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Molecular basis for the disruption of Keap1-Nrf2 interaction via Hinge & Latch mechanism.

ABSTRACT: The Keap1-Nrf2 system is central for mammalian cytoprotection against various stresses and a drug target for disease prevention and treatment. One model for the molecular mechanisms leading to Nrf2 activation is the Hinge-Latch model, where the DLGex-binding motif of Nrf2 dissociates from Keap1 as a latch, while the ETGE motif remains attached to Keap1 as a hinge. To overcome the technical difficulties in examining the binding status of the two motifs during protein-protein interaction (PPI) simultaneously, we utilized NMR spectroscopy titration experiments. Our results revealed that latch dissociation is triggered by low-molecular-weight Keap1-Nrf2 PPI inhibitors and occurs during p62-mediated Nrf2 activation, but not by electrophilic Nrf2 inducers_. This study demonstrates that Keap1 utilizes a unique Hinge-Latch mechanism for Nrf2 activation upon challenge by non-electrophilic PPI-inhibiting stimuli, and provides critical insight for the pharmacological development of next-generation Nrf2 activators targeting the Keap1-Nrf2 PPI.

SUBMITTER: Horie Y

PROVIDER: S-EPMC8121781 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Molecular basis for the disruption of Keap1-Nrf2 interaction via Hinge & Latch mechanism.

Horie Yuta Y Suzuki Takafumi T Inoue Jin J Iso Tatsuro T Wells Geoffrey G Moore Terry W TW Mizushima Tsunehiro T Dinkova-Kostova Albena T AT Kasai Takuma T Kamei Takashi T Koshiba Seizo S Yamamoto Masayuki M

Communications biology 20210514 1

The Keap1-Nrf2 system is central for mammalian cytoprotection against various stresses and a drug target for disease prevention and treatment. One model for the molecular mechanisms leading to Nrf2 activation is the Hinge-Latch model, where the DLGex-binding motif of Nrf2 dissociates from Keap1 as a latch, while the ETGE motif remains attached to Keap1 as a hinge. To overcome the technical difficulties in examining the binding status of the two motifs during protein-protein interaction (PPI) sim ...[more]

PMID: 33990683

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Project description:Background & aimsOxidative stress is recognized as a major driver of non-alcoholic steatohepatitis (NASH) progression. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, as well as detoxification, and thus appear to be attractive targets for the treatment of NASH.MethodsMolecular modeling and X-ray crystallography were used to design S217879 - a small molecule that could disrupt 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 (MCDD) and diet-induced obesity NASH (DIO NASH) models.ResultsMolecular 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 peripheral blood mononuclear cells. In MCDD mice, S217879 treatment for 2 weeks led to a dose-dependent reduction in NAFLD activity score 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 a clear reduction in both NAS and liver fibrosis. αSMA and Col1A1 staining, as well as quantification of liver hydroxyproline levels, confirmed the reduction in liver fibrosis in response to S217879. RNA-sequencing analyses revealed major alterations in the liver transcriptome in response to S217879, with activation of NRF2-dependent gene transcription and marked inhibition of key signaling pathways that drive disease progression.ConclusionsThese results highlight the potential of selective disruption of the NRF2-KEAP1 interaction for the treatment of NASH and liver fibrosis.Impact and implicationsWe report the discovery of S217879 - a potent and selective NRF2 activator with good pharmacokinetic properties. By disrupting the KEAP1-NRF2 interaction, S217879 triggers the upregulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved in NASH disease progression, leading ultimately to the reduction of both NASH and liver fibrosis progression in mice.

Project description:BackgroundNuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.Methods and findingsHere we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.ConclusionsThis is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.

Project description:RationaleSepsis syndrome is characterized by inappropriate amplified systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates a pleiotropic cytoprotective defense program including antioxidants and protects against several inflammatory disorders by inhibiting oxidative tissue injuries. However, the role of enhanced Nrf2 activity in modulating innate immune responses to microbial infection and pathogenesis of sepsis is unclear.ObjectivesTo determine whether Nrf2 in myeloid leukocytes alters inflammatory response and protects against sepsis.MethodsMice with deletion of Nrf2 or kelch-like ECH-associated protein (Keap1) in myeloid leukocyte cells and respective floxed controls were subjected to cecal ligation and puncture-induced sepsis and were assessed for survival, organ injury, systemic inflammation, and bacteremia. Using LPS-stimulated peritoneal macrophages, Toll-like receptor (TLR) 4 surface trafficking and downstream signaling events were analyzed.Measurements and main resultsMortality, organ injury, circulating levels of inflammatory mediators, and bacteremia were markedly reduced in LysM-Keap1(-/-) compared with respective floxed controls (Keap1(f/f) or Nrf2(f/f)) and significantly elevated in LysM-Nrf2(-/-) mice after cecal ligation and puncture. Peritoneal macrophages from septic LysM-Keap1(-/-) mice showed a greater bacterial phagocytic activity compared with LysM-Nrf2(-/-) and floxed controls. LPS stimulation resulted in greater reactive oxygen species-induced cell surface transport of TLR4 from trans-Golgi network and subsequent TLR4 downstream signaling (recruitment of MYD88 and TRIF, phosphorylation of IkB and IRF3, and cytokine expression) in macrophages of LysM-Nrf2(-/-) compared with LysM-Keap1(-/-) mice and floxed controls.ConclusionsOur study shows that Nrf2 acts as a critical immunomodulator in leukocytes, controls host inflammatory response to bacterial infection, and protects against sepsis.

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Molecular basis for the disruption of Keap1-Nrf2 interaction via Hinge & Latch mechanism.

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Molecular basis for the disruption of Keap1-Nrf2 interaction via Hinge & Latch mechanism.

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