Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling

Abstract

The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a K d of ϳ6 M, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 M RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.The transcription factor NF-E2-related factor 2 (Nrf2) 2 is a promising target for the treatment of oxidative and inflammatory stress-related disorders, such as neurodegenerative and microvascular diseases (1-5). Nrf2 regulates the expression of several cytoprotective anti-oxidative and anti-inflammatory proteins by binding to the cis-acting antioxidant response element (ARE) within gene promoters. Nrf2 itself is regulated by the kelch-like ECH-associated protein 1 (Keap1), which is a substrate recognition subunit for a cullin3-based ubiquitin E3 ligase and functions as a sensor for oxidative and electrophilic stress. Structural elements of the Keap1 protein include the C-terminal Nrf2-binding kelch domain, the intervening region, and the broad complex-Tramtrack-Bric-a-Brac domain. Keap1 binds as a dimer via its two Kelch domains to one molecule of Nrf2, specifically to the high affinity ETGE and the low affinity DLG motives at the N terminus of Nrf2. Without stressors, this leads to the ubiquitinylation and subsequent proteolytic degradation of the transcription factor. In the presence of oxidative or electrophilic stress, cysteine residues within the intervening region and broad complex-Tramtrack-Bric-a-Brac domain of Keap1 become modified. According to the hinge and latch model, this weakens the interaction between Keap1 and the DLG motif of Nrf2 but does not lead to a release of Nrf2 (6, 7). The conformation cycling model postulates a stabilization of the Keap1/Nrf2 interaction by the Keap1 modifica...

Transcription Factor NRF2 as a Therapeutic Target for Chronic Diseases: A Systems Medicine Approach

“…The docking to KEAP1 of the low-affinity DLG motif of NRF2 has also been characterized (Tong et al, 2007). Based on these interactions, peptidomimetic compounds were the first example of PPI inhibitors with significantly improved selectivity over electrophiles (Hu et al, 2013;Marcotte et al, 2013;Winkel et al, 2015). These inhibitors show weak activity in cells, and a new provocative strategy has now been reported based on the use of cyclic peptides.…”

Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice

“…Recently, we have characterized two siRNAs (Table 1) against murine Keap1, siKeap1-1 and siKeap1-2, which are able to reduce Keap1 mRNA levels leading to activation of Nrf2 [21]. In the present study these siRNAs were modified by introducing 2’-O-methyl groups and phosphorothioate to allow chronic in vivo administration.…”

“…The integrity of RNA samples was controlled by RNA nano assay (Agilent 2100 BioAnalyzer) and transferred to Atlas Biolabs (Berlin) for analysis. Amplification, labeling of probes, Affymetrix GeneChip hybridizations, comparative microarray analysis, and subsequent statistical analysis were performed using Mouse Genome 430 2.0 GeneChips (Affymetrix), as described previously [21]. All Affymetrix microarray data have been deposited in MIAME compliant format at NCBI-GEO under the accession number GSE80956.…”

“…In the present study we assessed the effects of inducible Nrf2 activation in the hepatic metabolism of adult mice under a Western diet. Due to the lack of suitable pharmacological tool compounds [21], we activated Nrf2 in liver using siRNAs against Keap1 as recently described [22]. Our data suggest that the chronic activation of Nrf2 in liver has no major effect on metabolism under the challenge of a Western diet.…”

Identification of compounds that inhibit the binding of Keap1a/Keap1b Kelch DGR domain with Nrf2 ETGE/DLG motifs in zebrafish

“…From the BLAST analysis, the crystal structure from Mus musculus (PDB ID: 5CGJ) was chosen as a template for homology modelling of DGR domains of both zebrafish Keap1a and Keap1b proteins. The 5CGJ crystal structure of M. musculus Keap1 complexed with (3S)‐1‐[4‐[(2,3,5,6‐tetramethylphenyl) sulfonylamino]‐1‐naphthyl] pyrrolidine‐3‐carboxylic acid (RA389) was retrieved from the PDB . RA389 exhibits a non‐covalent binding with Keap1.…”