization potential of agrochemicals can be assessed using laboratory methods such as the keratinocyte activation assay so that their use in regulatory toxicology might replace experimental animal testing. Here, we evaluated the skin sensitization potential of 11 agrochemicals by using an antioxidant response element-nuclear factor erythroid 2 luciferase assay in KeratinoSens and LuSens cells and applying a skin sensitization adverse outcome pathway (AOP). The KeratinoSens and LuSens assays consistently evaluated the skin sensitization potential of 10/11 agrochemicals with reference to animal testing databases. Benomyl, pretilachlor, fluazinam, terbufos, butachlor, and carbosulfan were correctly detected as sensitizers, and glufosinate ammonium, oxiadiazon, tebuconazole, and etofenprox were correctly detected as non-sensitizers. For diazinon, the skin sensitizing potential was positive in the KeratinoSens assay but not in the LuSens assay. These results suggest that the evaluation of in vitro skin sensitization using the AOP mechanism can be applied to assess active agrochemicals.

A hallmark of aging and neurodegenerative diseases is a disruption of proteome homeostasis (\"proteostasis\") that is caused to a considerable extent by a decrease in the efficiency of protein degradation systems. The ubiquitin proteasome system (UPS) is the major cellular pathway involved in the clearance of small, short-lived proteins, including amyloidogenic proteins that form aggregates in neurodegenerative diseases. Age-dependent decreases in proteasome subunit expression coupled with the inhibition of proteasome function by aggregated UPS substrates result in a feedforward loop that accelerates disease progression. Nuclear factor erythroid 2- like 1 (NFE2L1) is a transcription factor primarily responsible for the proteasome inhibitor-induced \"bounce-back effect\" regulating the expression of proteasome subunits. NFE2L1 is localized to the endoplasmic reticulum (ER), where it is rapidly degraded under basal conditions by the ER-associated degradation (ERAD) pathway. Under conditions leading to proteasome impairment, NFE2L1 is cleaved and transported to the nucleus, where it binds to antioxidant response elements (AREs) in the promoter region of proteasome subunit genes, thereby stimulating their transcription. In this review, we summarize the role of UPS impairment in aging and neurodegenerative disease etiology and consider the potential benefit of enhancing NFE2L1 function as a strategy to upregulate proteasome function and alleviate pathology in neurodegenerative diseases.

Tumorigenesis entails circumventing cell-intrinsic regulatory mechanisms while avoiding extrinsic immune surveillance and other host defence systems. Nevertheless, how tumour cells\' ability to eliminate misfolded proteins affects immune surveillance remains poorly understood. In this study, we find that overexpression of murine tripartite motif-containing protein 30a (TRIM30a) sensitises tumour cells to natural killer (NK) cells-mediated cytolysis. TRIM30a has no effect on tumour cell proliferation or apoptosis in vitro. However, TRIM30a-overexpressing tumour cells grow substantially slower than control tumour cells in immune-competent mice but not in NK cell-depleted mice. [Correction added on 04 October 2023, after first online publication: \'NK-depleted\' has been changed to \'NK cell-depleted\' in the preceding sentence.] Mechanistically, TRIM30a overexpression impedes the clearance of misfolded proteins and increases the production of reactive oxygen species induced by proteotoxic stress, implying that TRIM30a impairs protein quality control (PQC) systems in tumour cells. Furthermore, TRIM30a reduces expression of genes encoding proteasome subunits and antioxidant proteins. Our study demonstrates that TRIM30a is a potential tumour suppressor and immune modulator that promotes tumour cytolysis by NK cells, and suggests that an enhanced PQC and antioxidant capacity is an integral part of the immune escape mechanism during tumorigenesis.

Sickle cell disease (SCD) is the most common β-hemoglobinopathy caused by various mutations in the adult β-globin gene resulting in sickle hemoglobin production, chronic hemolytic anemia, pain, and progressive organ damage. The best therapeutic strategies to manage the clinical symptoms of SCD is the induction of fetal hemoglobin (HbF) using chemical agents. At present, among the Food and Drug Administration-approved drugs to treat SCD, hydroxyurea is the only one proven to induce HbF protein synthesis, however, it is not effective in all people. Therefore, we evaluated the ability of the novel Bach1 inhibitor, HPP-D to induce HbF in KU812 cells and primary sickle erythroid progenitors. HPP-D increased HbF and decreased Bach1 protein levels in both cell types. Furthermore, chromatin immunoprecipitation assay showed reduced Bach1 and increased NRF2 binding to the γ-globin promoter antioxidant response elements. We also observed increased levels of the active histone marks H3K4Me1 and H3K4Me3 supporting an open chromatin configuration. In primary sickle erythroid progenitors, HPP-D increased γ-globin transcription and HbF positive cells and reduced sickled erythroid progenitors under hypoxia conditions. Collectively, our data demonstrate that HPP-D induces γ-globin gene transcription through Bach1 inhibition and enhanced NRF2 binding in the γ-globin promoter antioxidant response elements.

Sickle cell disease (SCD) is a monogenic inheritable disease characterized by severe anemia, increased hemolysis, and recurrent, painful vaso-occlusive crises due to the polymerization of hemoglobin S (HbS)-generated oxidative stress. Up until now, only four drugs are approved for SCD in the US. However, each of these drugs affects only a limited array of SCD pathologies. Importantly, curative therapies, such as gene therapy, or hematopoietic stem cell transplantation are not available for every patient because of their high costs, availability of donor matching, and their serious adverse effects. Therefore, there is an unmet medical need for novel therapeutic strategies that target broader SCD sequelae. SCD phenotypic severity can be alleviated by increasing fetal hemoglobin (HbF) expression. This results in the inhibition of HbS polymerization and thus sickling, and a reduction in oxidative stress. The efficacy of HbF is due to its ability to dilute HbS levels below the threshold required for polymerization and to influence HbS polymer stability in RBCs. Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein-1 (Keap1)-complex signaling is one of the most important cytoprotective signaling controlling oxidative stress. Nrf2 is present in most organs and, after dissociation from Keap1, it accumulates in the cytoplasm, then translocates to the nucleus where it binds to the antioxidant response element (ARE) sequences and increases the expression of various cytoprotective antioxidant genes. Keeping this in mind, various researchers have proposed a role of multiple agents, more importantly tert-Butylhydroquinone (tBHQ), curcumin, etc., (having electrophilic properties) in inhibiting keap1 activity, so that Nrf2 can translocate to the nucleus to activate the gamma globin gene, thus maintaining alpha-hemoglobin-stabilizing protein (AHSP) and HbF levels. This leads to reduced oxidative stress, consequently minimizing SCD-associated complications. In this review, we will discuss the role of the Keap-1-Nrf2 complex in hemoglobinopathies, especially in SCD, and how this complex might represent a better target for more effective treatment options.

Host-derived cellular pathways can provide an unfavorable environment for virus replication. These pathways have been a subject of interest for herpesviruses, including the betaherpesvirus human cytomegalovirus (HCMV). Here, we demonstrate that a compound, ARP101, induces the noncanonical sequestosome 1 (SQSTM1)/p62-Keap1-Nrf2 pathway for HCMV suppression. ARP101 increased the levels of both LC3 II and SQSTM1/p62 and induced phosphorylation of p62 at the C-terminal domain, resulting in its increased affinity for Keap1. ARP101 treatment resulted in Nrf2 stabilization and translocation into the nucleus, binding to specific promoter sites and transcription of antioxidant enzymes under the antioxidant response element (ARE), and HCMV suppression. Knockdown of Nrf2 recovered HCMV replication following ARP101 treatment, indicating the role of the Keap1-Nrf2 axis in HCMV inhibition by ARP101. SQSTM1/p62 phosphorylation was not modulated by the mTOR kinase or casein kinase 1 or 2, indicating ARP101 engages other kinases. Together, the data uncover a novel antiviral strategy for SQSTM1/p62 through the noncanonical Keap1-Nrf2 axis. This pathway could be further exploited, including the identification of the responsible kinases, to define the biological events during HCMV replication. IMPORTANCE Antiviral treatment for human cytomegalovirus (HCMV) is limited and suffers from the selection of drug-resistant viruses. Several cellular pathways have been shown to modulate HCMV replication. The autophagy receptor sequestosome 1 (SQSTM1)/p62 has been reported to interact with several HCMV proteins, particularly with components of HCMV capsid, suggesting it plays a role in viral replication. Here, we report on a new and unexpected role for SQSTM1/p62, in HCMV suppression. Using a small-molecule probe, ARP101, we show SQSTM1/p62 phosphorylation at its C terminus domain initiates the noncanonical Keap1-Nrf2 axis, leading to transcription of genes under the antioxidant response element, resulting in HCMV inhibition in vitro. Our study highlights the dynamic nature of SQSTM1/p62 during HCMV infection and how its phosphorylation activates a new pathway that can be exploited for antiviral intervention.

Numerous studies have reported that tangeretin is a polymethoxylated flavone with a variety of biological activates, but little research has been done on the antioxidant mechanism of tangeretin. Hence, we investigated the effect of tangeretin on the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway and its potential molecular mechanisms by in vitro and in silico research. The results of molecular docking suggested that tangeretin bound at the top of the central pore of Kelch-like ECH-associated protein 1 (Keap1) Kelch domain, and the hydrophobic and hydrogen bond interactions contributed to their stable binding. Herein, the regulation of Nrf2-ARE pathway by tangeretin was explored in the human embryonic kidney cell line HEK293T, which is relatively easy to be transfected. Upon binding to tangeretin, Nrf2 translocated to the nucleus of HEK293T cells, which in turn activated the Nrf2-ARE pathway. Luciferase reporter gene analysis showed that tangeretin significantly induced ARE-mediated transcriptional activation. Real-time PCR and Western blot assays showed that tangeretin induced the gene and protein expressions of Nrf2-mediated targets, including heme oxygenase 1 (HO-1), nicotinamide adenine dinucleotide phosphate (NADPH) quinone dehydrogenase 1 (NQO1), and glutamate-cysteine ligase (GCLM). In addition, tangeretin could effectively scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. In summary, tangeretin may be a potential antioxidant via activating the Nrf2-ARE pathway.

UNASSIGNED: Friedreich ataxia (FRDA) is a rare autosomal recessive degenerative disorder characterized by ataxia, dysarthria, diabetes, cardiomyopathy, scoliosis, and occasionally vision loss in late-stage disease. The discovery of the abnormal gene in FRDA and its product frataxin has provided insight into the pathophysiology and mechanisms of treatment.
UNASSIGNED: Although the neurologic phenotype of FRDA is well defined, there are currently no established pharmacological treatments. Omaveloxolone, a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, is currently under review by the Food and Drug Administration (FDA) and has the potential to be the first approved treatment for FRDA. In the present report, we have reviewed the basic and clinical literature on Nrf2 deficiency in FRDA, and evidence for the benefit of omaveloxolone.
UNASSIGNED: The present perspective suggests that omaveloxolone is a rational and efficacious therapy that is possibly disease modifying in treatment of FRDA.

The human body is continuously exposed to xenobiotics and internal or external oxidants. The health risk assessment of exogenous chemicals remains a complex and challenging issue. Alternative toxicological test methods have become an essential strategy for health risk assessment. As a core regulator of constitutive and inducible expression of antioxidant response element (ARE)-dependent genes, nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in maintaining cellular redox homeostasis. Consistent with the properties of Nrf2-mediated antioxidant response, Nrf2-ARE activity is a direct indicator of oxidative stress and thus has been used to identify and characterize oxidative stressors and redox modulators. To screen and distinguish chemicals or environmental insults that affect the cellular antioxidant activity and/or induce oxidative stress, various in vitro cell models expressing distinct ARE reporters with high-throughput and high-content properties have been developed. These ARE-reporter systems are currently widely applied in drug discovery and safety assessment. In the present review, we provide an overview of the basic structures and applications of various ARE-reporter systems employed for discovering Nrf2-ARE modulators and characterizing oxidative stressors.