Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1)

Bresciani, Alberto; Missineo, Antonino; Gallo, Mariana; Cerretani, Mauro; Fezzardi, Paola; Tomei, Licia; Cicero, Daniel O.; Altamura, Sergio; Santoprete, Alessia; Ingenito, Raffaele; Bianchi, Elisabetta; Pacifici, Robert E.; Dominguez, Celia; Muñoz-Sanjuán, Ignacio; Harper, Steven J.; Toledo-Sherman, Leticia; Park, Larry C.

doi:10.1016/j.abb.2017.08.003

Cited by 37 publications

(26 citation statements)

References 28 publications

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“…Kinetic fit of the sensorgram to a 1:1 Langmuir model was performed (red line) and the rate constants ka and kd were calculated to 1.1 x 10 6 M -1 s -1 and 1.58 x 10 -3 Figure 1A). 29,31,34 For compound 3, the determined Kd of 2.4 μM ( Table 1) was in similar range of the reported Kd values of 1 μM 29 and 0.8 μM 31 . The SPR determined affinity (Kd) of 4 was similar to the Ki value determined by FP (Table 1), although lower than the Kd reported in literature.…”

Section: Binding Activity In Spr Assaysupporting

confidence: 82%

“…Ghorab et al 45 ; 18: Sun et al 46 ; 19-21: Zhuang et al 35 Figure 1A). 22,24,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] Additionally, a number of inhibitors (3, 4, 6, 7, 9, 11, and 14) have been confirmed by X-ray crystallography to bind the Kelch domain (see examples in Figure 1B). 10 However, since the Kelch binding pocket is still rather large compared to classical enzyme or receptor pockets and contains multiple arginine residues ( Figure 1B), bulky ligands featuring carboxylic acids have been enriched in medicinal chemistry programs.…”

Section: Introductionmentioning

confidence: 98%

“…7 Furthermore, the pocket features five interconnected epitopes (P1-P5), of which at least P1, P4, and P5 are considered hot spots for ligand-binding ( Figure 1B). 22,26,27 28 , Hancock et al 24 ; 3: Hu et al 29 , Jnoff et al 30 , Bresciani et al 31 , PubChem 32 ; 4:…”

Section: Introductionmentioning

confidence: 99%

“…Marcotte et al 33 , Jiang et al 22 , Jain et al 34 , Zhuang et al 35 ; 5: Jiang et al 22 ; Jain et al 34 ; Bresciani et al 31 ; 6: Jain et al 34 ; 7: Winkel et al 36 , Szill et al 37 ; 8: Yasuda et al 38 ; 9: Davies et al 27 ; 10: Callahan et al 39 ; 11: Marcotte et al 33 ; 12: Bertrand et al 40 ; 13: Kazantsev et al 41 ; 14: Satoh et al 42 ; 15: Shimozono et al 43 ; 16: Xu et al 44 ; 17:…”

Section: Introductionunclassified

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A Comparative Assessment Study of Known Small-Molecule Keap1−Nrf2 Protein–Protein Interaction Inhibitors: Chemical Synthesis, Binding Properties, and Cellular Activity

et al. 2019

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Inhibiting the protein-protein interaction (PPI) between the transcription factor Nrf2 and its repressor protein Keap1 has emerged as a promising strategy to target oxidative stress in diseases, including CNS disorders. Numerous non-covalent small-molecule Keap1-Nrf2 PPI inhibitors have been reported to date, but many feature suboptimal physicochemical properties for permeating the blood-brain barrier, while others contain problematic structural moieties. Here, we present the first side-by-side assessment of all reported Keap1-Nrf2 PPI inhibitor classes using fluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR)-and further evaluate the compounds in an NQO1 induction cell assay and in counter tests for non-boronate ester building block 41 as the crucial carbon skeleton-building step. Synthesis of the enol triflate using the one-step NaHMDS-mediated enolization/PhNTf2-induced trapping procedure reported in the patent application 39 was not efficient in our hands, giving low yield (43% vs. 88% reported in literature 39) and significant byproduct formation. We found that using a freshly-made LiHMDS as an alternative base gave a cleaner reaction and excellent yield (quantitative). The converging SM reaction step between 38 and 41 gave several wellknown by-products, including the boronic acid, aryl boronate homo-coupling product and protodeboronation product, but could still afford the desired cross-coupling product 42 in good yield (69% vs. 33% reported in literature 39). Catalytic hydrogenation to deprotect the carboxylic acid and reduce the alkene double bond diastereoselectively furnished only the cis-cyclohexane 43 in accordance with literature; 39 this was revealed by nuclear Overhauser effect (NOE) NMR (Supporting Information Figure S1). This facial selectivity can be explained by a steric directing effect of the carboxybenzyl group. The cyclohexane carboxylic acid of 43 was finally coupled with 2-butylpyrrolidine and the pyrazole carboxylic acid deprotected to give 10 as a mixture of four stereoisomers. Attempted separation of the two diastereoisomers by preparative HPLC was unsuccessful. In the patent application, purification by HPLC is reported to give two different fractions, each containing all four stereoisomers in slightly different proportions, of which one was directly tested as a mixture. 39 Having this literature result as a reference point, we did not proceed with further purification of 10.

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Section: Binding Activity In Spr Assaysupporting

confidence: 82%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionunclassified

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A Comparative Assessment Study of Known Small-Molecule Keap1−Nrf2 Protein–Protein Interaction Inhibitors: Chemical Synthesis, Binding Properties, and Cellular Activity

et al. 2019

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“…Excessive ROS production results in biomolecule damage and cancer cell apoptosis, and ROS have been widely found to play a crucial role in apoptosis upon cancer treatment [28]. Nrf2 is a member of the basic leucine zipper transcription factor NF-E2 family, which coordinates the induction of antioxidant and phase II detoxifying enzymes [29]. Keap1 is a component of the Cullin 3-based E3 ubiquitin ligase complex that controls the stability and accumulation of Nrf2 [30].…”

Section: Discussionmentioning

confidence: 99%

Fasting Induces Hepatocellular Carcinoma Cell Apoptosis by Inhibiting SET8 Expression

Chen

et al. 2020

Oxidative Medicine and Cellular Longevity

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Background. Hepatocellular carcinoma (HCC) is a life-threatening cancer, and the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signalling pathway plays a crucial role in apoptosis resistance in cancer cells. Fasting is reported to mediate tumour growth reduction and apoptosis. SET8 is involved in cancer proliferation, invasiveness, and migration. However, whether SET8 participates in fasting-mediated apoptosis in HCC remains unclear. Methods. We used immunohistochemical staining to analyse the expression of SET8, Keap1, and Nrf2 in HCC tissues. Cell viability, apoptosis, and cellular reactive oxygen species (ROS) were assessed, and Western blot and qPCR analyses were used to examine the expression of Keap1/Nrf2 in HCC cells under fasting, SET8 overexpression, and PGC1α overexpression conditions. Mass spectrometry, coimmunoprecipitation, and confocal microscopy were used to determine whether PGC1α interacts with SET8. In vivo experiments were performed to verify the conclusions from the in vitro experiments. Results. Our data indicate that SET8 expression is associated with poor survival in HCC patients. Both in vitro and in vivo results demonstrated that fasting decreased cell viability and downregulated expression of SET8, Nrf2, and downstream effectors of Nrf2, while it upregulated Keap1 expression, mediated ROS accumulation, and induced HCC cell apoptosis. These results were similar to what is observed in SET8-deficient cells. Furthermore, SET8 was found to interact with PGC1α, and both PGC1α and H4K20me1, a downstream target of SET8, were found to be enriched at the Keap1 promoter region. These two factors were further determined to attenuate Keap1 promoter activity. Conclusions. The results of our study demonstrate that fasting induces HCC apoptosis by inhibiting SET8 expression and that SET8 interacts with PGC1α to activate the Nrf2/ARE signalling pathway by inhibiting Keap1 expression.

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Inhibiting the Keap1/Nrf2 Protein‐Protein Interaction with Protein‐Like Polymers

Carrow,

Hamilton,

Hopps

et al. 2024

Advanced Materials

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Successful and selective inhibition of the cytosolic protein‐protein interaction (PPI) between nuclear factor erythroid 2‐related factor 2 (Nrf2) and Kelch‐like ECH‐associating protein 1 (Keap1) can enhance the antioxidant response, and has a potential therapeutic effect in a range of settings including in neurodegenerative disease. Small molecule inhibitors have been developed, yet many have off‐target effects or are limited by poor cellular permeability. Peptide‐based strategies have been used to enhance specificity yet face challenges due to susceptibility to degradation and lack of cellular penetration. Herein, we overcome these barriers utilizing a polymer‐based proteomimetics. The protein‐like polymer (PLP) consists of a synthetic, lipophilic polymer backbone displaying water soluble Keap1‐binding peptides on each monomer unit forming a brush polymer architecture. The PLPs are capable of engaging Keap1 and displacing the cellular protective transcription factor Nrf2, which then translocates to the nucleus, activating the antioxidant response element (ARE). PLPs exhibit increased Keap1 binding affinity by several orders of magnitude compared to free peptides, maintain serum stability, are cell‐penetrant, and selectively activate the ARE pathway in cells, including in primary cortical neuronal cultures. Keap1/Nrf2‐inhibitory PLPs have the potential to impact the treatment of disease states associated with dysregulation of oxidative stress, such as neurodegenerative diseases.This article is protected by copyright. All rights reserved

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Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1)

Cited by 37 publications

References 28 publications

A Comparative Assessment Study of Known Small-Molecule Keap1−Nrf2 Protein–Protein Interaction Inhibitors: Chemical Synthesis, Binding Properties, and Cellular Activity

A Comparative Assessment Study of Known Small-Molecule Keap1−Nrf2 Protein–Protein Interaction Inhibitors: Chemical Synthesis, Binding Properties, and Cellular Activity

Fasting Induces Hepatocellular Carcinoma Cell Apoptosis by Inhibiting SET8 Expression

Inhibiting the Keap1/Nrf2 Protein‐Protein Interaction with Protein‐Like Polymers

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