Why Adam Sinned / music by Alex Rogers; words by Alex Rogers

Cover: drawing of a serpent tempting a bust of Eve with an apple; photo inset of singer Aida Overton Walker; Publisher: The Gotham-Attucks Music Co. (New York)https://egrove.olemiss.edu/sharris_b/1073/thumbnail.jp

Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases

The transcription factor NF-E2 p45-related factor 2 (NRF2; encoded by NFE2L2) and its principal negative regulator, the E3 ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1), are critical in the maintenance of redox, metabolic and protein homeostasis, as well as the regulation of inflammation. Thus, NRF2 activation provides cytoprotection against numerous pathologies including chronic diseases of the lung and liver; autoimmune, neurodegenerative and metabolic disorders; and cancer initiation. 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.This work was supported by grants SAF2015-71304-REDT and SAF2016-76520-R from the Spanish Ministry Economy and Competitiveness; P_37_732/2016 REDBRAIN from the European Regional Development Fund; Competitiveness Operational Program 2014–2020; US National Institutes of Health grant R35 CA197222; Cancer Research UK grant C20953/A18644; Medical Research Council grant MR/N009851/1; Biotechnology and Biological Sciences Research Council grant BB/L01923X/1; Tenovus Scotland grant T17/14; and grant 275147 from the Academy of Finland, Sigrid Juselius Foundation and Finnish Cancer Foundation.Peer reviewe

Transcription factor NRF2 as a therapeutic target for chronic diseases: a systems medicine approach

Systems medicine has a mechanism-based rather than a symptom- or organ-based approach to disease and identifies therapeutic targets in a nonhypothesis-driven manner. In this work, we apply this to transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) by cross-validating its position in a protein-protein interaction network (the NRF2 interactome) functionally linked to cytoprotection in low-grade stress, chronic inflammation, metabolic alterations, and reactive oxygen species formation. Multiscale network analysis of these molecular profiles suggests alterations of NRF2 expression and activity as a common mechanism in a subnetwork of diseases (the NRF2 diseasome). This network joins apparently heterogeneous phenotypes such as autoimmune, respiratory, digestive, cardiovascular, metabolic, and neurodegenerative diseases, along with cancer. Importantly, this approach matches and confirms in silico several applications for NRF2-modulating drugs validated in vivo at different phases of clinical development. Pharmacologically, their profile is as diverse as electrophilic dimethyl fumarate, synthetic triterpenoids like bardoxolone methyl and sulforaphane, protein-protein or DNA-protein interaction inhibitors, and even registered drugs such as metformin and statins, which activate NRF2 and may be repurposed for indications within the NRF2 cluster of disease phenotypes. Thus, NRF2 represents one of the first targets fully embraced by classic and systems medicine approaches to facilitate both drug development and drug repurposing by focusing on a set of disease phenotypes that appear to be mechanistically linked. The resulting NRF2 drugome may therefore rapidly advance several surprising clinical options for this subset of chronic diseases

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

Induction of heme oxygenase I (HMOX1) by HPP-4382: a novel modulator of Bach1 activity.

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

HPP-4382 increases cellular glutathione levels: NHLF cells grown in 96-well Optilux plates were treated with compounds for 4 hours (BSO, 200 µM; Sulphorafane, 10 µM; CDDO-Me, 0.1 µM; HPP-1014, 10 µM; CoPP, 10 µM; HPP-4382 3 µM) and glutathione levels were determined using the GSH/GSSG-Glo Assay Kit (Promega).

<p>Positive values show pairs of means that are significantly different. All samples in duplicate, error bars represent standard deviation compared to DMSO. *, <i>p</i><0.05; **, <i>p</i><0.01, ***; <i>p</i><0.001.</p

HPP-4382 alters occupancies of Nrf2 and Bach1 on the HMOX1 E2 promoter.

<p>(A) NHLF Cells were treated with 0.1 µM CDDO-Me or 1 µM HPP-4382 for 6 hours after which they were crosslinked with 1% formaldehyde in media, washed, and collected to be processed for chromatin immunoprecipitation as described in <i>Materials and Methods</i>. Precleared nuclear lysates were incubated with antibodies against Nrf2 or Bach1. Immune complexes were than isolated with E.coli tRNA/Protein A agarose beads, and the obtained purified DNA with subjected to qPCR using primers for HMOX1 E2 promoter. *, <i>p</i><0.05 compared to the untreated sample of same antibody; n.s.  =  not significant. (B) NHLF cells were exposed to 20 nM Nrf2, Keap1, or control siRNA for 48 hours. Cells were lysed and separated via SDS-PAGE then Western blotted with antibodies against Nrf2, Keap1, or tubulin. (C) Cells transfected with either Nrf2 or control siRNA were subjected to chromatin immunoprecipitation after treatment with 1 µM HPP-4382 for 6 hours. Precleared nuclear lysates were probed with antibodies against Nrf2 or Bach1; a third set was not probed (mock). *, <i>p</i><0.01; **, <i>p</i><0.05. (D) Cells transfected with either Keap1 or control siRNA were subjected to chromatin immunoprecipitation after treatment with either 10 µM HPP-1014, 10 µM CoPP, or 1 µM HPP-4382 for 6 hours. Precleared nuclear lysates were probed with antibodies against Nrf2 or Bach1; a third set was not probed (mock). All samples were performed in triplicate, error bars represent standard deviation. *, <i>p</i><0.01; **, <i>p</i><0.05 compared to untreated siCtrl for same antibody probe. , <i>p</i><0.01; , <i>p</i><0.05 compared to untreated siKeap1 for same antibody probe.</p

Identification of molecules that induce HMOX1 expression.

<p>(A) Human lung fibroblast cells were plated in 384-well Optilux plates and screened with compound libraries at 15 µM for 18 hours. Cells were then fixed, permeabilized, and probed with anti-HMOX1 antibody. Fluorescence intensity of HMOX1 staining was quantified with a GE InCell imager. Control charts were prepared using the statistical software JMP. HMOX1-staining intensities greater than the upper confidence limit were deemed hits. (B) Representative images of cells expressing HMOX1 following compound treatment. NHLF cells were cultured in 96-well Optilux plates as described in <i>Materials and Methods</i>. Cells were treated with indicated compound at selected concentrations for 18 hours after which HMOX1 expression was determined by immunofluoresence and quantified on a GE InCell imager. (C) Potency of CoPP, HPP-1014, and HPP-4382 were determined in NHLF cells. Cells were treated for 18 hours, after which they were fixed, permeabilized, and HMOX1 expression determined via immunofluoresence captured on a GE InCell imager.</p