Molecular basis for the disruption of Keap1-Nrf2 interaction via Hinge & Latch mechanism.
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ABSTRACT:
SUBMITTER: Horie Y
PROVIDER: S-EPMC8121781 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Keap1 is a highly redox-sensitive member of the BTB-Kelch family that assembles with the Cul3 protein to form a Cullin-RING E3 ligase complex for the degradation of Nrf2. Oxidative stress disables Keap1, allowing Nrf2 protein levels to accumulate for the transactivation of critical stress response genes. Consequently, the Keap1-Nrf2 system is extensively pursued for the development of protein-protein interaction inhibitors that will stabilize Nrf2 for therapeutic effect in conditions of neurodegeneration, inflammation, and cancer. Here we review current progress toward the structure determination of Keap1 and its protein complexes with Cul3, Nrf2 substrate, and small-molecule antagonists. Together the available structures establish a rational three-dimensional model to explain the two-site binding of Nrf2 as well as its efficient ubiquitination.
| S-EPMC4668279 | biostudies-literature
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.
| S-EPMC9971056 | biostudies-literature
Project description:Activation of the transcription factor Nrf2 via the Keap1-Nrf2-ARE signaling system regulates the transcription and subsequent expression of cellular cytoprotective proteins and plays a crucial role in preventing pathological conditions exacerbated by the overproduction of oxidative stress. In addition to electrophilic modulators, direct non-covalent inhibitors that interrupt the Keap1-Nrf2 protein-protein interaction (PPI) leading to Nrf2 activation have attracted a great deal of attention as potential preventive and therapeutic agents for oxidative stress-related diseases. Structural studies of Keap1-binding ligands, development of biochemical and cellular assays, and new structure-based design approaches have facilitated the discovery of small molecule PPI inhibitors. This perspective reviews the Keap1-Nrf2-ARE system, its physiological functions, and the recent progress in the discovery and the potential applications of direct inhibitors of Keap1-Nrf2 PPI.
| S-EPMC7207041 | biostudies-literature
Project description:Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target for the prevention of neurodegenerative diseases, including Alzheimer's disease (AD). Current Nrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimental effects. An understanding of the mechanisms mediating Nrf2 inhibition in neurodegenerative conditions may therefore direct the design of drugs targeted for the prevention of these diseases with minimal side-effects. Our study provides the first in vivo evidence that specific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity in response to the AD-initiating Aβ42 peptide, in correlation with Nrf2 activation. Comparatively, lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aβ42 toxicity by mechanisms independent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain also prevented synaptotoxicity mediated by naturally-derived Aβ oligomers in mouse cortical neurons. Overall, our findings highlight Keap1 specifically as an efficient target for the re-activation of Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors for the prevention of neurodegeneration in vivo.
| S-EPMC5333801 | biostudies-literature
Project description:The KEAP1-NRF2 pathway is the principal protective response to oxidative and electrophilic stresses. Under homeostatic conditions, KEAP1 forms part of an E3 ubiquitin ligase, which tightly regulates the activity of the transcription factor NRF2 by targeting it for ubiquitination and proteasome-dependent degradation. In response to stress, an intricate molecular mechanism facilitated by sensor cysteines within KEAP1 allows NRF2 to escape ubiquitination, accumulate within the cell, and translocate to the nucleus, where it can promote its antioxidant transcription program. Recent advances have revealed that KEAP1 contains multiple stress sensors and inactivation modalities, which together allow diverse cellular inputs, from oxidative stress and cellular metabolites to dysregulated autophagy, to regulate NRF2 activity. This integration of the KEAP1-NRF2 system into multiple cellular signaling and metabolic pathways places NRF2 activation as a critical regulatory node in many disease phenotypes and suggests that the pharmaceutical modulation of NRF2's cytoprotective activity will be beneficial for human health in a broad range of noncommunicable diseases.
| S-EPMC7296212 | biostudies-literature
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.
| S-EPMC1584412 | biostudies-literature
Project description:INrf2(Keap1) functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to stress, Nrf2 is released from INrf2 and translocates inside the nucleus leading to activation of cytoprotective proteins critical in protection against adverse effects including cancer. We demonstrate here a novel role of heat shock protein 90 (Hsp90) in control of the INrf2 and Nrf2 activation. Hsp90 interacted with INrf2 that leds to stabilization of INrf2 during heat shock stress. Domain mapping showed the requirement of INrf2-NTR and the Hsp90-CLD region for interaction of Hsp90 with INrf2. Heat shock and antioxidants induced Hsp90, and casein kinase 2 (CK2) phosphorylated INrf2Thr55. This led to increased Hsp90-INrf2 interaction, dissociation of the Rbx1/Cul3·INrf2·Nrf2 complex, and activation of Nrf2. Inhibitors of CK2 and Hsp90, and mutation of INrf2Thr55 abolished the Hsp90-INrf2 interaction and downstream signaling. INrf2 is released from Hsp90 once the heat shock or antioxidant stress subsidized, thereby allowing INrf2 to interact with Nrf2 and facilitate Nrf2 ubiquitination and degradation. The results together demonstrate a novel role for the stress-induced Hsp90-INrf2 interaction in regulation of Nrf2 activation and induction of cytoprotective proteins.
| S-EPMC2978616 | biostudies-literature
Project description:Directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) has emerged as an attractive way to activate Nrf2, and Keap1-Nrf2 PPI inhibitors have been proposed as potential agents to relieve inflammatory and oxidative stress diseases. In this work, we investigated the diacetic moiety around the potent Keap1-Nrf2 PPI inhibitor DDO1018 (2), which was reported by our group previously. Exploration of bioisosteric replacements afforded the ditetrazole analog 7, which maintains the potent PPI inhibition activity (IC50 = 15.8 nM) in an in vitro fluorescence polarization assay. Physicochemical property determination demonstrated that ditetrazole replacement can improve the drug-like property, including elevation of pK a, log D, and transcellular permeability. Additionally, 7 is more efficacious than 2 on inducing the expression of Nrf2-dependent gene products in cells. This study provides an alternative way to replace the diacetic moiety and occupy the polar subpockets in Keap1, which can benefit the subsequent development of Keap1-Nrf2 PPI inhibitor.
| S-EPMC5018864 | biostudies-other
Project description: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. S217879 a small molecule disrupting the KEAP1-NRF2 interaction has been highly characterized using various molecular and cellular assays. It was then evaluated in two different NASH-relevant preclinical models, and RNAseq data were obtained with samples from Diet-induced Obesity NASH (DIO NASH) mouse model. 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
2022-09-07 | GSE212644 | GEO
Project description:Chemotherapy resistance is a major problem in non-small cell lung cancer (NSCLC) treatment. A major mechanism of chemoresistance involves stabilization of the NRF2 transcription factor. NRF2 levels are normally tightly regulated through interaction with KEAP1, an adaptor that targets NRF2 to the CUL3 E3 ubiquitin ligase for proteolysis. In NSCLC, aberrant NRF2 stabilization is best understood through mutations in NRF2, KEAP1, or CUL3 that disrupt their interaction. Biochemical studies, however, have revealed that NRF2 can also be stabilized through expression of KEAP1-interacting proteins that competitively sequester KEAP1 away from NRF2. Here, we have identified PIDD, as a novel KEAP1-interactor in NSCLC that regulates NRF2. We show that this interaction allows PIDD to reduce NRF2 ubiquitination and increase its stability. We also demonstrate that PIDD promotes chemoresistance in NSCLC cells both in vitro and in vivo, and that this effect is dependent on NRF2. Finally, we report that NRF2 protein expression in a NSCLC cohort exceeds the typical incidence of combined NRF2, KEAP1, and CUL3 mutations, and that NRF2 expression in this cohort is correlated with PIDD levels. Our data identify PIDD as a new NRF2 regulator, and suggest that variations in PIDD levels contribute to differential chemosensitivities among NSCLC patients.
| S-EPMC6712044 | biostudies-literature