Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism

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Abstract

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.

Introduction

To ameliorate oxidative and environmental stresses, a central transcription factor, Nrf2 (Nuclear factor-erythroid2-related factor 2), directs the transcription of protective genes by interacting with a cis-acting element (ARE/EpRE) in their promotors. The negative regulation of Nrf2 occurs by its interaction with a facilitator of ubiquitination, Keap1 (Kelch-like ECH-associated protein 1), which shepherds the transcription factor toward the polyubiquitination and degradation machinery of the cell before it can translocate into the nucleus. We reasoned that a small molecule inhibitor that blocks the Keap1/Nrf2 interaction might extend the half-life of the Nrf2 transcription factor and improve the outcome of a wide variety of diseases such as cancer, diabetes, atherosclerosis, Alzheimer’s disease and arthritis1 in which reduction of oxidative stress by protective genes plays an important therapeutic role.

Nrf2 contains an N-terminal regulatory domain, Neh2, encompassing two motifs which bind in a large, positively charged central cavity of the Kelch-DC domain at the C-terminus of Keap1. We reasoned, that this site of interaction could be blocked by a small molecule inhibitor, and would differ from the site of many of the other small molecule inducers of the ARE response genes that have been described, which modify Cys151 of the N-terminal BTB domain. This includes the quinoline-based inducer AI-1,2 the triterpenoid CDDO-methyl esters developed by Reata pharmaceuticals,3 and Tecfidera™, the dimethyl, monomethyl fumarates developed by Biogen Idec, Inc.4

Both motifs in the Neh2 region of Nrf2 have been shown to be essential for binding to the Kelch-DC and for the subsequent ubiquitination of the poly-lysine region between the two motifs.5 Crystal structures of each motif bound to the Kelch-DC have led to the proposal of a ‘hinge-latch’ ordered binding mechanism, in which binding of the high affinity motif is followed by binding of the low affinity site for proper geometric presentation of the internal poly-lysine sequence to the ubiquitin ligase. Both the higher affinity ‘hinge’ DxETGE peptide motif (PDB ID: 2FLU)6 and the lower affinity ‘latch’ DLG peptide motif (PDB ID: 2DYH)7 bind in the central cavity of the Kelch-DC and interact with serine, arginine and tyrosine side chains.

Recent biochemical experiments investigating this inhibitory mechanism have been described. The minimal peptide sequence sufficient for binding to the Keap1 Kelch-DC domain, a 9mer linear Nrf2 peptide (H-LDEETGEFL-OH), demonstrated moderate affinity (KD = 352 nM) using a surface plasmon resonance-based solution competition assay; its potency could be improved by removing the positive charge at the N-terminus.8 Using peptide displacement and fluorescence polarization as a readout, a high throughput screen was performed to identify small molecules that bind to the Kelch domain of Keap1 and block Nrf2 binding.9 This assay was used to screen the MLPCN library of 330,000 compounds at the Broad Institute and compounds have been identified which displace the peptide with μM Ic50s, as reported on PubChem Bioassay database AID: 58868310 and the best compounds inhibited at ∼0.4 μM Ic50.

We report here our own version of a high throughput screen for small molecules capable of inhibiting the Keap1–Nrf2 interaction. During this study we have screened the Evotec Lead Discovery library supplemented with compounds derived from virtual screening. Several classes of identified hit compounds have been validated and their binding modes to the Keap1 Kelch-DC domain characterized by various biophysical methods to understand the nature of the binding hot-spot. The ability of one compound series to induce the expression of ARE genes in a cell-based reporter assay demonstrates that the approach could work in an in vivo system and that this compound may serve as a tool for investigating the biology of this pathway.

Section snippets

HTS screen for compounds that block Nrf2/Kelch-DC interaction, chemical subclasses and SAR

Using a homogeneous confocal fluorescence anisotropy assay (two-dimentional fluorescence intensity distribution analysis, 2D-FIDA)11 in a miniaturized 2080-well plate format, 267,551 compounds from the Evotec Lead Discovery library along with 1911 compounds from virtual screening were tested for inhibition at 50 μM screening concentration. For the interaction of Keap1 Kelch-DC with the fluorescent Nrf2 peptide (NH2-LQLDEETGEFLPIQGK(MR121)-OH) an apparent KD of 1.6 nM was determined (Fig. 1A). As

Orthogonal biophysical assays complement and extend our understanding of the nature of compound binding

In this study we have used ESI-MS and NMR to demonstrate stoichiometry and specificity of the binding of small molecule ligands. The results are validated by their binding mode within the crystal structures. The structure of Cpd15 bound to Kelch-DC shows the two compounds binding in close proximity with different environments. Plotting the % occupancy versus unbound ligand concentration reveals that the two Cpd15 molecules bind in a non-cooperative fashion with a Hill coefficient of ∼1. Thus,

Protein cloning, expression and purification

The human Keap1 Kelch-DC (321-609) was amplified by PCR using the 5′ Nco I and 3′ Xho I cut sites and ligated into pET28b(+)(Novagen). Constructs were expressed in Escherichia coli BL21 Codon Plus (DE3)-RIL (Stratagene). Isotopic labeling for NMR experiments with 15N arginine amino-acid (Cambridge Isotope Laboratory, Inc., Andover, MA) was conducted in modified minimal M9 medium as described.15 Briefly, the expressing clone was inoculated in 5 ml modified M9 minimal media with kanamycin at 37 °C

Acknowledgments

We would like to acknowledge Jessica Friedman for establishing the Kelch-DC biochemical assay and LRL-Cat (Sector 31 at the advanced photon source) for collection of X-ray diffraction data.

The Kelch-DC/Cpd15 complex structure has been deposited in the Protein Data Bank with accession number 4IN4. The Kelch-DC-E540A, E542A/Cpd16 complex structure has been deposited in the protein data bank with accession number 4IQK.

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