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Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice

Open AccessPublished:December 16, 2022DOI:https://doi.org/10.1016/j.jhepr.2022.100651

      Highlights

      • S217879 is a potent and selective small molecule disrupting the KEAP1-NRF2 interaction leading to robust NRF2 pathway activation.
      • S217879 treatment prevents NASH progression in mice fed a methionine and choline-deficient diet.
      • S217879 treatment significantly improves established liver injury with clear reduction in both NAS score as well as liver fibrosis in DIO-NASH mice.
      • NRF2 activation triggers an up-regulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved with disease progression.

      Abstract

      Background & Aims

      Oxidative stress is recognized as a major driver of nonalcoholic steatohepatitis (NASH) progression. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, detoxification and appear therefore as attractive drug targets for the treatment of NASH.

      Methods

      Molecular modeling as well X-ray crystallography were used to design S217879 as a small molecule disrupting the KEAP1-NRF2 interaction. S217879 was highly characterized using various molecular and cellular assays. It was then evaluated in two different NASH-relevant preclinical models, namely the methionine and choline-deficient diet (MCD) and Diet-induced Obesity NASH (DIO NASH) mouse model.

      Results

      Molecular and cell-based assays confirmed that S217879 is a highly potent and selective NRF2 activator with marked anti-inflammatory properties as shown in primary human PBMCs. S217879 treatment for 2 weeks led in MCD mice to a dose-dependent reduction in NAFLD activity score (NAS) while significantly increasing liver Nqo1 mRNA levels, a specific NRF2 target engagement biomarker. In DIO NASH mice, S217879 treatment resulted in a significant improvement of established liver injury with clear reduction in both NAS score as well as liver fibrosis. Alpha SMA and Col1A1 staining as well as quantification of liver hydroxyproline levels confirmed the reduction in liver fibrosis in response to S217879. RNA seq analyses revealed major alterations in the liver transcriptome in response to S217879 with activation of NRF2-dependent gene transcription as well as a marked inhibition of key signaling pathways driving disease progression.

      Conclusions

      These results highlight the potential of selective disruption of NRF2-KEAP1 interaction for the treatment of NASH and liver fibrosis.

      Lay summary

      We report the discovery of S217879 as a potent and selective NRF2 activator with good pharmacokinetic properties. By disrupting the KEAP1-NRF2 interaction, S217879 triggers the up-regulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved with NASH disease progression leading ultimately to the reduction of both NASH and liver fibrosis progression in mice.

      Graphical abstract

      Keywords

      Abbreviations

      4-HNE
      4-hydroxynonenal
      ARE
      Antioxidant response element
      GSEA
      Gene Set Enrichment Analysis
      HSCs
      Hepatic stellate cells
      KEAP1
      Kelch-like ECH associated protein 1
      MCD
      Methionine and choline-deficient diet
      NAFLD
      Nonalcoholic fatty liver disease
      NAS
      NAFLD activity score
      NASH
      Nonalcoholic steatohepatitis
      NRF2
      Nuclear factor erythroid 2–related factor 2
      PBMCs
      Peripheral Blood Mononuclear Cells
      PPI
      Protein-protein interaction
      PSR
      Picro Sirius Red

      Introduction

      Nonalcoholic fatty liver disease (NAFLD) is a common and progressive disease mainly characterized by hepatic fat accumulation in the absence of alcohol consumption. NAFLD is strongly associated to obesity, metabolic syndrome, Type 2 Diabetes and dyslipidemia. NAFLD is subdivided into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) based on histological examination of liver biopsy and defined by the presence of inflammation and hepatocyte ballooning with various degrees of fibrosis [
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      ]. In contrast to NAFL, which is considered as a benign and reversible disease state, NASH accounts for an increased number of patients with cirrhosis, liver failure and hepatocellular carcinoma [
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      ]. NASH patients display an increased mortality compared to control population with a high cardiovascular risk. Long term follow-up studies revealed that fibrosis is the main driver of mortality in NASH [
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      ]. Several genes have been associated to the development of NAFLD and more generally liver diseases using Genome Wide Association Studies (See [
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      ] for review). From a mechanistic standpoint, it is believed that the accumulation of both triglycerides and pro-inflammatory and cytotoxic lipid oxidation side-products results in the formation of a necro-inflammatory milieu which triggers the activation of the main fibrogenic hepatic cell population, namely hepatic stellate cells (HSCs). Oxidative stress is largely recognized as a major driver of NASH progression[
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      ]. 4-HNEs are formed during lipid peroxidation of polyunsaturated fatty acids. Finally, there is a marked decrease of glutathione and hepatic antioxidant enzymes in NAFLD patients[
      • Masarone M.
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      ]. Indeed, the insulin resistance-mediated increase in oxidative phosphorylation is a major source of oxidative stress which triggers hepatocellular damage and further exacerbates insulin resistance. Satapati and colleagues have shown that hepatic oxidative stress and inflammation are associated with an elevated oxidative metabolism of saturated fatty acids in NAFLD[
      • Satapati S.
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      ]. Adaptation of mitochondrial function during NAFL is lost in NASH with increased ROS, lipid peroxidation products and decreased ATP content leading to necrosis fueling the development of steatohepatitis[
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      The nuclear factor erythroid 2–related factor 2 (NRF2), member of the cap’ n’ collar basic leucine zipper transcription factor family and its negative regulator, the E3 ligase adaptator Kelch-like ECH associated protein 1 (KEAP1) are master regulators of cellular resistance to oxidants (See Dodson et al for review[
      • Dodson M.
      • De La Vega M.R.
      • Cholanians A.B.
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      • Chapman E.
      • Zhang D.D.
      Modulating NRF2 in Disease: Timing Is Everything.
      ]). Under resting conditions, NRF2 is sequestered within the cytoplasm. The N-terminal domain of the KEAP1 homodimer binds one molecule of NRF2 leading to ubiquitination by the E3 ligase complex, namely CUL3/RBX1 and ultimately to proteasomal degradation [
      • Tong K.I.
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      • Tanaka T.
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      Keap1 Recruits Neh2 through Binding to ETGE and DLG Motifs: Characterization of the Two-Site Molecular Recognition Model.
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      Different electrostatic potentials define ETGE and DLG motifs as hinge and latch in oxidative stress response.
      ]. Under oxidative conditions, increased levels of electrophilic molecules result in the covalent modifications of highly reactive cysteine residues within KEAP1 triggering conformational changes and the release of CUL3 [
      • Zhang D.D.
      • Hannink M.
      Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress.
      ,
      • Zhang D.D.
      • Lo S.-C.
      • Cross J.V.
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      • Hannink M.
      Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex.
      ,
      • Eggler A.L.
      • Small E.
      • Hannink M.
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      Cul3-mediated Nrf2 ubiquitination and antioxidant response element (ARE) activation are dependent on the partial molar volume at position 151 of Keap1.
      ,
      • Wakabayashi N.
      • Dinkova-Kostova A.T.
      • Holtzclaw W.D.
      • Kang M Il
      • Kobayashi A.
      • Yamamoto M.
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      Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.
      ]. NRF2 is then released, translocated into the nucleus where it accumulates and dimerizes with small MAF proteins to activate transcription of genes containing the so-called antioxidant response element (ARE)[
      • Itoh K.
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      • Takahashi S.
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      ,
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      • Casula L.
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      • Cao A.
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      hMAF, a small human transcription factor that heterodimerizes specifically with Nrf1 and Nrf2.
      ]. NRF2 regulates the transcription of more than 250 genes bearing an ARE involved in antioxidant cellular defense, xenobiotic metabolism and detoxification, carbohydrate and lipid metabolism, protein degradation as well as inflammation[
      • Hayes J.D.
      • Dinkova-Kostova A.T.
      The Nrf2 regulatory network provides an interface between redox and intermediary metabolism.
      ]. It is therefore not surprising that the NRF2 signaling pathway is considered as a valid target for a number of acute and chronic diseases which have oxidative stress and inflammation as key biological drivers[
      • Cuadrado A.
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      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      ]. Increased oxidative stress is a hallmark of chronic liver diseases which explains why NRF2 biology has been extensively probed in the liver (See [
      • Tang W.
      • Jiang Y.F.
      • Ponnusamy M.
      • Diallo M.
      Role of Nrf2 in chronic liver disease.
      ] for review 2014). Interestingly, Nrf2-deficient mice display an increased susceptibility to the development of NASH and fibrosis when placed on high fat diet for 24 weeks[
      • Meakin P.J.
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      • Sharma R.S.
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      • Walsh S.V.
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      Susceptibility of Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a high-fat diet is associated with oxidative stress, perturbation of the unfolded protein response, and disturbance in the expression of metabolic enzymes but not with insulin resistance.
      ]. Biochemical and molecular analyses revealed that NRF2 protects against NASH by, at least in part regulating oxidative stress and suppressing de novo lipogenesis, ER stress and inflammation[
      • Meakin P.J.
      • Chowdhry S.
      • Sharma R.S.
      • Ashford F.B.
      • Walsh S.V.
      • McCrimmon R.J.
      • et al.
      Susceptibility of Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a high-fat diet is associated with oxidative stress, perturbation of the unfolded protein response, and disturbance in the expression of metabolic enzymes but not with insulin resistance.
      ]. Similar findings namely marked increased in steatosis, inflammation and oxidative stress were obtained in Nrf2 KO mice fed a methionine and choline-deficient (MCD) diet[
      • Chowdhry S.
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      • Walsh S.V.
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      Loss of Nrf2 markedly exacerbates nonalcoholic steatohepatitis.
      ,
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      Deletion of nuclear factor-E2-related factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice.
      ]. Conversely, sustained NRF2 activation in Keap1 gene knock-down mice prevented MCD-induced liver injury[
      • Okada K.
      • Warabi E.
      • Sugimoto H.
      • Horie M.
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      • Ueda T.
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      Nrf2 inhibits hepatic iron accumulation and counteracts oxidative stress-induced liver injury in nutritional steatohepatitis.
      ]. Furthermore, NRF2 pharmacological activation of using potent electrophilic compounds such as 1-[2-cyano3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im), TBE-31, Sulforaphane and Omaveloxolone were shown to limit NASH progression and fibrosis in multiple preclinical models[
      • Okada K.
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      • Sugimoto H.
      • Horie M.
      • Tokushige K.
      • Ueda T.
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      Nrf2 inhibits hepatic iron accumulation and counteracts oxidative stress-induced liver injury in nutritional steatohepatitis.
      ,
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      NF-E2-related factor 2 inhibits lipid accumulation and oxidative stress in mice fed a high-fat diet.
      ,
      • Sharma R.S.
      • Harrison D.J.
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      Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2).
      ,
      • Reisman S.A.
      • Ferguson D.A.
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      Omaveloxolone and TX63682 are hepatoprotective in the STAM mouse model of nonalcoholic steatohepatitis.
      ]. Finally, NRF2 protein levels have been shown to be significantly reduced in livers from NASH but not NAFL obese patients[
      • Azzimato V.
      • Jager J.
      • Chen P.
      • Morgantini C.
      • Levi L.
      • Barreby E.
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      Liver macrophages inhibit the endogenous antioxidant response in obesity-associated insulin resistance.
      ,
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      • Guillou H.
      • et al.
      The histone demethylase Phf2 acts as a molecular checkpoint to prevent NAFLD progression during obesity.
      ]. Taken together, all these studies strongly support NRF2 targeting for the treatment of NASH and liver fibrosis. While a number of electrophilic NRF2 agonists delivered initially promising results for various therapeutic indications in preclinical models, they all suffer from reduced safety margin mainly due to their limited selectivity and pleiotropic pharmacology preventing their clinical evaluation in large randomized trials for NASH for instance. Bardoxolone has been shown to interact with more than 500 molecular species[
      • Yore M.M.
      • Kettenbach A.N.
      • Sporn M.B.
      • Gerber S.A.
      • Liby K.T.
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      ] and to inhibit endothelin-1 signaling pathway and increased cardiovascular risk in CKD patients[
      • de Zeeuw D.
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      • Chin M.P.
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      ]. The availability of KEAP1 X-ray structure[
      • Li X.
      • Zhang D.
      • Hannink M.
      • Beamer L.J.
      Crystal structure of the Kelch domain of human Keap1.
      ] opened the door to the discovery of second generation of NRF2 activators with a completely different way of engaging the target via the direct disruption of its interaction with KEAP1[

      Schmoll D, Engel C, Technologies HG-DDT, 2017 undefined. The Keap1–Nrf2 protein–protein interaction: a suitable target for small molecules. Elsevier n.d.

      ]. This led to the identification of potent and selective compounds but with limited oral bioavailability[
      • Davies T.G.
      • Wixted W.E.
      • Coyle J.E.
      • Griffiths-Jones C.
      • Hearn K.
      • McMenamin R.
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      Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery.
      ]. Here, we report the discovery and characterization of S217879, a potent and selective small molecule disrupting the KEAP1-NRF2 interaction with good pharmacokinetic properties upon oral administration in rodents. Then, we evaluated its potential as a novel treatment for NASH and liver fibrosis in two different preclinical models.

      Material and methods

      S217879

      Compound was synthesized by Servier medicinal chemistry department and was determined to be >98% pure by HPLC and/or NMR analysis (chemical synthesis described within supplementary information).

      LPS-induced cytokine secretion assay

      Human Peripheral Blood Mononuclear Cells (PBMCs) were provided by Lonza (#CC-2702). hPBMCs (150,000 cells/well) were plated in 96 well plates in RPMI1640 medium supplemented with 10% Fetal Calf Serum, 2% Glutamax and 2% Penicillin-Streptomycin cocktail (Life Technologies). Cells were then preincubated at 37°C for 4h with various concentrations of S217879 or vehicle (DMSO, 0.1%). hPBMCs are then stimulated with LPS (10ng/mL) for 4h. At the end of the stimulation period, cytokines secretion was evaluated using MagPix multiplex kits (Biorad) according to manufacturer’s instructions.

      Animals

      Mice were maintained on a 12:12 h light/dark cycle at 21 ± 2°C and had ad libitum access to tap water and standard (A04) or MCD or AMLN diet. All procedures were performed according to the ethical protocol that has been approved by the Servier Institutional Animal Care and Use Committee in accordance with the French regulations (Decree n° 2013-118 from 01 February 2013 relative to the protection of animals used for scientific purposes and 4 for orders).

      Methionine and Choline Deficient diet (MCD)

      8 weeks old male C57BL6/J mice (Janvier labs, France) were randomly assigned to either control diet (A04 diet, SAFE) or the Methionine and Choline-deficient diet (MCD, BROGAARDEN, open source diets) for 2 weeks. Mice on MCD diet received immediately either S217879 (3 or 30mg/kg/day) or vehicle (hydroxyethyl cellulose (HEC) 1%) by gavage for 2 weeks. At the end of the study, mice were fasted for 4 hours before termination. Intracardiac blood samples were collected on anesthetized animals (using Isoflurane). Liver samples were collected for biochemical, histological and gene expression analyses.

      DIO NASH model

      5-6 weeks old C57BL/6JRj mice were fed with AMLN diet (D09100301, Research Diet, US) (40% fat (18% trans-fat), 40% carbohydrate (20% fructose), and 2% cholesterol) for 33 weeks prior to initiation of treatment protocol. Prior to treatment, all mice underwent liver biopsy for confirmation and stratification of liver steatosis and fibrosis, using the nonalcoholic fatty liver disease activity score (NAS) and fibrosis staging system as previously described[
      • Kristiansen M.N.B.
      • Veidal S.S.
      • Rigbolt K.T.G.
      • Tølbøl K.S.
      • Roth J.D.
      • Jelsing J.
      • et al.
      Obese diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy.
      ]. Only mice with fibrosis stage ≥ 1 and steatosis score ≥ 2, were included in the study. DIO NASH mice were kept on AMLN diet and received either S217879 (30mg/kg, PO, QD) or vehicle (HEC 1%) for 8 weeks. A terminal blood sample was collected from the tail vein in non-fasted mice and used for plasma biochemistry. Animals were sacrificed by cardiac puncture under isoflurane anesthesia. Liver samples were processed as described below.

      Histological analyses

      Formalin-fixed, paraffin-embedded livers were sliced into 3-μm sections. Hematoxylin and Eosin (H&E) staining was performed to investigate liver histology and Picro Sirius Red staining was used for liver fibrosis. Type 1 collagen (Southern Biotech, #1310-01), Galectin-3 and alpha smooth muscle actin (αSMA, Abcam, Ab124964) immunohistochemistry were performed using standard procedures. NAFLD Activity Score (NAS) and fibrosis stage were determined by two double-blinded persons using the NASH CRN scoring system [
      • Kleiner D.E.
      • Brunt E.M.
      • Van Natta M.
      • Behling C.
      • Contos M.J.
      • Cummings O.W.
      • et al.
      Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
      ]. For hepatocellular steatosis, livers were classified into scores 0 to 3 (0: <5% of hepatocytes presenting steatosis, 1: 5 to 33% of hepatocytes presenting steatosis, 2: 33 to 66% of hepatocytes presenting steatosis and 3: > 66% of hepatocytes presenting steatosis). For inflammation, livers were scored into grades 0 to 3 (0: non inflammatory foci, 1: 1 inflammatory focus, 2: 2 to 4 inflammatory foci, 3: >4 inflammatory foci). Fibrosis was scored into stages from 0 to 4 (0: no fibrosis, 1: perisinusoidal or periportal fibrosis, 2: perisinusoidal and periportal fibrosis, 3: bridging fibrosis or septa, 4: cirrhosis).

      Biochemical analyses

      Plasma parameters were determined with an automatic biochemical analyzer (Indiko Clinical Chemistry Analyzer, Thermofisher). Liver TG levels were measured using commercially available kit (Roche Diagnostics) after homogenization and extraction as described[
      • Morel P.S.
      • Duvivier V.
      • Bertin F.
      • Provost N.
      • Hammoutene A.
      • Hubert E.L.
      • et al.
      Procollagen C-Proteinase Enhancer-1 (PCPE-1) deficiency in mice reduces liver fibrosis but not NASH progression.
      ]. For hydroxyproline levels, liver samples are homogenized in 6 M HCl and hydrolyzed to degrade collagen. Samples are centrifuged and the hydroxyproline content is measured in duplicates in the supernatant, using a colorimetric assay (Quickzyme Biosciences) according to the manufacturer’s instructions.

      Gene expression studies by QPCR

      Total RNA was extracted using Qiagen RNeasy Lipid extraction kit following manufacturer’s instructions. Total RNA was treated with DNase I (Qiagen) at 37°C for 30 minutes, followed by inactivation at 75°C for 5 minutes. Real time quantitative PCR (RT-qPCR) assays were performed using an Applied Biosystems QuantStudio 7 Flex System. Total RNA (1 μg) was reverse transcribed with random hexamers using Hight-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, ThermoFisher Scientific) following the manufacturer’s protocol. Gene expression levels were determined with Taqman Universal Master mix (2x) and using Taqman assays (Applied Biosystems). 18S transcript was used as an internal control to normalize the variations for RNA amounts. Gene expression levels are expressed relative to 18S mRNA levels. All primers used in this study were provided by Thermofisher.

      Liver RNA Sequencing

      Total RNA was extracted using Qiagen RNA extraction kits following manufacturer’s instructions. RNA concentrations were obtained using nanodrop or a fluorometric Qubit RNA assay (Life Technologies, Grand Island, New York, USA). The quality of the RNA (RNA integrity number) was determined on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) as per the manufacturer’s instructions. To construct the libraries, 400 ng of high quality total RNA sample (RIN >8) was processed using TruSeq Stranded mRNA kit (Illumina) according to manufacturer instructions. Briefly, after purification of poly-A containing mRNA molecules, mRNA molecules are fragmented and reverse-transcribed using random primers. Replacement of dTTP by dUTP during the second strand synthesis will permit to achieve the strand specificity. Addition of a single A base to the cDNA is followed by ligation of Illumina adapters. Libraries were quantified by qPCR using the KAPA Library Quantification Kit for Illumina Libraries (KapaBiosystems, Wilmington, MA) and library profiles were assessed using the DNA High Sensitivity LabChip kit on an Agilent Bioanalyzer. Libraries were sequenced on an Illumina NovaSeq 6000 instrument using 150 base-lengths read V2 chemistry in a paired-end mode. Sequence reads weere trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. Unique gene hit counts were calculated by using the featureCounts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. Counts have been normalized in TPM using standard formula.

      RNA Seq data analyses

      Data analyses were carried out using R system software (http://www.R-project.org, V4.0.2) packages including those of Bioconductor or original R code. Gene hit counts were used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the S217879-treated samples and vehicle samples was performed. The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 and absolute log2 fold change > 1 (FC > 2) were called as differentially expressed genes between conditions. Enrichment analysis was performed by applying the fast gene set enrichment analysis (fgsea function from fgsea R package v.1.16.0, pre-ranked mode) against two separated genesets collections, HALLMARK and a combination of KEGG and REACTOME curated pathways. Representations of data and results have been generated with ComplexHeatmap v.2.6.2 and ggplot2 v.3.3.5 packages. Data are available under the GSE212644 accession number.

      Statistical analysis

      For comparison of 2 groups, an unpaired Student t test was used (GraphPad Prism® software) after verification of the normal distribution of data. For more than 2 groups, a one-way of variance was performed followed by a Dunnett’s multiple comparison test. For body weight, a two-way analysis of variance was performed followed by a Tukey’s test. For histological parameters, a statistical model ANOVA with strain and diet as fixed effects on a score parameter (NAS, Steatosis, Fibrosis or inflammation), followed by lsmeans estimations (SAS software). Significance threshold was 5%. Results are expressed as mean ± SEM.

      Data availability

      All the data presented in this manuscript are available from the corresponding author upon request.

      Results

      Non-covalent inhibitors of the KEAP1 Kelch/NRF2 protein-protein interaction may increase selectivity with reduced cytotoxicity translating into larger therapeutic index. Several groups have reported the discovery of such molecules. Most of them exhibit promising binding affinities. However, they usually suffer from poor ADME-PK properties limiting their use for probing NRF2 biology in vivo. Davies and colleagues reported the discovery of KI-696 (1) (Figure 1), a potent compound which activates the NRF2 antioxidant response in cellular models. In vivo target engagement required iv infusion due to its poor oral bioavailability[
      • Davies T.G.
      • Wixted W.E.
      • Coyle J.E.
      • Griffiths-Jones C.
      • Hearn K.
      • McMenamin R.
      • et al.
      Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery.
      ]. KI-696 binding to the Kelch domain has been confirmed by X-ray crystallography [
      • Tran K.T.
      • Pallesen J.S.
      • Solbak S.M.
      • Narayanan D.
      • Baig A.
      • Zang J.
      • et al.
      A Comparative Assessment Study of Known Small-Molecule Keap1-Nrf2 Protein-Protein Interaction Inhibitors: Chemical Synthesis, Binding Properties, and Cellular Activity.
      ]. More recently, the discovery of a novel non-covalent chemotype with an acceptable pharmacokinetic profile for oral dosing was reported[
      • Ma B.
      • Lucas B.
      • Capacci A.
      • Lin E.Y.S.
      • Jones J.H.
      • Dechantsreiter M.
      • et al.
      Design, synthesis and identification of novel, orally bioavailable non-covalent Nrf2 activators.
      ]. Preliminary in vivo results of the best compound (2) (Figure 1) revealed target engagement as measured by NRF2-driven gene induction from 10mg/kg onwards[
      • Ma B.
      • Lucas B.
      • Capacci A.
      • Lin E.Y.S.
      • Jones J.H.
      • Dechantsreiter M.
      • et al.
      Design, synthesis and identification of novel, orally bioavailable non-covalent Nrf2 activators.
      ]. The X-ray structure of compound 1 and compound 2 Kelch complexes revealed the possibility of macrocyclization which is generally known to modify the entropic contribution to binding[
      • Mallinson J.
      • Collins I.
      Macrocycles in new drug discovery.
      ]. In addition, variation of the linker’s position and substitution allows for the fine tuning of the physicochemical properties without disrupting the interactions with the protein. We decided to replace the benzoxathiazepine and benzoyl scaffolds of 1 and 2 with benzoxathiazine part hoping that in these compounds will have a binding mode exhibiting the same key interactions as the parent molecules, with improved hydrophobic interactions (Tyr334, Phe557). We hypothesized that position 6 of the benzoxathiazine’s benzene ring provides a suitable vector for linking the aromatic ring with benztriazole’s N-alkyl group tolerating diverse substitution pattern on the linker. Substutition of the benzyl position next to the benzoxathiazine nitrogene atom also proved to be an effective tool to finetune the interaction between benzoxathiazine oxygenes and the protein. Optimization of the macrocyclics’ structure was focused on the linker part and led to the synthesis and subsequent selection of compound 3 (See Figure 1; Compound 3 is referred as S217879 throughout the manuscript) as candidate. Binding to KEAP1 Kelch domain was confirmed with direct surface plasmon resonance (SPR) assay (Kd = 4.15nM; Supplementary Table 1). The X-ray cocrystal structure confirmed that the benzoxathiazine ring’s position allowed improved interactions with hydrophobic residues (Tyr334, Phe557, Figure 1C). Then, we assessed the ability of S217879 to trigger NRF2 nuclear translocation in U2OS cells using a beta galactosidase complementation assay. Andrographolide, a plant-derived diterpenoid was used at 10 μM as positive control. Andrographolide triggers NRF2 nuclear translocation by forming adducts with Cysteine 151 within KEAP1[
      • Wen Wong D.P.
      • Ng M.Y.
      • Leung J.Y.
      • Boh B.K.
      • Lim E.C.
      • Tan S.H.
      • et al.
      Regulation of the NRF2 transcription factor by andrographolide and organic extracts from plant endophytes.
      ]. S217879 induced a concentration-dependent increase in NRF2 translocation in U2OS cells with an EC50 of 23nM (Figure 2A). In line with these results, S217879 significantly activated a reporter gene driven by an ARE in HepG2 cells in a concentration-dependent manner with EC50 of 18nM (Figure 2B). Since NRF2 is a master regulator of cellular resistance to oxidants (See Dodson et al for review[
      • Dodson M.
      • De La Vega M.R.
      • Cholanians A.B.
      • Schmidlin C.J.
      • Chapman E.
      • Zhang D.D.
      Modulating NRF2 in Disease: Timing Is Everything.
      ]), we next tested S217879 ability to reduce H2O2-stimulated ROS production in HepG2 cells. As expected, H2O2 triggered a significant increase in ROS production which was significantly reduced by S217879 treatment (Figure 2C). Taken together, these results indicate that S217879 is a potent NRF2 activator in vitro.
      Figure thumbnail gr1
      Figure 1Identification of S217879 as a novel NRF2 activator. A Chemical structure of Kelch-NRF2 interaction disruptors: (1) PPI compound with in vivo target engagement established upon IV infusion[
      • Davies T.G.
      • Wixted W.E.
      • Coyle J.E.
      • Griffiths-Jones C.
      • Hearn K.
      • McMenamin R.
      • et al.
      Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery.
      ] (2) PPI compound with in vivo target engagement established upon oral administration[
      • Ma B.
      • Lucas B.
      • Capacci A.
      • Lin E.Y.S.
      • Jones J.H.
      • Dechantsreiter M.
      • et al.
      Design, synthesis and identification of novel, orally bioavailable non-covalent Nrf2 activators.
      ] (3) Chemical structure of novel PPI compound with good developability profile aka S217879. B Overlay of Xray structures 5GNU (compound 1, grey) and compound 3 (cyan). C Crystal structure of the Kelch domain of KEAP1 bound to S217879 (resolution: 1.3 Angström).
      Figure thumbnail gr2
      Figure 2S217879 is a potent NRF2 activator in cell-based assays. A S217879 triggers NRF2 translocation in U2OS cells in a concentration-dependent manner (2 experiments performed in triplicates; data expressed as mean ± SEM). B Activation of ARE-driven transcription in HepG2 cells in response to S217879 (2 experiments performed in triplicates; data expressed as mean ± SEM). C S217879 pretreatment (1μM overnight, DMSO 0.1% used as vehicle) limits H2O2-triggered ROS generation in HepG2 cells (n=5 per condition, data expressed as mean ± SEM). Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test (###: p<0.001 H2O2-triggered vs. untriggered cells, ***: p<0.001 S217879-treated vs. vehicle).
      Next, S217879 was evaluated in a broad selectivity panel comprising 110 targets. In this panel, S217879 was tested at 10 μM, a roughly 2,000-fold higher concentration with respect to KEAP1 binding. Interestingly, none of these targets were significantly activated or inhibited in response to S217879 strongly supporting its selectivity (Supplementary Table 2).
      Since NRF2 activation has been shown to inhibit LPS-driven inflammation independently of redox control in both monocytes and macrophages[
      • Kobayashi E.H.
      • Suzuki T.
      • Funayama R.
      • Nagashima T.
      • Hayashi M.
      • Sekine H.
      • et al.
      Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription.
      ], we next evaluated the anti-inflammatory properties of S217879 in human primary PBMCs. In human PBMCs, S217879 treatment for 6h resulted in a concentration-dependent increase in Nqo1 gene expression with EC50 of 16nM (Figure 3A) in line with its cell-based potency determined in both HepG2 and U2OS cells (Figure 2). As expected, LPS triggered a robust and significant increase in IL-1β, IL-6 and MCP-1 secretion (Figure 3B-D). Pretreatment for 4h with S217879 led to a significant and concentration dependent inhibition of LPS-induced cytokine release as measured by IL1β, MCP-1 and IL6 MagPix multiplex assays (Figure 3B-D). Off note, both IL-1β and MCP-1 secretions were significantly reduced by S217879 treatment (IC50<30nM in line with NRF2 cell-based potency) in contrast to IL-6 which was slightly and not significantly reduced at higher concentrations (Figure 3B-D). NRF2 has been shown to negatively interfere with the NF-κB pathway which regulates the expression of a wide spectrum of inflammatory genes[
      • Kobayashi E.H.
      • Suzuki T.
      • Funayama R.
      • Nagashima T.
      • Hayashi M.
      • Sekine H.
      • et al.
      Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription.
      ]. Why IL6 regulation seems to be less sensitive to NRF2 remains unknown. Nevertheless, these data indicate that S217879 displays significant anti-inflammatory properties in human PBMCs (Figure 3B-D) while strongly engaging the NRF2 signaling pathway (Figure 3A).
      Figure thumbnail gr3
      Figure 3Inhibition of LPS-driven inflammatory response by S217879 in PBMCs. A Nqo1 gene upregulation in human PBMCs in response to increasing concentrations of S217879 (6h incubation; DMSO 0.1% used as vehicle). B-D Inhibition of LPS-stimulated cytokines secretion in human PBMCs exposed to increasing concentrations of S217879 (4h pre-incubation; DMSO 0.1% used as vehicle). Data expressed as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test. ##: p<0.01 and ###: p<0.001 LPS-triggered vs. untriggered cells. *: p<0.05, **: p<0.01 and ***: p<0.001 S2178789-treated vs. vehicle.
      In vitro ADME and safety parameters were profiled comprehensively predicting an orally available and safe compound (Supplementary Table 3). The ability of S217879 to activate NRF2 signaling pathway in vivo was next evaluated in C57BL6 mice. Male C57Bl6 mice received a single administration of S217879 (30mg/kg) or vehicle (HEC 1%) by gavage. Drug plasma exposure was quantified over time and NRF2 target engagement was assessed in the liver by measuring Nqo1 mRNA levels by qPCR. S217879 drugs levels rise rapidly following oral administration with Cmax reaching 3.2 μM (Supplementary Fig. 1). Plasma concentrations declined rapidly (AUC=3.8 μM.h) while Nqo1 mRNA levels in the liver increased in a time-dependent manner and reaching up to 10-fold increase over vehicle 24h post oral administration (Supplementary Fig. 1A). Similar results were obtained in the kidney (data not shown). Off note, S217879 treatment (single or chronic administration) was found to increase the hepatic expression of numerous NRF2 target genes such as Gclc, Gstm1, Gpx2 (Supplementary Fig. 1B, Figure 7, Supplementary Figs. 7–8). These results indicate that S217879 oral administration leads to NRF2 pathway activation in vivo.
      Having characterized S217879 as a potent and selective NRF2 activator in vitro and in vivo, we next evaluated its potential for the treatment of NASH using two well-established preclinical models. First, the methionine and choline deficient diet mice model was selected as a screening model to test the ability of S217879 to prevent or limit disease progression. This model is highly reproducible and mice rapidly develop NASH-like liver phenotype just after few weeks (see [
      • Santhekadur P.K.
      • Kumar D.P.
      • Sanyal A.J.
      Preclinical models of non-alcoholic fatty liver disease.
      ] for review). Furthermore, this model was initially used to genetically validate NRF2 as a potential target for NASH[
      • Chowdhry S.
      • Nazmy M.H.
      • Meakin P.J.
      • Dinkova-Kostova A.T.
      • Walsh S.V.
      • Tsujita T.
      • et al.
      Loss of Nrf2 markedly exacerbates nonalcoholic steatohepatitis.
      ,
      • Sugimoto H.
      • Okada K.
      • Shoda J.
      • Warabi E.
      • Ishige K.
      • Ueda T.
      • et al.
      Deletion of nuclear factor-E2-related factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice.
      ,
      • Okada K.
      • Warabi E.
      • Sugimoto H.
      • Horie M.
      • Tokushige K.
      • Ueda T.
      • et al.
      Nrf2 inhibits hepatic iron accumulation and counteracts oxidative stress-induced liver injury in nutritional steatohepatitis.
      ]. As expected, mice fed the MCD diet lost rapidly weight compared to mice receiving the control diet (Figure 4A). Two weeks of exposure to MCD diet led to significant liver injury with sharp increase in plasma liver enzymes (both ALT and AST) (Figure 4B-C). Histological analyses confirmed the development of NASH with severe steatosis and hepatic inflammation reaching a NAFLD activity score (NAS) above 4 (Figure 4E, Supplementary Table 5) in line with previous reports[
      • Machado M.V.
      • Michelotti G.A.
      • Xie G.
      • De Almeida T.P.
      • Boursier J.
      • Bohnic B.
      • et al.
      Mouse Models of Diet-Induced Nonalcoholic Steatohepatitis Reproduce the Heterogeneity of the Human Disease.
      ]. While treatment with S217879 at 3 and 30 mg/kg/day had no effect on total body weight versus vehicle (Figure 4A), it significantly improved liver histology with a dose-dependent reduction in NAS score (Figure 4E, Supplementary Fig. 2). A reduction in both steatosis and lobular inflammation scores was recorded at highest dose (Supplementary Table 5). Interestingly, this reduction in NAS score was not followed by a reduction of plasma liver enzymes (Figure 4B-C). These data are consistent with previous results obtained by Okada and colleagues who reported an impact of constitutive NRF2 activation on liver enzymes only after 13 weeks on MCD diet[
      • Sugimoto H.
      • Okada K.
      • Shoda J.
      • Warabi E.
      • Ishige K.
      • Ueda T.
      • et al.
      Deletion of nuclear factor-E2-related factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice.
      ]. Biochemical analyses confirmed a dose-dependent reduction in liver triglycerides in response to S217879 treatment in MCD diet-fed mice (Supplementary Fig. 3) in agreement with our histological analyses (Figure 4E, Supplementary Table 5). As expected, S217879-mediated reduction in NAS score occurred at doses which triggered significant NRF2 target engagement as measured by the dose dependent increase in liver Nqo1 mRNA levels (Figure 4F, Supplementary Fig. 4) in line with the dose dependent increase in drug exposure (Supplementary Table 3). It is noteworthy that the MCD diet itself triggered the upregulation of antioxidant response as shown previously[
      • Lickteig A.J.
      • Fisher C.D.
      • Augustine L.M.
      • Cherrington N.J.
      Genes of the antioxidant response undergo upregulation in a rodent model of nonalcoholic steatohepatitis.
      ]. Gene expression studies confirmed the clear upregulation of the antioxidant response in response to S217879 with significant Gpx2 gene induction. (Supplementary Fig. 4). In addition, S217879 treatment led to a significant inhibition of proinflammatory genes expression such as Ccl5, Cd68, Il1b and Il6 (Supplementary Fig. 4). These results are consistent with the anti-inflammatory properties of S217879 described in PBMCs (Figure 3). Finally, analyses of genes involved in de novo lipogenesis such as Fasn, Acaca and Scd1 revealed a lack of effect of S217879 on their expression levels (Supplementary Fig. 4). Sugimoto and colleagues failed to demonstrate a major impact of NRF2 activation on fatty acid metabolism genes in MCD-fed mice [
      • Sugimoto H.
      • Okada K.
      • Shoda J.
      • Warabi E.
      • Ishige K.
      • Ueda T.
      • et al.
      Deletion of nuclear factor-E2-related factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice.
      ]. It is noteworthy that we did observe a small but significant increase in liver weight in response to S217879 treatment (maximum already reached at 3mg/kg). In a separate experiment, we performed a full dose response analysis to determine the minimal effective dose in this model (see Supplementary Fig. 5). Indeed, S217879 treatment prevented disease progression in a dose-dependent manner as shown by reduced NAS score and induction of Nqo1 liver expression (supplementary Figure 5AB). Minimal effective dose was set at 1mg/kg/day in this model. Taken together, these results suggest that S217879-mediated NRF2 activation may provide hepatoprotective properties.
      Figure thumbnail gr4
      Figure 4S217879 reduced NASH progression in MCD mice in a dose-dependent manner. A Body weight evolution throughout the study. B Plasma ALT levels at termination. C Plasma AST levels at termination. D Liver weight at termination. E NAFLD activity scores. F Liver Nqo1 mRNA levels expressed as fold induction over control (A04 fed group). Data expressed as mean ± SEM (n=15 per group). Statistical significance was assessed by one-way ANOVA on log transformed data followed by a Holm’s adjustment for the comparisons of each dose of S217879 vs. MCD vehicle group. ##: p<0.01, ###: p<0.001: MCD vs. A04 (control diet). ***: p<0.001, **: p<0.01 S217879-treated vs. vehicle. NS: non-significant.
      Despite its utility for drug discovery, the MCD mouse model poorly recapitulates the human NASH etiology with its substantial weight loss and lack of insulin resistance (See [
      • Santhekadur P.K.
      • Kumar D.P.
      • Sanyal A.J.
      Preclinical models of non-alcoholic fatty liver disease.
      ,
      • Rinella M.E.
      • Green R.M.
      The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance.
      ] for review). Nevertheless, results from the basic characterization of the MCD model in response to S217879 (Figure 4, Supplementary Figs. 2–5) prompted us to further document the potential of S217879 for the treatment of NASH in the DIO NASH mouse model in a therapeutic setting. This model has been shown to have a good clinical translatability with respect to the histopathological, transcriptional and metabolic aspects of the human disease[
      • Hansen H.H.
      • Ægidius H.M.
      • Oró D.
      • Evers S.S.
      • Heebøll S.
      • Eriksen P.L.
      • et al.
      Human translatability of the GAN diet-induced obese mouse model of non-alcoholic steatohepatitis.
      ]. 5-6 weeks old C57BL/6JRj mice were fed with DIO NASH diet for 33 weeks prior to initiation of treatment protocol. Following a liver biopsy for inclusion (see methods), mice were randomized to receive either S217879 (30mg/kg, PO, QD) or vehicle (HEC 1%) for 8 weeks while being maintained on DIO NASH diet. S217879 treatment had no impact on food intake (Supplementary Fig. 6) and body weight (Figure 5A). It led to a significant reduction of established liver injury as measured by reduced ALT and AST levels (Figure 5C-D). However, we noticed again a small but significant increase in liver weight in response to treatment (Figure 5B). Biochemical analyses indicated a reduction in liver triglyceride levels (Figure 5E). Histological analyses revealed a significant reduction in lobular inflammation resulting in a significant reduction in NAS score (Figure 5G-H, Supplementary Table 6). These data were confirmed by the decrease of Galectin-3 staining (Figure 5F), a marker of hepatic inflammation[
      • Henderson N.C.
      • Mackinnon A.C.
      • Farnworth S.L.
      • Poirier F.
      • Russo F.P.
      • Iredale J.P.
      • et al.
      Galectin-3 regulates myofibroblast activation and hepatic fibrosis.
      ,
      • Iacobini C.
      • Menini S.
      • Ricci C.
      • Fantauzzi C.B.
      • Scipioni A.
      • Salvi L.
      • et al.
      Galectin-3 ablation protects mice from diet-induced NASH: A major scavenging role for galectin-3 in liver.
      ]. Interestingly, S217879 treatment led to a marked reduction in liver fibrosis progression as demonstrated by PSR staining (Figure 5 G-H, Supplementary Table 6). This reduction in liver fibrosis was independently confirmed by a significant reduction in liver hydroxyproline content (vehicle 0.12 ± 0.017, S217879 0.06 ± 0.014 μg/mg liver, p<0.001) and reduced liver Collagen 1A1 and αSMA staining (Figure 6A-B). Taken together, these results indicate that S217879 treatment led to significant improvements of established NASH and decreased liver fibrosis.
      Figure thumbnail gr5
      Figure 5S217879 reduced NASH progression and liver fibrosis in DIO NASH mice model. A Body weight at the end of the study. B Liver weight expressed as percentage of total body weight. C Plasma ALT levels. D Plasma AST levels. E Liver TGs. F Liver Gal3 positive area. Data expressed as mean ± SEM (n=14 per group). G H&E and PSR-stained liver sections. H Summary of histopathological NAFLD activity (left graph) scoring and Fibrosis Stage (left graph) of pre- and post-study biopsies. For each group, the number of animals with a higher (worsening), same or lower (improvement) in score at post-compared to pre-study is indicated by the height of the bar. A-F: Significant differences were analyzed using Student’s t test. H Significance of number of animals with a lower score versus vehicle was assessed using Fisher’s Exact Test followed by correction for multiple comparisons using the Bonferroni method. ***: p<0.001, **: p<0.01 and *: p<0.05 S217879-treated vs. vehicle.
      Figure thumbnail gr6
      Figure 6Markers of liver fibrosis are reduced in response to S217879 treatment in DIO NASH mice. A Col1A1 and αSMA stained liver sections at termination (n=14 per group). B Quantification of Col1A1 and αSMA positive areas by morphometry. Liver hydroxyproline levels quantified after extraction and centrifugation using a colorimetric assay. Data expressed as mean ± SEM. Significant differences were analyzed using Student’s t test. ***: p<0.001 and **: p<0.01 S217879-treated vs. vehicle.
      In order to dissect out the molecular mechanisms by which S217879-mediated NRF2 activation resulted into reduced NASH and fibrosis progression, RNA Seq analysis was performed on liver total RNA at the end of the study. 740 genes were found differentially expressed (361 up and 379 down) between S217879 and vehicle-treated animals (Log2 fold change >1, FDR-adjusted p-value<0.05, Figure 7A). These transcriptional changes were further explored by gene set enrichment analysis (Figure 7B). As expected, GSEA confirmed that the NRF2-mediated oxidative stress response was ranked among the most affected pathway in response to treatment (“Reactive Oxygen Species Pathway”, Figure 7B) with strong enrichment in NRF2 target genes found up-regulated (Figure 7BC, Supplementary Figs. 7–8). The clear and marked up-regulation of the overall NRF2 pathway as shown by increased expression of Nqo1, Gstm1, Gclc and Gpx2 expression (Supplementary Figs. 7–8) demonstrates again that S217879 is a potent NRF2 activator in vivo. More interestingly, key pathways involved in NASH pathophysiology related to inflammation (“inflammatory response”, “TNFA signaling”) and fibrosis (“Epithelial Mesenchymal Transition”) were found significantly suppressed in response to treatment (Figure 7B-E). In line with the reduction of liver inflammation (Figure 5), we did record the inhibition of several proinflammatory genes such as Mapk4 (Supplementary Figs. 7–8). Furthermore, a large number of genes associated to fibrosis and stellate cell activation were found down-regulated such as Col1a1, Itgax, Lox and Bmp8b (Supplementary Figs. 7–8). Again, this is consistent with the significant reduction in liver fibrosis measured in response to treatment. Finally, the expression of several key genes involved in de novo lipogenesis were also found significantly down-regulated (Supplementary Fig. 8) which is consistent with the anti-steatotic effects of S217879 in this model (Figure 5E). Taken together, our RNA Seq data confirmed that NRF2 activation leads to the up-regulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved with disease progression leading ultimately to the reduction of both NASH and liver fibrosis progression.
      Figure thumbnail gr7
      Figure 7S217879-mediated NRF2 activation leads to the up-regulation of the antioxidant response and the suppression of a wide spectrum of genes involved in disease progression. A Heatmap of Differentially Expressed Genes from S217879-treated DIO NASH mice vs. vehicle (Log2 fold change cutoff >1; FDR-adjusted p-value cutoff: 0.05). B Gene Set Enrichment Analysis of differentially expressed genes from DIO NASH mice treated with S217879 or vehicle (HEC 1%). Enriched pathways from gene ontology analysis. NES, normalized enrichment score. C-E Gene sets from Hallmark and Reactome revealed upregulated and down-regulated pathways (enrichment plots). C: Reactive Oxygen species, D: Inflammatory response and E: Collagen formation.

      Discussion

      In this manuscript, we reported the identification and detailed characterization of S217879 as a novel, potent and selective compound activating the NRF2 pathway. This compound selectively binds to KEAP1 leading to the disruption of the KEAP1-NRF2 interaction resulting in NRF2 nuclear translocation and subsequent gene regulation. Moreover, we found that S217879 exhibits an excellent selectivity profile (Supplementary Table 2) resulting into an excellent safety profile in vitro (LD50 in HepG2 cells >30μM) in contrast to potent first-generation compound such as Bardoxolone (LD50 in HepG2 cells <1μM). One key feature of this molecule is its good oral bioavailability which is a major differentiation with respect to PPI compounds previously identified which displayed good potency and cellular activity but suffered from poor absorption, limited distribution and low metabolic stability (See [
      • Cuadrado A.
      • Rojo A.I.
      • Wells G.
      • Hayes J.D.
      • Cousin S.P.
      • Rumsey W.L.
      • et al.
      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      ] for review). By contrast, we showed that single oral administration S217879 resulted in a marked target engagement as measured by increased Nqo1 mRNA levels in the liver (supplementary Figure 1AB) but also in the kidney (data not shown). Furthermore, we found 217879 to be efficacious in the MCD mice model at doses as low as 1mg/kg/day. These data strongly indicate that S217879 is an excellent compound to probe NRF2 biology in preclinical disease models.
      In this study, we have shown using two different preclinical models that selective NRF2 activation is beneficial for the treatment of NASH and liver fibrosis. S217879 treatment led to marked reduction in both steatosis and inflammation resulting in a lower NAS score (Figure 4, Figure 5, Figure 6). Furthermore, S217879 treatment led to a marked reduction on liver fibrosis in DIO-NASH mice as measured by reduced PSR, Col1A1, αSMA staining as well reduced hydroxyproline content (Figure 5, Figure 6). These data are consistent with histological improvements observed in response to first generation NRF2 activators such as TBE-31 and Omaveloxolone in high fat high fructose-fed mice[
      • Sharma R.S.
      • Harrison D.J.
      • Kisielewski D.
      • Cassidy D.M.
      • McNeilly A.D.
      • Gallagher J.R.
      • et al.
      Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2).
      ] and STAM mice[
      • Reisman S.A.
      • Ferguson D.A.
      • Lee C.Y.I.
      • Proksch J.W.
      Omaveloxolone and TX63682 are hepatoprotective in the STAM mouse model of nonalcoholic steatohepatitis.
      ], respectively. To our knowledge, this is the first report of beneficial properties of a non-covalent NRF2 activator for the treatment NASH and fibrosis. RNA Seq analyses indicated that NRF2 activation led to the upregulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved with disease progression (Supplementary Figs. 7–8, Figure 7). We did observe a pronounced impact on genes belonging to de novo lipogenesis pathway in line with previous observations[
      • Meakin P.J.
      • Chowdhry S.
      • Sharma R.S.
      • Ashford F.B.
      • Walsh S.V.
      • McCrimmon R.J.
      • et al.
      Susceptibility of Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a high-fat diet is associated with oxidative stress, perturbation of the unfolded protein response, and disturbance in the expression of metabolic enzymes but not with insulin resistance.
      ,
      • Slocum S.L.
      • Skoko J.J.
      • Wakabayashi N.
      • Aja S.
      • Yamamoto M.
      • Kensler T.W.
      • et al.
      Keap1/Nrf2 pathway activation leads to a repressed hepatic gluconeogenic and lipogenic program in mice on a high-fat diet.
      ,
      • Knatko E.V.
      • Tatham M.H.
      • Zhang Y.
      • Castro C.
      • Higgins M.
      • Dayalan Naidu S.
      • et al.
      Downregulation of Keap1 Confers Features of a Fasted Metabolic State.
      ]. This is in line with the marked reduction in liver triglyceride levels (Figure 5E). In addition to the expected impact on inflammation (Figure 5F), one of the most remarkable results obtained in this manuscript is the anti-fibrotic properties of S217879 in vivo as measured at the histological, biochemical and gene expression levels (Figure 5, Figure 6, Figure 7). This is of major interest since long term follow-up studies revealed that fibrosis is the main driver of mortality in NASH [
      • Angulo P.
      • Kleiner D.E.
      • Dam-Larsen S.
      • Adams L.A.
      • Bjornsson E.S.
      • Charatcharoenwitthaya P.
      • et al.
      Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease.
      ,
      • Hagström H.
      • Nasr P.
      • Ekstedt M.
      • Hammar U.
      • Stål P.
      • Hultcrantz R.
      • et al.
      Risk for development of severe liver disease in lean patients with nonalcoholic fatty liver disease: A long‐term follow‐up study.
      ]. NRF2 activation may trigger the reduction of liver fibrosis via multiple mechanisms. First, hepatocyte-specific activation of NRF2 has recently been shown to control fibrogenesis during NASH by regulating, at least in part, the antioxidant stress response thereby reducing DNA damage and apoptosis[
      • Mohs A.
      • Otto T.
      • Schneider K.M.
      • Peltzer M.
      • Boekschoten M.
      • Holland C.H.
      • et al.
      Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis.
      ]. Second, NRF2 activation may limit the profibrotic macrophages-derived inflammation by negatively interfering with both NFκB and YAP/NLRP3 pathways[
      • Kobayashi E.H.
      • Suzuki T.
      • Funayama R.
      • Nagashima T.
      • Hayashi M.
      • Sekine H.
      • et al.
      Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription.
      ,
      • Wang P.
      • Ni M.
      • Tian Y.
      • Wang H.
      • Qiu J.
      • You W.
      • et al.
      Myeloid Nrf2 deficiency aggravates non-alcoholic steatohepatitis progression by regulating YAP-mediated NLRP3 inflammasome signaling.
      ]. Finally, NRF2 has been shown to protect hepatic stellate cells from TGF-β-mediated cell activation[
      • Prestigiacomo V.
      • Suter-Dick L.
      Nrf2 protects stellate cells from Smad-dependent cell activation.
      ] suggesting direct anti-fibrotic properties. Our data are consistent with a model in which S217879-induced NRF2 activation reduces NASH progression and slows down the development of liver fibrosis by targeting both parenchymal and non-parenchymal cells (immune cells and stellate cells mainly) as suggested by cell-specific gain (Keap1 gene deletion) or loss of function (NRF2 gene deletion) studies[
      • Mohs A.
      • Otto T.
      • Schneider K.M.
      • Peltzer M.
      • Boekschoten M.
      • Holland C.H.
      • et al.
      Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis.
      ,
      • Wang P.
      • Ni M.
      • Tian Y.
      • Wang H.
      • Qiu J.
      • You W.
      • et al.
      Myeloid Nrf2 deficiency aggravates non-alcoholic steatohepatitis progression by regulating YAP-mediated NLRP3 inflammasome signaling.
      ], respectively. Additional studies using single cell RNA Seq analyses or spatial transcriptomics are required to further document the molecular mechanisms at the cellular level and determine the potential contribution of other relevant cell populations such as Liver Sinusoidal Endothelial Cells[
      • Furuta K.
      • Guo Q.
      • Hirsova P.
      • Ibrahim S.H.
      Emerging Roles of Liver Sinusoidal Endothelial Cells in Nonalcoholic Steatohepatitis.
      ] to S217879-mediated beneficial properties on NASH and fibrosis. Finally, NRF2 activation has been shown to improve gut barrier integrity[
      • Singh R.
      • Chandrashekharappa S.
      • Bodduluri S.R.
      • Baby B.V.
      • Hegde B.
      • Kotla N.G.
      • et al.
      Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway.
      ] which may to some extent contribute to the beneficial impact on NASH progression[
      • Mouries J.
      • Brescia P.
      • Silvestri A.
      • Spadoni I.
      • Sorribas M.
      • Wiest R.
      • et al.
      Microbiota-driven gut vascular barrier disruption is a prerequisite for non-alcoholic steatohepatitis development.
      ].
      NRF2 activity is reduced during aging[
      • Schmidlin C.J.
      • Dodson M.B.
      • Madhavan L.
      • Zhang D.D.
      Redox regulation by NRF2 in aging and disease.
      ] and considered as an attractive target for a number of chronic diseases including but not limited to autoimmune, respiratory, neurodegenerative, and cardio-metabolic diseases (See [
      • Cuadrado A.
      • Rojo A.I.
      • Wells G.
      • Hayes J.D.
      • Cousin S.P.
      • Rumsey W.L.
      • et al.
      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      ] for review). However, it has also been shown to exert both anti-tumorigenic and pro-tumorigenic actions. It is believed that low NRF2 activity may facilitate the initiation of carcinogenesis whereas constant high activity may drive cancer progression and resistance to chemotherapy[
      • Kensler T.W.
      • Wakabayashi N.
      Nrf2: friend or foe for chemoprevention?.
      ]. It is noteworthy that more than 10% of all hepatocellular carcinoma (HCC) present a mutation in either KEAP1 or NRF2[
      • Schulze K.
      • Imbeaud S.
      • Letouzé E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ]. Interestingly, we found that treatment with S217879 led to small but significant increase in liver weight in both MCD and DIO NASH models (Figure 4, Figure 5B). Similar observations were also made in both rats and mice on chow diet exposed to similar drug levels (data not shown). These results are reminiscent of the phenotype of KEAP1-deficient mice suggesting that constitutive NRF2 activation may trigger hepatomegaly[
      • Mohs A.
      • Otto T.
      • Schneider K.M.
      • Peltzer M.
      • Boekschoten M.
      • Holland C.H.
      • et al.
      Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis.
      ,
      • Okawa H.
      • Motohashi H.
      • Kobayashi A.
      • Aburatani H.
      • Kensler T.W.
      • Yamamoto M.
      Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity.
      ,
      • Komatsu M.
      • Kurokawa H.
      • Waguri S.
      • Taguchi K.
      • Kobayashi A.
      • Ichimura Y.
      • et al.
      The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.
      ]. It is noteworthy that constitutive hepatocyte specific NRF2 activation was shown to significantly reduce NASH-associated HCC development in mice with hepatocyte-specific NEMO deficiency[
      • Mohs A.
      • Otto T.
      • Schneider K.M.
      • Peltzer M.
      • Boekschoten M.
      • Holland C.H.
      • et al.
      Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis.
      ]. Furthermore, He and colleagues reported that NRF2 forced expression leads to hepatomegaly by, at least in part, increasing the expression of several growth factors such as PDGF and EGF in an AKT-dependent manner[
      • He F.
      • Antonucci L.
      • Yamachika S.
      • Zhang Z.
      • Taniguchi K.
      • Umemura A.
      • et al.
      NRF2 activates growth factor genes and downstream AKT signaling to induce mouse and human hepatomegaly.
      ]. By contrast, we failed to detect any impact on cell proliferation in vivo and genes related to cell cycle/proliferation (including the gene signature identified by He and colleagues[
      • He F.
      • Antonucci L.
      • Yamachika S.
      • Zhang Z.
      • Taniguchi K.
      • Umemura A.
      • et al.
      NRF2 activates growth factor genes and downstream AKT signaling to induce mouse and human hepatomegaly.
      ] (Aurka, Foxm1, Ccnb2, Cdc25b and Cdc25c)) were not significantly modulated upon treatment with S217879 (Figure 7). Furthermore, in vitro studies performed with S217879 tested up to 10μM (roughly 500-fold higher than NRF2 cell potency) failed to reveal any impact on cell proliferation using HepG2 cells. Finally, we did not detect any AKT phosphorylation in both hepatocytes and HepG2 cells in response to high concentrations of S217879 (data not shown). Obviously, pharmacological activation of NRF2 with a small molecule in vivo is not equivalent to NRF2 forced expression[
      • He F.
      • Antonucci L.
      • Yamachika S.
      • Zhang Z.
      • Taniguchi K.
      • Umemura A.
      • et al.
      NRF2 activates growth factor genes and downstream AKT signaling to induce mouse and human hepatomegaly.
      ] or constitutive NRF2 activation by KEAP1 gene deletion[
      • Mohs A.
      • Otto T.
      • Schneider K.M.
      • Peltzer M.
      • Boekschoten M.
      • Holland C.H.
      • et al.
      Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis.
      ,
      • Okawa H.
      • Motohashi H.
      • Kobayashi A.
      • Aburatani H.
      • Kensler T.W.
      • Yamamoto M.
      Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity.
      ,
      • Komatsu M.
      • Kurokawa H.
      • Waguri S.
      • Taguchi K.
      • Kobayashi A.
      • Ichimura Y.
      • et al.
      The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.
      ]. As a matter of fact, using a different genetic strategy, Kohler and colleagues created a mouse line with constitutive NRF2 activation in hepatocytes. Surprisingly, these mice did not have any increase in liver weight compared to wild type despite significant increase in NRF2-regulated gene expression[
      • Köhler U.A.
      • Kurinna S.
      • Schwitter D.
      • Marti A.
      • Schäfer M.
      • Hellerbrand C.
      • et al.
      Activated Nrf2 impairs liver regeneration in mice by activation of genes involved in cell-cycle control and apoptosis.
      ]. Taken together, these independent studies suggest that different levels of NRF2 activation due to either constitutive expression or pharmacological activation may result in different phenotypes. Interestingly, Chan recently reported that NRF2 pharmacological activation may enhance liver regeneration in mice by supporting hepatocellular hypertrophy[
      • Chan B.K.Y.
      • Elmasry M.
      • Forootan S.S.
      • Russomanno G.
      • Bunday T.M.
      • Zhang F.
      • et al.
      Pharmacological Activation of Nrf2 Enhances Functional Liver Regeneration.
      ]. Preliminary evaluation of S217879 safety profile using non-GLP dose range finding studies in both rats and non-human primates did not reveal any major toxicity. Histological analyses confirmed hepatocellular hypertrophy in rodents as measured by reduced nuclei density (data not shown). Additional studies including GLP-toxicology are required to confirm these preliminary findings and determine the long-term safety of S217879 mediated NRF2 activation.

      Funding

      This work was funded by Institut de Recherches Servier.

      Conflicts of interest

      All the authors are/were employees of Servier.

      Authors contributions

      KS and PD coordinated the work. SC, OB, AG, CI, AH, JR and KL performed the in vitro and in vivo studies. VD, NP, KS, PD, CV, SB, VP and LMV contributed to experimental design and data interpretation. CW supervised the medicinal chemistry program (design and synthesis) with the help of AK and NM. DD and VM contributed the characterization of S217879. FM performed bioinformatic analyses. CW and PD drafted the manuscript.

      Data availability statement

      The data that support the findings of this study are available from the corresponding author upon reasonable request.

      Acknowledgments

      We would like to thank Michael Feigh and the team at GUBRA (Hørsholm, Denmark) for their excellent support regarding the DIO NASH study.

      Appendix A. Supplementary data

      The following is the supplementary data to this article:

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