A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1-Nrf2 protein-protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure-activity relationships support its use as a lead for our ongoing optimization.

Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z′ factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.

K elch‐like ECH ‐associated protein 1 ( K eap1)‐nuclear factor erythroid 2‐related factor 2 ( N rf2)‐antioxidant response elements ( ARE ) pathway represents one of the most important cellular defense mechanisms against oxidative stress and xenobiotic damage. Activation of N rf2 signaling induces the transcriptional regulation of ARE‐dependent expression of various detoxifying and antioxidant defense enzymes and proteins. Keap1‐Nrf2‐ARE signaling has become an attractive target for the prevention and treatment of oxidative stress‐related diseases and conditions including cancer, neurodegenerative, cardiovascular, metabolic, and inflammatory diseases. Over the last few decades, numerous N rf2 inducers have been developed and some of them are currently undergoing clinical trials. Recently, overactivation of N rf2 has been implicated in cancer progression as well as in drug resistance to cancer chemotherapy. Thus, N rf2 inhibitors could potentially be used to improve the effectiveness of cancer therapy. Herein, we review the signaling mechanism of K eap1‐ N rf2‐ ARE pathway, its disease relevance, and currently known classes of small molecule modulators. We also discuss several aspects of K eap1– N rf2 interaction, N rf2‐based peptide inhibitor design, and the screening assays currently used for the discovery of direct inhibitors of K eap1‐ N rf2 interaction. © 2012 Wiley Periodicals, Inc. Med Res Rev., 32, No. 4, 687‐726, 2012

The Keap1Nrf2 interaction plays important roles in regulation of Nrf2 activity and induction of chemopreventive enzymes. To better understand the interaction and to determine the minimal Nrf2 sequence required for Keap1 binding, we synthesized a series of Nrf2 peptides containing ETGE motif and determined their binding affinities to the Kelch domain of Keap1 in solution using a surface plasmon resonance-based competition assay. The equilibrium dissociation constant for the interaction between 16mer Nrf2 peptide and Keap1 Kelch domain in solution (KDsoultion) was found to be 23.9 nm, which is 10X lower than the surface binding constant (KDsoultion) of 252 nm obtained for the direct binding of Keap1 Kelch domain to the immobilized 16mer Nrf2 peptide on a surface plasmon resonance sensor chip surface. The binding affinity of Nrf2 peptides to Keap1 Kelch domain was not lost until after deletion of eight residues from the N-terminus of the 16mer Nrf2 peptide. The 9mer Nrf2 peptide has a moderate binding affinity with a of 352 nm and the affinity was increased 15X upon removal of the positive charge at the peptide N-terminus by acetylation. These results suggest that the minimal Nrf2 peptide sequence required for Keap1 binding is the 9mer sequence of LDEETGEFL.

An improved practical synthetic method was developed for the preparation of selenocarboxylates of amino acids through the reaction of the corresponding activated esters with sodium hydrogen selenide in alcoholic or aqueous medium. The protected cc-amino selenocarboxylates reacted readily with sulfonyl azide to form N-(alpha-aminoacyl)sulfonamides in high yields. The commonly used protecting groups in amino acid and peptide chemistries are well tolerated under these reaction conditions. No protecting groups are needed for the side chains of Arg, Met, Set, Tyr, and Trp. (C) 2009 Published by Elsevier Ltd.

Keap1, an oxidative stress “sensor” protein, forms a complex with the transcription factor Nrf2 and regulates the expression of the cytoprotective genes. The interaction between Keap1 and Nrf2 plays an important role in several diseases, including cancer, inflammation, and neurodegeneration. However, emerging data is contradictory, indicating a poor understanding of this regulation. To better probe this process, we developed and performed a high throughput screen (HTS) to identify non-reactive inhibitors to disrupt the interaction between Keap1 and Nrf2 using a biochemical fluorescence polarization (FP) assay. Of the 337,116 tested compounds from the NIH MLPCN library, we found 460 hits with a hit rate of 0.14%. Eight compounds were confirmed in the following retest and the IC50s of two of them were approximately 2 μM. These two potent hits exhibited direct binding activity to Keap1 protein by thermal shift assay and by competitive surface plasmon resonance (SPR). One of the two hits would be expected to be readily converted into an active Michael acceptor in cells and was not investigated further. The remaining hit (hit1) had three chiral centers with undefined configurations. We undertook stereoselective synthesis and identified the most active stereoisomer. Only one of the eight possible stereoisomers was active. A series of analogs was synthesized and evaluated in both biochemical assays and cell-based reporter assays. One compound, a single enantiomer of the original hit, was identified as the probe ML334 (SID 152344591, CID 56840728). ML334 binds to the Keap1 protein with Kd of 1.0 μM in competitive SPR experiments, and competes with a Nrf2 peptide with an IC50 of 1.6 μM in FP assay. Moreover, ML334 disrupts Keap1-Nrf2 interaction at the cellular level as demonstrated by an induction of Nrf2 nuclear translocation and an upregulation of ARE controlled reporter gene. ML334 does not show detectable cytotoxicity up to 26 μM in HEK293 and HepG2 cells. Experiments with pre-incubation of ML334 with Keap1 and subsequent filtration demonstrated that ML334 is a reversible inhibitor of Keap1-Nrf2 interaction. In addition, incubation of ML334 with GSH did not show the formation of GSH adduct. To the best of our knowledge, ML334 is the first non-covalent small molecule inhibitor of the Keap1-Nrf2 interaction, distinctive from other known inducers of Nrf2. This first in-class inhibitor will be used to elucidate the role of Keap1-Nrf2 interactions in multiple disease areas and to address safety concerns about chemoprotection.