BTB Domain Of Keap1 Research Articles

Characterization of the modification of Kelch-like ECH-associated protein 1 by different fumarates

Fumarates (fumaric acid esters), primarily dimethyl fumarate (DMF) and monoethyl fumarate (MEF) and its salts, are orally administered systemic agents used for the treatment of psoriasis and multiple sclerosis. It is widely believed that the pharmaceutical activities of fumarates are exerted through the Keap1-Nrf2 pathway. Although it has been revealed that DMF and MEF differentially modify specific Keap1 cysteine residues and result in the differential activation of Nrf2, how the modification of DMF and MEF impacts the biochemical properties of Keap1 has not been well characterized. Here, we found that both DMF and MEF can only modify the BTB domain of Keap1 and that only C151 is accessible for covalent binding in vitro. Dynamic fluorescence scanning (DSF) assays showed that the modification of DMF to Keap1 BTB increased its thermal stability, while the modification of MEF dramatically decreased its thermal stability. Further crystal structures revealed no significant conformational variation between the DMF-modified and MEF-modified BTBs. Overall, our biochemical and structural study provides a better understanding of the covalent modification of fumarates to Keap1 and may suggest fundamentally different mechanisms adopted by fumarates in regulating the Keap1-Nrf2 pathway.

Electrophilic thymol isobutyrate from Inula nervosa Wall. (Xiaoheiyao) ameliorates steatosis in HepG2 cells via Nrf2 activation

• Electrophilic components from Xiaoheiyao are knocked out using magnetic GSH probe. • Electrophilic components have the potential to ameliorate steatosis in HepG2 cells. • 10-Isobutyryloxy-8,9-epoxythymol isobutyrate is discovered as a bioactive compound. • 10-Isobutyryloxy-8,9-epoxythymol isobutyrate improves steatosis via Nrf2 activation. Xiaoheiyao , the rhizomes of Inula nervosa Wall., is a culinary spice known to contain some electrophilic Nrf2 activators that could potentially intervene in nonalcoholic fatty liver disease (NAFLD). In this study, the electrophilic components from Xiaoheiyao were annotated by comprehensive cheminformatics analyses on UHPLC-MS/MS data after being knocked out with glutathione (GSH)-immobilized magnetic microspheres. Their intervention effects against hepatic steatosis were investigated in free fatty acid (FFA)-treated HepG2 cells. The electrophilic components in Xiaoheiyao were found to be the major bioactive components preventing hepatic steatosis in FFA-treated HepG2 cells. In particular, 10-isobutyryloxy-8,9-epoxythymol isobutyrate (XHY-1) improved hepatic lipid metabolism disorder, Krebs cycle activity, oxidative phosphorylation, and the cellular and mitochondrial redox status. Moreover, XHY-1 activates the Nrf2-ARE signaling pathway through the up-regulation of Nrf2 expression and promotion of Nrf2 nuclear translocation. Molecular docking further showed that XHY-1 could conjugate with Cys151 in the BTB domain of Keap1 through the epoxy group. In conclusion, the electrophilic components of Xiaoheiyao , especially XHY-1, attenuated hepatic steatosis in FFA-treated HepG2 cells through activation of the Nrf2-ARE signaling pathway.

Potential Cancer- and Alzheimer's Disease-Targeting Phosphodiesterase Inhibitors from Uvaria alba: Insights from In Vitro and Consensus Virtual Screening.

Inhibition of the major cyclic adenosine monophosphate-metabolizing enzyme PDE4 has shown potential for the discovery of drugs for cancer, inflammation, and neurodegenerative disorders such as Alzheimer’s disease. As a springboard to explore new anti-cancer and anti-Alzheimer’s chemical prototypes from rare Annonaceae species, the present study evaluated anti-PDE4B along with antiproliferative and anti-cholinesterase activities of the extracts of the Philippine endemic species Uvaria alba using in vitro assays and framed the resulting biological significance through computational binding and reactivity-based experiments. Thus, the PDE4 B2B-inhibiting dichloromethane sub-extract (UaD) of U. alba elicited antiproliferative activity against chronic myelogenous leukemia (K-562) and cytostatic effects against human cervical cancer (HeLa). The extract also profoundly inhibited acetylcholinesterase (AChE), an enzyme involved in the progression of neurodegenerative diseases. Chemical profiling analysis of the bioactive extract identified 18 putative secondary metabolites. Molecular docking and molecular dynamics simulations showed strong free energy binding mechanisms and dynamic stability at 50-ns simulations in the catalytic domains of PDE4 B2B, ubiquitin-specific peptidase 14, and Kelch-like ECH-associated protein 1 (KEAP-1 Kelch domain) for the benzylated dihydroflavone dichamanetin (16), and of an AChE and KEAP-1 BTB domain for 3-(3,4-dihydroxybenzyl)-3′,4′,6-trihydroxy-2,4-dimethoxychalcone (8) and grandifloracin (15), respectively. Density functional theory calculations to demonstrate Michael addition reaction of the most electrophilic metabolite and kinetically stable grandifloracin (15) with Cys151 of the KEAP-1 BTB domain illustrated favorable formation of a β-addition adduct. The top-ranked compounds also conferred favorable in silico pharmacokinetic properties.

Structural insights into the multiple binding modes of Dimethyl Fumarate (DMF) and its analogs to the Kelch domain of Keap1.

The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription function has been implicated in the protection of neurodegenerative diseases. The cytoplasmic protein, Kelch-like ECH-associated protein 1 (Keap1), negatively regulates Nrf2. The Keap1-Nrf2 pathway is a potential therapeutic target for tackling free-radical damage. Dimethyl fumarate (DMF) is currently an approved drug for the treatment of relapsing multiple sclerosis. Recent studies showed that DMF modifies the reactive cysteines in the BTB domain of Keap1 and thus activates Nrf2 transcription function. Intriguingly, our crystal structure studies revealed that DMF also binds to the β-propeller domain (Keap1-DC) of Keap1. The crystal structure of the complex, refined to 1.54Å resolution, revealed unexpected features: DMF binds (a) to the Nrf2-binding site (bottom region of Keap1-DC, site 1) with moderate interaction, and (b) to the top region of Keap1-DC, near to the blade II (site 2). The specificity of the binding 'site 2' was found to be unique to blade II of the β-propeller domain. The newly identified 'site 2' region in Keap1-DC may have a different functional role to regulate Nrf2. Moreover, the crystal structures of Keap1-DC in complex with the DMF analogs, including monoethyl fumarate, fumarate, and itaconate, also exhibited similar binding modes with Keap1-DC. Binding studies confirmed that DMF binds, in a nanomolar range, to the Keap1-DC region as well as the BTB domain of Keap1. Furthermore, the competitive binding assay in the presence of the Nrf2 peptide affirmed the direct binding of DMF at the Nrf2-binding region of Keap1-DC. Overall, our studies suggest that the drug molecule, DMF, binds at multiple sites of Keap1 and thus potentially activates Nrf2 function through covalent as well as the noncovalent mode of action, to combat oxidative stress. DATABASE: Structural data are available in RCSB-protein data bank database(s) under the accession numbers 6LRZ, 7C60, and 7C5E.

Formononetin ameliorates oxaliplatin-induced peripheral neuropathy via the KEAP1-NRF2-GSTP1 axis

Management of oxaliplatin-induced peripheral neuropathy (OIPN) has proven challenging owing to the concern that any OIPN-preventing agents may also decrease the efficacy of the chemotherapeutic agent and fail to reverse established neuronal damage. Nevertheless, targeting redox signaling pathways constitutes a promising therapy in OIPN and we have previously demonstrated the protective role of nuclear factor erythroid-2 related factor 2 (NRF2) in this disorder. Here, we investigated the protective properties of formononetin (FN), a clinical preparation extract, in OIPN. RNA interference experiments revealed that FN protects against OIPN directly through activation of the NRF2 pathway. Further expression profile sequencing showed that FN exerts its protective effect via the NRF2 downstream-oxaliplatin metabolism enzyme, GSTP1. We also demonstrated that FN does not influence the chemotherapeutic function of oxaliplatin, as NRF2 exhibits a different drug metabolic enzyme activation state downstream in colorectal cell lines than that in neurons. Following synthesis of Bio-FN to screen the target binding proteins, we found that FN selectively binds to His129 and Lys131 in the BTB domain of KEAP1. In vivo experiments revealed that FN-induced activation of the NRF2 signaling pathway alleviated the nociceptive sensations in mice. Our findings highlight a new binding mechanism between KEAP1 and isoflavones for activation of the NRF2 system and suggest that pharmacological or therapeutic activation of the NRF2-GSTP1 axis may serve as an effective strategy to prevent or attenuate the progression of OIPN.

Effects of 3-Bromo-4,5-dihydroisoxazole Derivatives on Nrf2 Activation and Heme Oxygenase-1 Expression.

Natural and synthetic electrophilic compounds have been shown to activate the antioxidant protective Nrf2 (nuclear factor erythroid 2‐related factor 2)/heme oxygenase‐1 (HO‐1) axis in cells and tissues. Here, we tested the ability of different isoxazoline‐based electrophiles to up‐regulate Nrf2/HO‐1. The potency of activation is dependent on the leaving group at the 3‐position of the isoxazoline nucleus, and an additional ring on the molecule limits the Nrf2/HO‐1 activating properties. Among the synthetized compounds, we identified 3‐bromo‐5‐phenyl‐4,5‐dihydroisoxazole 1 as the derivative with best activating properties in THP‐1 human monocytic cells. We have confirmed that the target of our compounds is the Cys151 of the BTB domain of Keap1 by using mass spectrometry analyses and X‐ray crystallography. Our findings demonstrate that these compounds affect the Nrf2/HO‐1 axis and highlight a positive activity that can be of relevance from a therapeutic perspective in inflammation and infection.

Britanin Ameliorates Cerebral Ischemia-Reperfusion Injury by Inducing the Nrf2 Protective Pathway.

Oxidative stress is considered the major cause of tissue injury after cerebral ischemia. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is one of the most important defensive mechanisms against oxidative stresses and has been confirmed as a target for stroke treatment. Thus, we desired to find new Nrf2 activators and test their neuronal protective activity both in vivo and in vitro. The herb-derived compound, Britanin, is a potent inducer of the Nrf2 system. Britanin can induce the expression of protective enzymes and reverse oxygen-glucose deprivation, followed by reperfusion (OGD-R)-induced neuronal injury in primary cortical neurons in vitro. Furthermore, the administration of Britanin significantly ameliorated middle cerebral artery occlusion-reperfusion (MCAO-R) insult in vivo. We report here the crystal structure of the complex of Britanin and the BTB domain of Keap1. Britanin selectively binds to a conserved cysteine residue, cysteine 151, of Keap1 and inhibits Keap1-mediated ubiquitination of Nrf2, leading to induction of the Nrf2 pathway. Britanin is a potent inducer of Nrf2. The complex crystal structure of Britanin and the BTB domain of Keap1 help clarify the mechanism of Nrf2 induction. Britanin was proven to protect primary cortical neurons against OGD-R-induced injury in an Nrf2-dependant way. Additionally, Britanin had excellent cerebroprotective effect in an MCAO-R model. Our results demonstrate that the natural product Britanin with potent Nrf2-activating and neural protective activities both in vitro and in vivo could be developed into a cerebroprotective therapeutic agent. Antioxid. Redox Signal. 27, 754-768.

Effects of Novel Nitric Oxide-Releasing Molecules against Oxidative Stress on Retinal Pigmented Epithelial Cells.

Oxidative stress is a hallmark of retinal degenerations such as age-related macular degeneration and diabetic retinopathy. Enhancement of heme oxygenase-1 (HO-1) activity in the retina would exert beneficial effects by protecting cells from oxidative stress, therefore promoting cell survival. Because a crosstalk exists between nitric oxide (NO) and HO-1 in promotion of cell survival under oxidative stress, we designed novel NO-releasing molecules also capable to induce HO-1. Starting from curcumin and caffeic acid phenethyl ester (CAPE), two known HO-1 inducers, the molecules were chemically modified by acylation with 4-bromo-butanoyl chloride and 2-chloro-propanoyl chloride, respectively, and then treated in the dark with AgNO3 to obtain the nitrate derivatives VP10/12 and VP10/39. Human retinal pigment epithelial cells (ARPE-19) subjected to H2O2-mediated oxidative stress were treated with the described NO-releasing compounds. VP10/39 showed significant (p < 0.05) antioxidant and protecting activity against oxidative damage, in comparison to VP10/12, which in turn showed at 100 μM concentration a slight but significant cell toxicity. Only VP10/39 significantly (p < 0.05) induced HO-1 in ARPE-19, most likely through covalent bond formation at Cys151 of the Keap1-BTB domain, as revealed from molecular docking analysis. In conclusion, the present data indicate VP10/39 as a promising candidate to protect ARPE-19 cells against oxidative stress.

Characterization of novel small-molecule NRF2 activators: Structural and biochemical validation of stereospecific KEAP1 binding

BackgroundSemi-synthetic oleanane triterpenoid antioxidant inflammation modulators (tpAIMs) are small molecules that interact with KEAP1 cysteine residue 151 (C151) and activate NRF2. Exploration of the structure-activity relationship between the tpAIMs and KEAP1 is limited by the predominantly hydrocarbon nature of the oleanane triterpenoid pentacyclic ring structure. Therefore, we used novel, chemically-tractable, synthetic antioxidant inflammation modulators (sAIMs) to probe the stereoselectivity of the ligand-protein interaction. MethodsWe measured several parameters of NRF2 activation to assess the potency of sAIM enantiomers with natural (tpAIM-like) 4(S),5(S),10(R) or unnatural 4(R),5(R),10(S) configurations. Additionally, we determined the crystal structure of the KEAP1 BTB domain in complex with two different sAIMs. ResultsWe found that the potencies of sAIM enantiomers in the natural configuration were similar to those of the tpAIM, RTA 405. Strikingly, sAIM enantiomers in the unnatural configuration were 10- to 40-fold less potent than their natural counterparts. Crystallographic studies of sAIMs in complex with the KEAP1 BTB domain demonstrated that these ligands form a covalent bond with C151 and revealed the presence of additional hydrogen bonds, Van der Waals interactions, and pi-stacking interactions. ConclusionsAlthough KEAP1 C151 is required for NRF2 activation by tpAIMs and sAIMs, interactions with other KEAP1 residues are critical for the stereospecific recognition and potency of these ligands. General significanceThis work demonstrates that reversible cyanoenone Michael acceptors, such as the tpAIMs and sAIMs, can be specifically tuned to regulate redox sensitive cysteine residues on key signaling molecules, an approach with significant promise for innovative drug development.

Pterisolic Acid B is a Nrf2 Activator by Targeting C171 within Keap1-BTB Domain.

The use of chemoprotective agents to minimize the side effects of the chemotherapy, primarily via activation of the Nrf2 pathway, is an emerging research field, which has attracted broad attention from both academia and pharmaceutical industry. Through high-throughput chemical screens we have disclosed that pterisolic acid B (J19), a naturally occuring diterpenoid, is an effective Nrf2 activator. We have also identified a more potent natural product analogue J19-1 by semisynthesis and the subsequent biochemical evaluations revealed that J19-1 activates the Nrf2 pathway by covalently modifying Cys171 of keap1, which inhibits Nrf2 degradation mediated by Keap1-Cul3 complexes. Ultimately, we have demonstrated that J19-1 shows significant cytoprotective effect against cisplatin-induced cytotoxicity in HKC cells.

Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO.

The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.

The Discovery of Direct Noncovalent Bonded Interaction between Keap1 and Diethyl Maleate through Molecular Dynamics Simulations

Keap1 negatively regulates the function of Nrf2 that is a major activator of genes encoding phase 2 detoxifying enzymes via sequestering cytoplasmic Nrf2 and subsequent degradation through the proteasome system. Reactive cysteine residues of Keap1 could be modified by Michael reaction acceptor molecules. Previous studies have shown that adduction at Cys151 by diethyl maleate (DEM) can give rise to a significant conformational change in Keap1 that leads to the dissociation of Keap1 from CUL3, hence inhibits Nrf2 ubiquitylation. The BTB domain of Keap1 plays a crucial role in both forming self-dimerization and binding to CUL3. In order to better understanding the molecular mechanism how DEM interact with amino acid residues around Cys151, we performed two molecular dynamics (MD) simulations including Keap1-DEM complex and Keap1 alone (control group). Interestingly, we found that after a short period of lingering around Cys151, DEM ultimately stabilized in a gap between two specific helixes away from the cavity around Cys151 and induced a concomitant significant conformational change of BTB domain of Keap1. Similar phenomenon, however, was not observed in the control group. These results suggested that DEM could impair the normal function of Keap1 by inducing the conformational change of BTB domain via direct noncovalent bonded interaction. Our research provides a new insight into another way of interaction between Keap1 and DEM in spite of their known Michael addition reaction, by which novel phase2 enzyme inducer drugs with higher specificity could be discovered in the future

Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

Targeting Nrf2 signaling appears to be an attractive approach for the treatment of maladaptive cardiac remodeling and dysfunction; however, pharmacological modulation of the Nrf2 pathway in the cardiovascular system remains to be established. Herein, we report that a novel synthetic triterpenoid derivative, dihydro-CDDO-trifluoroethyl amide (dh404), activates Nrf2 and suppresses oxidative stress in cardiomyocytes. Dh404 interrupted the Keap1-Cul3-Rbx1 E3 ligase complex-mediated Nrf2 ubiquitination and subsequent degradation saturating the binding capacity of Keap1 to Nrf2, thereby rendering more Nrf2 to be translocated into the nuclei to activate Nrf2-driven gene transcription. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 was resistant to dh404-induced stabilization of Nrf2 protein. In addition, dh404 did not dissociate the interaction of Nrf2 with the Keap1-Cul3-Rbx1 E3 ligase complex. Thus, it is likely that dh404 inhibits the ability of Keap1-Cul3-Rbx1 E3 ligase complex to target Nrf2 for ubiquitination and degradation via modifying Cys-151 of Keap1 to change the conformation of the complex. Moreover, dh404 was able to stabilize Nrf2 protein, to enhance Nrf2 nuclear translocation, to activate Nrf2-driven transcription, and to suppress angiotensin II (Ang II)-induced oxidative stress in cardiomyocytes. Knockdown of Nrf2 almost blocked the anti-oxidative effect of dh404. Dh404 activated Nrf2 signaling in the heart. Taken together, dh404 appears to be a novel Nrf2 activator with a therapeutic potential for cardiac diseases via suppressing oxidative stress.

Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex

The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.