Transcription factor NF-E2 p45-related factor 2 (NRF2, gene name NFE2L2) and its principal negative regulator, the E3 ligase adapter Kelch-like ECH-associated protein 1 (KEAP1), play a critical role in the development and progression of chronic diseases of the lung and liver, autoimmune, neurodegenerative and metabolic disorders, and also cancer. NRF2 activation provides cytoprotection against numerous pathologies characterized by chronic inflammation, metabolic alterations and redox disturbances. One NRF2 activator has received clinical approval and several electrophilic modifiers of the cysteine-based sensor KEAP1 and inhibitors of its interaction with NRF2 are now in clinical development. However, challenges regarding target specificity, pharmacodynamic properties, efficacy, and safety remain.

Oxidative stress is generated by reactive oxygen species (ROS) produced in response to metabolic activity and environmental factors. Increased oxidative stress is associated with the pathophysiology of a broad spectrum of inflammatory diseases. Cellular response to excess ROS involves the induction of antioxidant response element (ARE) genes under control of the transcriptional activator Nrf2 and the transcriptional repressor Bach1. The development of synthetic small molecules that activate the protective anti-oxidant response network is of major therapeutic interest. Traditional small molecules targeting ARE-regulated gene activation (e.g., bardoxolone, dimethyl fumarate) function by alkylating numerous proteins including Keap1, the controlling protein of Nrf2. An alternative is to target the repressor Bach1. Bach1 has an endogenous ligand, heme, that inhibits Bach1 binding to ARE, thus allowing Nrf2-mediated gene expression including that of heme-oxygenase-1 (HMOX1), a well described target of Bach1 repression. In this report, normal human lung fibroblasts were used to screen a collection of synthetic small molecules for their ability to induce HMOX1. A class of HMOX1-inducing compounds, represented by HPP-4382, was discovered. These compounds are not reactive electrophiles, are not suppressed by N-acetyl cysteine, and do not perturb either ROS or cellular glutathione. Using RNAi, we further demonstrate that HPP-4382 induces HMOX1 in an Nrf2-dependent manner. Chromatin immunoprecipitation verified that HPP-4382 treatment of NHLF cells reciprocally coordinated a decrease in binding of Bach1 and an increase of Nrf2 binding to the HMOX1 E2 enhancer. Finally we show that HPP-4382 can inhibit Bach1 activity in a reporter assay that measures transcription driven by the human HMOX1 E2 enhancer. Our results suggest that HPP-4382 is a novel activator of the antioxidant response through the modulation of Bach1 binding to the ARE binding site of target genes.

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.

Bone destructive diseases such as periodontitis are common worldwide and are caused by excessive osteoclast formation and activation. Receptor activator of nuclear factor-κB ligand (RANKL) is essential factor for osteoclastogenesis. This triggers reactive oxygen species (ROS), which has a key role in intracellular signaling as well exerting cytotoxicity. Cells have protective mechanisms against ROS, such as nuclear factor E2-related factor 2 (Nrf2), which controls the expression of many antioxidant enzyme genes. Conversely, BTB and CNC homology 1 (Bach1), a competitor for Nrf2, transcriptionally represses the expression of anti-oxidant enzymes. Previously, we demonstrated that RANKL induces Bach1 nuclear import and attenuates the expression of Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular ROS signaling and osteoclastogenesis. However, it remains unknown if Bach1 inhibitors attenuate osteoclastogenesis. In this study, we hypothesized that Bach1 inhibition would exert an anti-osteoclastogenic effects via diminishing of intracellular ROS signaling through augmented antioxidation. We used RAW 264.7 cells as osteoclast progenitor cells. Using flow cytometry, we found that Bach1 inhibitors attenuated RANKL-mediated ROS generation, which resulted in the inhibition of osteoclastogenesis. Local injection of a Bach1 inhibitor into the calvaria of male BALB/c mice blocked bone destruction induced by lipopolysaccharide. In conclusion, we demonstrate that Bach1 inhibitor attenuates RANKL-mediated osteoclastogenesis and bone destruction in mice by inducing the expression of Nrf2-regulated antioxidant enzymes that consequently decrease intracellular ROS levels. Bach1 inhibitors have potential in inhibiting bone destructive diseases such as periodontitis, rheumatoid arthritis and osteoporosis.

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.

We report for the first time a novel class of compounds that specifically modulate the Bach1 transcriptional repressor pathway. In cellula, the compounds selectively inhibit the activity of the transcriptional repressor Bach1 resulting in transcription of a network of antioxidant and cytoprotective genes including HMOX1. HPP-1971, a member of this class, is a potent and selective Bach1 inhibitor that induces HMOX1 > 40-fold with a potency of 408 nM in human lung fibroblasts. To assess activity in vivo, we tested HPP-1971 in the Goldblatt model of renovascular hypertension. Sprague Dawley rats were implanted with in dwelling pressure telemeter probes and subsequently underwent sham surgery or placement of a 0.25 mm silver clip around the left renal artery. HPP-1971 treatment, dosed orally at 1,3,10 and 30 mpk, commenced three days following clip surgery, and continued for 18 days. At study completion, blood pressure, clipped kidney weight, renal HMOX1 enzyme activity and plasma aldosterone levels were measured (see Table). HPP-1971 attenuated both kidney atrophy and the increase in blood pressure in a dose dependent manner with significant differences seen at 3, 10 and 30 mpk (p<.0001). HMOX1 enzyme activity in the clipped kidney increased with treatment, reaching a maximum of 5.7 ± 0.9 nM/hr/mg at 30 mpk relative to sham operated animals (p<.0001). Plasma aldosterone levels increased in 2K1C animals compared to sham controls but were reduced by HPP-1971 treatment. These findings define a novel role for Bach1 suppressors in counteracting the influence of the RAAS system on hypertension and kidney atrophy in the 2K1C model of renovascular hypertension.

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