The identification and characterization of non-reactive inhibitor of Keap1-Nrf2 interaction through HTS using a fluorescence polarization assay

Wang L, Lewis T, Zhang YL, et al.

Publication Details

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.

Assigned Assay Grant #: 1 R03 MH093197-01

Screening Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber

Chemistry Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber

Assay Submitter & Institution: Longqin Hu, Rutgers, The State University of New Jersey

PubChem Summary Bioassay Identifier (AID): 504540

Probe Structure & Characteristics

Image ml334f1

1. Recommendations for Scientific Use of the Probe

We have developed a small-molecule probe, ML334 that inhibits the interaction between the oxidative sensor Keap1 and the transcriptional factor Nrf2. This probe will enable researchers to have a deeper understanding of the Nrf2-induced cytoprotective response in numerous human diseases and represents a novel class of inhibitors for further optimization towards drug development.

Nrf2-induced cytoprotective gene expression is a sensitive, yet complicated response to environmental stress. The interaction of Keap1-Nrf2 plays a central role in the regulation of Nrf2 activation. The current model for this interaction is that two distinct binding sites in the Neh2 domain of Nrf2, termed ETGE and DLG motifs interact with the Kelch domain of Keap1 (1). The two motifs have different binding affinities for the Kelch domain and are required for Nrf2 activation. p21, a member of cyclin-dependent kinase inhibitor family (CKI), also associates with Nrf2 through DLG motif and competes with Keap1 for Nrf2 binding. This competition results in an upregulation of the Nrf2 signaling pathway (2). There are several small molecules that act as Nrf2 inducers and they dissociate Nrf2 from Keap1 binding. However, these molecules do not directly interact with either ETGE motif or DLG motif, but they covalently modify the active cysteine residues of Keap1, leading to conformational changes in Keap1 that are responsible for the dissociation (3, 4). Other signaling pathways also influence Nrf2 activation through posttranscriptional modification. It has been shown that the phosphorylation of Nrf2 by MAP kinase promotes the release of Nrf2 from Keap1 (5), while the phosphorylation of Nrf2 by Fyn kinase facilitates the Nrf2 nuclear export (6). ML334 is identified as a competitive inhibitor of Keap1 binding to ETGE motif. It is important to evaluate if ML334 also can inhibit the interaction between Keap1 and DLG motif. ML344 is a direct inhibitor and will be a useful tool to further elucidate the regulation of Nrf2 activation under both basal and induced condition. These studies would determine the significance of these pathways in vivo and, importantly, their suitability as pharmacological targets for modulating Nrf2 signaling.

The Keap1-Nrf2 interaction is widely studied as an effective target for Nrf2 involved chemoprevention in numerous therapeutic areas. This concept is supported by the studies in gene-knockout mouse models (7, 8). Currently known Nrf2 inducers are covalent modifiers of the cysteines found in Keap1. The lack of specificity of these compounds raises the concerns over the use of these Nrf2 inducers as chemoprotective agents. Recent reports on mutations in KEAP1 gene and NRF2 gene in multiple cancers brings up other safety concerns (9, 10). These mutations lead to a constitutive expression of cytoprotective genes that is similar to the response induced by Nrf2 inducers and contributes to chemoresistance during therapy. Studies in the following areas would shed light on addressing the safety concern. Do genetic alterations mimic the pharmacological modification? Do they evoke the same magnitude and duration of response and the same batteries of downstream genes? To what extent might unabated exposure to enzyme inducers enhance a cancer phenotype? ML334, a non-covalent and specific inhibitor of Keap1-Nr2 interaction, is superior to the previously known covalent inhibitors for these studies

2. Materials and Methods

See subsections for a detailed description of the materials and methods used for each assay.

Materials and Reagents

  • The sequence of human Kelch domain of Keap1 is cloned into expression vector pET15b and transformed into E.coli BL21 (DE3) strain. The 34 kDa protein is purified through Ni-NTA affinity column due to 6× his tag. Purified protein is stored in the buffer containing 20 mM HEPES (pH7.4), 5mM DTT at -80 °C.
  • The sequence of the peptides used in fluorescence polarization (FP) assay is based on ETGE motif within the Neh2 domain of Nrf2 protein. A 9mer peptide LDEETGEFL is synthesized on solid phase. A derivative of this peptide with N-terminal fluorescein isothiocyanate (FITC) label and C-terminal amide capping (FITC-9mer-Nrf2-NH2) is used as the probe and another derivative with N-acetylation (Ac-LDEETGEFL-OH) is used as positive control in FP assay.
  • CellTiter-Glo® Luminescent Cell Viability Assay Kit was purchased from Promega (Catalog No. G7573; Madison, WI).
  • LiveBLAzer™-FRET B/G Loading Kit for beta-lactamase activity detection was purchased from Invitrogen (Catalog No. K1095, Carlsbad, CA)
  • Gal-Screen Beta-Galactosidase Reporter Gene Assay System was purchased from Applied Biosystems (Catalog No. T1027, Foster City, CA)
  • Sypro Orange is purchased from Sigma (Catalog No. S5692-50UL, Saint Louis, MI)
  • tert-Butylhydroquinone (tBHQ) is purchased from Sigma (Catalog No. 112941, Saint Louis, MI)
  • MG132 is purchased from Selleck (Catalog No. S2619, Houston, TX)
  • All the other chemicals are purchased from Sigma

Cell line and culture condition

The following cell lines were used in this study:

  • CellSensor™ ARE-bla HepG2 cell line (Invitrogen, Cat# K1208) is a stable cell line expressing beta-lactamase reporter gene regulated by ARE in the promoter region. The culture media and condition was based on the manufacture's recommendation.
  • PathHunter U2OS cell line (DiscoveryRx, Cat# 93-0821C3) is a human osteosarcoma cell line engineered by DiscoveryRx for nuclear translocation assay. The culture media and condition was based on the manufacture's recommendation.
  • HEK293 and HepG2 were purchased from ATCC.

2.1. Assays

2.1.1. Primary Assay (Primary HTS, AID 504523)

A Fluorescence polarization assay was used for the primary HTS (20). The assay was conducted in 1536-well format in assay ready plates (ARPs). ARPs were prepared by dispensing 7.5 nL/well of 10 mM compound in DMSO to Aurora black plates (Aurora, Catalog No. 00019180BX) using Echo acoustic dispenser (Labcyte, Sunnyvale, CA). Three solutions, including Kelch domain of Keap1 protein, positive control Ac-9mer-Nrf2 and assay buffer (10 mM HEPES, pH 7.4, 3.4mM EDTA, 150 mM NaCl, 0.005% Tween-20) were dispensed simultaneously to ARPs according to plate map to a final volume of 4μL/well using BioRAPTR (Beckman, Brea, CA). After 10 min incubation of Keap1 with compounds at room temperature, 4μL/well of FITC-9mer-Nrf2-NH2 probe was then dispensed onto assay plates using Combi NL (Thermo Scientific, Waltham, MA). The assay plates were incubated at room temperature again for 1 h before reading on uHTS Microplate Imager ViewLux (PerkinElmer, Waltham, MA) with excitation wavelength of 480 nm and emission wavelength of 535 nm with dichroic mirror of D505fp/D535. The FP signals were normalized against DMSO and the positive control using Assay Analyzer in Genedata (v7.0.3). Any compound with more than 3 times of standard deviation (3*SD) of DMSO wells in both replicate was scored as actives and cherry-picked for retest.

2.1.2. Confirmatory Assay (AID 588683, AID 651798, AID 651801, AID 651806 and AID 651834)

The activity of the compounds was confirmed in FP assay in 8-point dose. The assay procedure was similar to primary HTS except that compounds were transferred using a pin tool (Cybio, Woburn, MA) and the assay volume was 40 μL/well. The data was normalized and the percent (%) activity was determined at each concentration in GeneData. The concentration response curves (CRCs) were generated with GeneData Condeseo.

2.1.3. Thermal shift assay (Secondary Assay: AID 651823 and AID 651829)

The binding of the compounds to Keap1 protein was tested in a thermal shift assay. 5 μL/well of the Kelch domain of the Keap1 protein mixed with detection dye Sypro orange was dispensed into a qPCR plate (Roche, Catalog No. 05102430001) using Combi NL. 25 nL/well of the compounds in dose was transferred to the assay plates using the pin tool. The assay plates were set on a plate shaker for 5 second at a speed of 2,000 rpm. After 10 minutes of incubation at room temperature to promote compound binding to Keap1, the plates were read on LightCycler 480 using a protein melting protocol. The fluorescence signal was recorded at a temperature range of 25∼85 °C with a temperature ramping rate of 3.6 °C/min at an excitation of 465 nm and emission of 580 nm. Tm values for each well were derived through Roche protein-melting analysis software.

2.1.4. Counterscreen for thermal shift assay (Secondary Assay: AID 651858)

To eliminate the false positive from thermal shift assay, a counterscreen was performed by looking at the Tm of the compounds in assay buffer. Compounds with a readable Tm in the counterscreen were removed from the hit list.

2.1.5. Cytotoxicity assay (Secondary Assay: AID 651860 and AID 651863)

Compound toxicity was assessed in two wildtype cell lines, HEK293 and HepG2, using CellTitre-Glo (Promega, Catalog No.G7573) reagent. This assay detects cellular ATP levels that correlate with the number of viable cells. The cells were cultured in DMEM media in a humidified 37 °C/5% CO2 incubator and plated onto Corning 384-well white assay plates (Corning, Catalog No. 3712) with 5000 cells/well in 40 μL of media. After 24h incubation, the cells were treated with 100nL/well of compounds for 48h. MG132 (PubChem SID 125164700) at 16 μM was used as a positive control. The data was normalized against DMSO and the positive control in Genedata Assay Analyzer and the curves were fitted in Genedata Condeseo.

2.1.6. Nrf2 nuclear translocation assay (Secondary Assay: AID 651833)

The cellular activity of the compounds was tested in a cell-based nuclear translocation assay. This assay uses the Enzyme Fragment Complementation (EFC) technology in which the beta-galactosidase (beta-gal) enzyme has been split into two inactive fragments. A small 4 kDa complementing fragment of beta-gal, termed PK by DiscoveryRx is fused recombinantly to the Nrf2 protein. The larger portion of beta-gal, termed EA, is anchored in the nucleus. The inhibition of Keap1-Nrf2 interaction results in a nuclear translocation of Nrf2 fusion. The nuclear accumulation of Nrf2 fusion complements the two fragments PK-EA and forms a functional enzyme. The activity of beta-galactosidase is then quantitatively detected using the chemiluminescent substrate in the PathHunter® Detection Kit. At the experiment level, the engineered U2OS cells were cultured in MEM media in a humidified 37 °C/5% CO2 incubator and plated onto Corning 384-well white assay plates (Corning, Catalog No. 8867BC) with 10,000 cells/well in 20 μL of media. After overnight incubation, the media was discarded and replaced with 25 μL/well of HBSS buffer with Ca++, and Mg++ (Invitrogen, Catalog No. 24020117). The cells then treated with 100nL/well of compounds for 3h. tert-Butyl hydroquinone (tBHQ, PubChem SID 137276090) at 200 μM was used as a positive control. The beta-galactosidase activity was detected with GalScreen detection reagents (ABI, Catalog No.T1027) on EnVison. The data was normalized against DMSO and the positive control in Genedata Assay Analyzer and the curves were fitted in Genedata Condeseo.

2.1.7. Cell-based beta-lactamase reporter assay (Secondary Assay: AID 651807)

The inhibition of the compounds to Keap1-Nrf2 interaction was also tested in Nrf2 transactivation assay in ARE-bla HepG2 cells (Invitrogen, Catalog No. K1208). This assay is for detecting the subsequent step of Nrf2 nuclear translocation which is Nrf2 induced gene expression controlled by ARE. In ARE-bla HepG2 cells, reporter gene beta-lactamase gene is controlled by ARE. Therefore, the inhibition of Keap1-Nrf2 interaction is directly correlated with an increase of the beta-lactamase activity. The cells were cultured in DMEM media in a humidified 37 °C/5% CO2 incubator and plated onto Corning 384-well black-wall, clear-bottom assay plates (Corning, catalog No. 3712) with 12,500 cells/well in 30 μL of media. After 5 h incubation, the assay plates were treated with the compounds for 15 h in incubator. tBHQ (PubChem SID 137276090) at 150 μM was used as a positive control. The beta-lactamase activity was detected with LiveBLAzer™ FRET – B/G Loading Kit from Invitrogen (Catalog No. 1089). The fluorescence labeled substrate CCF4-AM was added on the cells. After 2h reaction, the plates were read on EnVison at an excitation of 409 nm and the emissions of 460 nm and 530 nm. The data was expressed by a ratio of 460nm/530nm. The inhibition of compounds results in a decrease of FRET signal and an increase of the ratio. The ratio of 460 nm/530 nm was normalized against DMSO and the positive control in Genedata Assay Analyzer and the curves were fitted in Genedata Condeseo.

2.1.8. Competitive SPR assay (Secondary Assay: AID 651876)

A solution based competitive SPR assay was used to guide SAR activity (21). In this assay, a biotinylated 16mer peptide from Keap1 binding region of Nrf2 was captured by streptavidin on CM5 chip surface (300RU). 40 nM of the Kelch domain of Keap1 protein preincubated with tested compounds was injected over the immobilized Nrf2 peptide surface for a 1-min association time and a 3-min dissociation time at 25 °C with a flow rate of 50 μL/min. The sensor chip surface was regenerated with 1M NaCl with a flow rate of 100 μL/min for 1 min followed by two consecutive 1-min washes with the running buffer at a flow rate of 100 μL/min. The binding signal of Keap1 to Nrf2 was recorded using BIAcore 3000 (GE Healthcare Life Sciences). The concentration of unbound Keap1 was calculated based on its standard curve. The fraction of bound Keap1 (fb) was plotted against the concentration of the compounds.

2.1.9. Reversibility assay (Mechanism of action study)

To determine if the leading compounds covalently modify Keap1 like other Nrf2 inducers, we performed a reversibility test. In this experiment, the Kelch domain of Keap1 protein was first incubated with compound at a concentration of 10 times of Kd. The binding of the compound to Keap1 protein was confirmed using FP assay and thermal shift assay. The incubated sample was then diluted into FP buffer with a dilution factor of 10 and concentrated using an Amicon ultrar-0.5ml centrifugal filter (cutoff-30K) for 3 times. The binding ability to Keap1 protein was evaluated using FP assay and thermal shift assay in comparison to un-treated Keap1 protein control. The detailed assay condition, refer 2.1.2 for FP assay and 2.1.3 for thermal shift assay.

2.2. Probe Chemical Characterization

After preparation as described in Section 2.3, the probe (ML334) was analyzed by UPLC, 1H and 13C NMR spectroscopy, and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy were consistent with the structure of the probe, and UPLC indicated an isolated purity of >95%. The absolute stereochemistry of the probe was determined by both asymmetric synthesis and X-ray crystallography of one of its diastereomers (Section 2.3) and isomeric purity was determined to be >95% by chiral chromatography.

The solubility of the probe (ML334) was experimentally determined to be > 100 μM in phosphate buffered saline (PBS, pH 7.4, 23 °C) solution. Plasma protein binding (PPB) was determined to be 95% bound in human plasma. The probe is moderately stable in human plasma, with approximately 75% remaining after a 5-hour incubation period. The compound was found to be stable in glutathione (GSH) with 98% remaining after 48 hours.

The stability of the probe (ML334) in PBS (0.1% DMSO) was measured and the probe is stable as 94% is still present after 24 hours of incubation (Figure 1).

Figure 1. Stability of the Probe (ML334) in PBS Buffer (pH 7.4, 23°C).

Figure 1

Stability of the Probe (ML334) in PBS Buffer (pH 7.4, 23°C).

Table 1. Summary of Known Probe Properties in PubChem.

Table 1

Summary of Known Probe Properties in PubChem.

2.3. Probe Preparation

Scheme 1. Synthesis of the Probe (ML334).

Scheme 1Synthesis of the Probe (ML334)

Commercially available (S)-1,2,3,4-tetrahydro-1-isoquinoline carboxylic acid was protected with carbobenzyloxy chloride and the carboxylic acid was reduced with diborane. Displacement of the resulting alcohol with phthalimide under Mitsunobu conditions followed by reduction produced the deprotected amine. Ring opening of cis-cyclohexane dicarboxylic anhydride with the amine produced two isomers which were readily separated by silica gel chromatography, ML334 being the less polar of the two isomers on silica gel.

3. Results

3.1. Dose Response Curves for Probe

Figure 2. Dose-dependent Activity of the Probe (ML334).
Figure 2. Dose-dependent Activity of the Probe (ML334).

Figure 2Dose-dependent Activity of the Probe (ML334)

ML334 (SID 137276180, CID 56840728) was tested in the primary assay and several secondary assays. Concentration response curves were generated with Genedata Condeseo and show normalized percent activity for the individual doses. Thermal shift data was generated in Excel. Competitive SPR curve was generated in SigmaPlot. Florescence polarization assay (AID 651806), IC50= 1.6 μM (A); Thermal shift assay (AID 651829) delta Tm>12°C at a concentration of 38 μM. The curve was not fit to avoid confusion as the calculated Kd was the Kd at the Tm of 51°C instead of the Kd at room temperature (B); Solution based competitive SPR (AID 651876) Kd=1μM (C); Cell-based nuclear translocation assay (AID 651833), EC50=13 μM (D); Cell-based beta-lactamase reporter assay (AID 651807), EC50 =18 μM (E); Cytotoxicity assay in HEK293 cells after 48h treatment (AID 651863), EC50 >26 μM (F); Cytotoxicity assay in HepG2 cells after 48h treatment (AID 651860) EC50 >26 μM (G). =replicate 1, △=replicate 2

3.2. Cellular Activity

The cellular activity of ML334 was evaluated in two reporter assays.

The first assay detects Nrf2 nuclear translocation using beta-galactosidase fragment complementation technology. In this assay, tBHQ (tert-butylhydroquinone), which is converted in cells to a covalent inhibitor, was used as the positive control. Upon ML334 treatment for 15 h, a dose dependent increase of beta-galactosidase activity was observed (Figure. 2D). This indicated that ML334 was able to disrupt Keap1-Nrf2 interaction and transfer Nrf2 fusion with beta-galactosidase fragment into the nucleus; the EC50 of ML334 in this assay was 13 μM. We also checked the inhibition of ML334 on endogenous Keap1-Nrf2 complex. In this assay, the reporter of beta-lactamase gene is regulated by ARE on the promoter region and beta-lactmase activity is correlated to Nrf2 transactivation. We found that ML334 induces Nrf2 transactivation with an EC50 of 18 μM (Figure. 2E). These data clearly demonstrate that ML334 is cell penetrable and is able to dissociate Nrf2 from Keap1 to upregulate cytoprotective gene expression.

ML334-induced cytotoxicity was assessed in human kidney cells (HEK293) and human liver cells (HepG2) using MG132 (PubChem SID 125164700) as positive control. Both cell lines were treated with ML334 in a concentration serial up to 26 μM for 48 h, and no cell killing was detected (Figure. 2F and 2G). These cellular experiments demonstrate that ML334 is a superior tool compound with good potency and low toxicity to elucidate the function of Keap1-Nrf2 pathway in cells.

3.3. Profiling Assays

No profiling assays were carried out.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Investigation into relevant prior art entailed searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO), and World Intellectual Property Organization (WIPO) databases. Abstracts were obtained for all references returned and were analyzed for relevance to the current project. The searches were performed on, and are current as of May 8th, 2012.

All small molecule inhibitors are electrophilic species which react with free cysteinyl residues of Keap1 (Figure 3). The molecules are classified into six types of compounds, isothiocyanates, phenol or flavonoid antioxidants, Michael acceptors, organosulfur compounds, electrophiles bearing a leaving group, and heavy metal (As, Se) species (3, 13). Phenols and flavonoids are oxidized in vivo to reactive Michael acceptors. Due to this inherent reactivity, target selectivity is a major issue. In fact, one of the most advanced Michael acceptors, CDDO, is a multifunctional agent responsible for numerous activities (24). To ascertain the importance of the Keap1-Nrf2 system, more selective, ideally non-covalent, inhibitors are needed.

Figure 3. Electrophilic Keap1 inhibitors.

Figure 3

Electrophilic Keap1 inhibitors.

Thus far there are no reported small molecule non-covalent inhibitors, though peptidic inhibitors have been recently reported but are not expected to have cellular activity (25, 26). While covalent inhibition of targets has been experiencing renewed interest of late (27), issues such as selectivity and allergic sensitivity will have to be addressed. With the discovery of ML334 various researchers will now be able to ascertain if indeed non-covalent inhibition of the Keap1-Nrf2 pathway has advantages over covalent inhibition.

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