Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization

Sogabe, Satoshi; Sakamoto, Kotaro; Kamada, Yusuke; Kadotani, Akito; Fukuda, Yasunori; Sakamoto, Junichi

doi:10.1016/j.bbrc.2017.03.038

Cited by 9 publications

(7 citation statements)

References 31 publications

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“…PPI inhibition assay development using TR-FRET. Keap1/Nrf2 PPI inhibition and Bcl6/F1325 PPI inhibition counter assays were performed using TR-FRET as previously described 52,53 to determine the potency of selected inhibitors using 384-well white flat-bottom small volume plates (Greiner Bio-One). Assay buffer consisting of 50 mM Tris-HCl (pH 7.5, FujiFilm Wako Pure Chemical), 100 mM NaCl (Nacalai Tesque), 0.01% Tween-20 (Bio-Rad), 1 mM dithiothreitol (FujiFilm Wako Pure Chemical), and 0.01% bovine serum albumin (Merck Millipore) was used to dilute the reagents.…”

Section: Preparation Of Human Keap1 and Bcl6 Proteinmentioning

confidence: 99%

Identification of novel inhibitors of Keap1/Nrf2 by a promising method combining protein–protein interaction-oriented library and machine learning

Shimizu

Yonezawa

Sakamoto³

et al. 2021

Sci Rep

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Protein–protein interactions (PPIs) are prospective but challenging targets for drug discovery, because screening using traditional small-molecule libraries often fails to identify hits. Recently, we developed a PPI-oriented library comprising 12,593 small-to-medium-sized newly synthesized molecules. This study validates a promising combined method using PPI-oriented library and ligand-based virtual screening (LBVS) to discover novel PPI inhibitory compounds for Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2). We performed LBVS with two random forest models against our PPI library and the following time-resolved fluorescence resonance energy transfer (TR-FRET) assays of 620 compounds identified 15 specific hit compounds. The high hit rates for the entire PPI library (estimated 0.56–1.3%) and the LBVS (maximum 5.4%) compared to a conventional screening library showed the utility of the library and the efficiency of LBVS. All the hit compounds possessed novel structures with Tanimoto similarity ≤ 0.26 to known Keap1/Nrf2 inhibitors and aqueous solubility (AlogP < 5). Reasonable binding modes were predicted using 3D alignment of five hit compounds and a Keap1/Nrf2 peptide crystal structure. Our results represent a new, efficient method combining the PPI library and LBVS to identify novel PPI inhibitory ligands with expanded chemical space.

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Section: Preparation Of Human Keap1 and Bcl6 Proteinmentioning

confidence: 99%

Identification of novel inhibitors of Keap1/Nrf2 by a promising method combining protein–protein interaction-oriented library and machine learning

Shimizu

Yonezawa

Sakamoto³

et al. 2021

Sci Rep

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“…Because of its high binding potency and short sequence length, the ETGE containing motif provides a good starting point for research of Keap1–Nrf2 inhibitors. A cell-penetrant ETGE-derived peptide conjugated with trans-activating transcriptional activator (TAT) sequence confirmed the Nrf2 activation effects, and then a number of peptide inhibitors were reported to explore the interface of Keap1–Nrf2 PPI, − revealing the five subpockets model of the Keap1 cavity. , To date, a lot of small molecule Keap1–Nrf2 inhibitors have been reported, − and some of the inhibitors have achieved high potency. − …”

Section: Introductionmentioning

confidence: 99%

Discovery of a Potent Kelch-Like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1–Nrf2) Protein–Protein Interaction Inhibitor with Natural Proline Structure as a Cytoprotective Agent against Acetaminophen-Induced Hepatotoxicity

Zhang

et al. 2019

J. Med. Chem.

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The transcription factor Nrf2 is a key regulator of cytoprotective system, and enhancing Nrf2 activity can protect cells from various insults and threats. Directly disrupting Keap1−Nrf2 protein−protein interactions has been regarded as a promising way to activate Nrf2. We reported here the first identification of amino acids as preferred substituents to design potent Keap1−Nrf2 inhibitors. Comprehensive structure−activity analysis identified Pro as a preferred substituent, obtaining a potent inhibitor 35 with an IC 50 of 43 nM in the competitive fluoresce polarization (FP) assay and a K d value of 53.7 nM for Keap1 protein in the isothermal titration calorimetry (ITC) assay. The Pro analogue 35 exhibited tight and prolonged Keap1 binding in vitro and in cells, and treatment with 35 activated Nrf2-regulated cytoprotective response and antagonized acetaminophen-induced liver injury both in cellular and in vivo models. This work not only provides a useful tool to further explore the therapeutic potential of Keap1−Nrf2 inhibition but also enriches the diversity of chemical structures suitable for the Keap1−Nrf2 interface.

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“…In RAC1 (Rac family small GTPase 1) 51 , the driver and compensators all appear to be involved in modifying the binding, cleavage, and release of GDP. In KEAP1 (kelch like ECH associated protein (1) 52 , 53 , driver G333C and compensators S508P and R554K appear at the binding interface of KEAP1 and an engineered peptide shown to inhibit the interaction with NRF2 (NFE2L2; nuclear factor, erythroid 2-like (2). It has been shown that G333C mutants of KEAP1 are unable to repress NRF2 activity 40 , further demonstrating the functional importance of these sites.…”

Section: Resultsmentioning

confidence: 99%

Deep phylogeny of cancer drivers and compensatory mutations

2020

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Driver mutations (DM) are the genetic impetus for most cancers. The DM are assumed to be deleterious in species evolution, being eliminated by purifying selection unless compensated by other mutations. We present deep phylogenies for 84 cancer driver genes and investigate the prevalence of 434 DM across gene-species trees. The DM are rare in species evolution, and 181 are completely absent, validating their negative fitness effect. The DM are more common in unicellular than in multicellular eukaryotes, suggesting a link between these mutations and cell proliferation control. 18 DM appear as the ancestral state in one or more major clades, including 3 among mammals. We identify within-gene, compensatory mutations for 98 DM and infer likely interactions between the DM and compensatory sites in protein structures. These findings elucidate the evolutionary status of DM and are expected to advance the understanding of the functions and evolution of oncogenes and tumor suppressors.

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Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization

Cited by 9 publications

References 31 publications

Identification of novel inhibitors of Keap1/Nrf2 by a promising method combining protein–protein interaction-oriented library and machine learning

Identification of novel inhibitors of Keap1/Nrf2 by a promising method combining protein–protein interaction-oriented library and machine learning

Discovery of a Potent Kelch-Like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1–Nrf2) Protein–Protein Interaction Inhibitor with Natural Proline Structure as a Cytoprotective Agent against Acetaminophen-Induced Hepatotoxicity

Deep phylogeny of cancer drivers and compensatory mutations

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