Design, Synthesis, and Initial Evaluation of Affinity-Based Small

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Design, Synthesis, and Initial Evaluation of Affinity-based Small-Molecule Probes for Fluorescent Visualization and Specific Detection of Keap1 Mengchen Lu, Hai-shan Zhou, Qi-Dong You, and Zhengyu Jiang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00775 • Publication Date (Web): 13 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016

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Journal of Medicinal Chemistry

Design, Synthesis, and Initial Evaluation of Affinity-based Small-Molecule Probes for Fluorescent Visualization and Specific Detection of Keap1 Mengchen Lu,a Hai-shan Zhou,a Qi-Dong You,*a,b and Zhengyu Jiang*a,b a

State Key Laboratory of Natural Medicines and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China. b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China. ABSTRACT: Keap1 is a pluripotent protein which plays a predominant role in cellular homeostasis and stress responses. Given that the cellular environment is quite dynamic and versatile, further investigation of the function of Keap1 depends on tools for specific and real-time detection of Keap1. Herein, we report the development of functional affinity-based small-molecule probes which can overcome some shortcomings of current methods and be applied in further studying the function of Keap1.

INTRODUCTION Kelch-like ECH-associated protein-1 (Keap1) is a pluripotent protein, which plays an important role in stress responses, especially in oxidative stress. Firstly, Keap1 is an important adaptor component in the Cullin 3 (Cul3)-based E3 ligase, which is the primary regulator of NF-E2-related factor-2 (Nrf2) through mediating the ubiquitination of Nrf2.1, 2 Nrf2 is the master regulator of the cellular defense mechanism by eliciting an adaptive defense system under oxidative stress. By transcriptional activation of a battery of antioxidant response element (ARE)-bearing genes, the activation of the Nrf2mediated defense system can maintain the cellular homeostasis and promote the cell survival under stress conditions.3 Besides, Keap1 also functions as the sensor of the cellular redox status.4 The hypersensitive cysteine residues of Keap1 can monitor the cellular environment whether it is under the redox-balancing state or not, and it will switch the Nrf2mediated response off or on depending on the redox state. Specifically, under normal conditions, Keap1 targets Nrf2 for ubiquitination, leading to subsequent degradation of Nrf2 by the 26S proteasome by acting a substrate adaptor component of the Cul3-E3 ubiquitin ligase. Under stress conditions, excessive reactive oxygen species and electrophilic agents can covalently modify these cysteine residues, leading to the conformation change of the Cul3-Keap1-Nrf2 complex. As a result, the Keap1-mediated Nrf2 depression is abolished and Nrf2-induced antioxidant system is turned on.5, 6 Moreover, Keap1 can also interact with a lot of other substrates. The interaction between Keap1 and the selective autophagy substrate p62 reveals the role of Keap1 in mediating the crosstalk between selective autophagy and oxidative stress.7-9 Keap1-Cul3 E3 ligase can also mediate ubiquitination of B-cell CLL/lymphoma 2 (Bcl-2), which directly associates the antioxidant protection with cell survival. In addition, there are a lot of other important proteins10 that have been identified as Keap1 binding partners, including heat shock protein-90 (Hsp90)11, DJ-1 that associate with cancer and Parkinson’s

disease (PD)12, and IκB kinase β (IKKβ) that functions as a fine-tuning controller of the nuclear factor (NF)-κB.13, 14 These findings fully confirmed the predominant role of Keap1 in cellular homeostasis and stress responses, and also inspired the development of Keap1 inhibitors.15, 16 Several small molecules that exhibit tight and selective binding to Keap1 have been reported,17-23 and their potency, especially the activation of the Nrf2 antioxidant response have been validated in cellular and in vivo models.23-27 Considering the cellular environment is quite dynamic and versatile, the further exploration of function and therapeutic potential of Keap1 under various conditions is quite urgent. These achievements depend on the tools for specific and realtime detection of the Keap1 protein. However, current available methods have quite a few deficiencies. The primary antibody-based detecting method, immunofluorescence stain, cannot be used to detect live cells. Using construct encoding fusion proteins of Keap1 with the bioluminescent protein needs transfections procedures. Besides, the bioluminescent part may affect Keap1’s normal function and may not reflect the inact state of Keap1. Using functional small-molecule probe can overcome these shortcomings. It can be used for the in situ profiling of Keap1, visualization of the dynamic changes in live cells and specific detection of Keap1. To this end, we report the design, synthesis and application of functional small-molecule ligands of Keap1 to fluorescently visualize and specifically detect Keap1. Previously, we have been exploring potent small molecules which interact with the Nrf2-binding site on Keap1 and compete with the interaction between Nrf2 and Keap1.17, 24 This series of Keap1 ligands fulfilled the substrate binding pocket in Keap1 and showed nanomole binding affinity with Keap1. It proved a good starting point for the rational design of affinity-based small-molecule probes. Herein, on the basis of the potent small-molecule ligands of Keap1, we report the design, synthesis and application of functional affinity-based smallmolecule probes to fluorescently visualize and specifically

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detect Keap1, which can be applied in further research of Keap1.

RESULTS AND DISCUSSIONS Keap1 substrate binding cavity that has been well studied provided the anchoring site for small molecules. It is composed of five sub-pockets (P1 to P5), and the polar subpockets, P1 and P2, are the hot-spots for Keap1 binding.28, 29 The primary substrate, Nrf2, occupies the polar hot-spots by the ‘ETGE’30, 31 and ‘DIDLG’32 motifs in which two acidic residues are included. Inspired by the natural substrate Nrf2, we introduced the diacetic moiety into the template 1 (PDB code: 4IQK)33 to mimic the natural protein binding partner. It significantly enhanced the binding affinity from a Kd value of 1.69 µM to 9.91 nM,17 which was similar with the binding potency of Nrf2. Given the high binding potency and satisfactory match to Keap1, it can be used as a good starting point to develop the functional small-molecule probes targeting Keap1.

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amino. Reduction of the nitro group afforded the corresponding amine 5, which reacted with dihydropyran-dione followed by tert-butyl (2-aminoethyl) carbamate, yielding compound 7 with the linker. After that, the two aliphatic carboxylic acid side chains which were crucial for binding were introduced into the imino group of the two sulfamides. By removing the Boc and the Bn protecting groups, we finally got the target compound 10, ready to be acylated with activated esters of reporter groups. Compared to 1, which exhibited an IC50 of 28.6 nM in a fluorescence polarization (FP) competition assay in our previous study17, compound 10 showed an IC50 of 30.7 nM, suggesting that the introduction of the linker hardly caused any loss of the Keap1 binding affinity.

Figure 1. Structure-based design of the affinity-based small molecule probe of Keap1. Chemical structures of 1(A) and 2 (B). Binding surface depiction of 1 (PDB code: 4IQK) and 2. Hydrogen bonds are represented as green dashed lines and electrostatic interactions are represented as yellow dashed lines. The carbon atoms in small molecules and the Keap1 residues are colored cyan and purple, respectively.

To initiate studies on fluorescent visualization and specific detection of Keap1, we required the introduction of functional groups into the ligand. Previously, we have investigated the binding mode of compound 2 with Keap1 and found that the two methoxy substituted benzene rings linked by the sulfamides occupy the outside P4 and P5 sub-pockets.17, 24 The methoxy groups extend towards outside of the binding pocket, which may have the capacity to tolerate other functional groups without compromising the Keap1 binding affinity. The co-crystal structure of compound 1’s analogue with Keap1 further confirmed this binding mode (PDB code: 4XMB).18 Besides, on the basis of our previous study,24 this p-acetamido substituents on the side chain phenyl rings was beneficial for Keap1 binding and physicochemical properties. Thus we introduced the linker with amide group to substitute the pmethoxy at the benzsulfamide ring and the attachment of reporter groups was envisaged via the terminal active amino group on the linker. By incorporating different reporter groups through the linker with appropriate length and nature, probes with various functions can be achieved (Figure 1). The synthetic root is depicted in Scheme 1. The starting material, 4-nitro-1-naphthylamine, was synthesized as previously reported.17, 34 Introduction of the 4-methoxybenzenesulfonyl chloride into one of the two aminos of naphthalene-1,4diamine gave the intermediate compound 3. Subsequently, the 4-nitrobenzenesulfonyl chloride was introduced into the other

Scheme 1. Synthesis of the linker. Reagents and conditions: (a) NH2OH·HCl, KOH/MeOH/EtOH, 60 °C, 4 h , 90%; (b) H2, Pd/C, THF, RT, 4 h; (c) 4-methoxy-phenylsulfonyl chloride, Na2CO3, THF, N2, 0 °C, 5 h, 82%; (d) 4-nitrophenylsulfonyl chloride, toluene, pyridine, 100 °C, N2, 5 h, 93%; (e) H2, Pd/C, THF, RT, 4 h, 94%; (f) dihydro-2H-pyran-2,6(3H)-dione, toluene, 110 °C, N2, 5 h, 75%; (g) tert-butyl (3-aminopropyl) carbamate, triethylamine, PyBOP, THF, RT, 8 h, 79%; (h) benzyl 2-bromoacetate, K2CO3, DMF, RT, 8 h, 87%; (i) TFA, THF, 60 °C, 5 h , 88%; (j) H2, Pd/C, MeOH, 60 °C, 5 h, 87%.

Biotin is universally used to immobilise or conjugate bioactive compounds, via its high-affinity interaction with avidin or streptavidin.35 The Biotin-Streptavidin system has proven to be particularly useful in the detection and localization of antigens, glycoconjugates, and nucleic acids by employing biotinylated antibodies, lectins, or nucleic acid probes. In order to detect the Keap1 protein, we obtained probe 1 through conjugating the biotin group with compound 10 (Figure 2A). First, we examined the binding affinity of probe 1 with Keap1. The IC50 in the FP competition assay was 74 nM (Figure 2B), still in the same range of 1, which indicated that the introduction of the biotin group maintained the Keap1 binding affinity. To further validate the cellular interaction between probe 1 and Keap1, the affinity pull-down assay, which was widely used to identify novel protein-protein interactions, was applied in this


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study. Probe 1 was used as the bait and Keap1 is the prey. As shown in Figure 2, the cellular Keap1 was successfully preyed and enriched by probe 1, confirming that probe 1 can tightly bind to the cellular Keap1 protein.

Figure 2. The validation of the Keap1 binding potency of probe 1. (A). The chemical structure of probe 1. (B). Dose-response inhibition curve of probe 1 determined by the FP-based binding assay. (C). Western blot analysis of probe 1 bound to the streptavidin coupled magnetic beads after incubation with NCM460 colonic cell lysates. Keap1 was detected with an anti-Keap1 primary antibody.

time of the ligand and a tight binding between probe 1 and the Keap1 protein. This result is similar with that of 1 and strongly suggested that probe 1 can be used to specifically detect Keap1 and properly demonstrated the suitability of probe 1 for further applications. Fluorescein is one of a family of fluorophores commonly used in biochemistry.35, 37 Fluorescently labelled ligands, due to their high sensitivity and ease of functionalization, are widely used in many pharmacological experiments, especially in the visually differentiating between specific and nonspecific binding and real-time monitoring of cellular processes.38 In order to track cellular Keap1 protein, the fluorescein isothiocyanate (FITC) group was attached on the active amino group of the linker chain in compound 10, which resulted in probe 2. The isothermal titration calorimetry (ITC) assay was applied to quantify the binding of probe 2 to Keap1 (Figure 4). The resulting Kd value was 29.1 nM. The ITC profiles clearly fit a reversible 1:1 binding model, suggesting that one molecule bound to one molecule of Keap1. The titration FP experiment was also used to determine both the maximum FP response and the dissociation constant Kd for the Keap1–probe complex. The results showed that probe 2 tightly bound to Keap1 with a Kd of 14.7 nM, which induced the large signal-to-noise ratio of FP signal, consistent with the ITC result.

The stable binding between probe 1 and the Keap1 protein ensured that probe 1 could be a potent tool to investigate the interaction with Keap1. For further biophysical characterization, the biolayer interferometry assay (BLI), a label-free technology for measuring biomolecular interactions36, was applied. In this study, the biotin-labeled small molecule probe was immobilized on the Super Streptavidin Biosensors through biotin–streptavidin complex as the ligand and the Keap1 Kelch domain protein as the analyte.

Figure 4. The Keap1 binding affinity of probe 2. (A). The chemical structure of probe 2. (B). The ITC titration profile of Keap1 Kelch Domain with probe 2. (C). Dose-response inhibition curve of probe 2 determined by the titration FP assay. Figure 3. Biolayer interferometry sensorgrams of the binding of varying concentrations of the Keap1 protein to the immobilized probe 1.

A concentration-dependent binding of Keap1 to the immobilized probe 1 was observed (Figure 3). The interaction between the surface-bound probe 1 and the Keap1 protein was measured in real time, which provided the ability to monitor binding specificity and quantify the concentration of the Keap1 protein, with precision and accuracy. The rate constants of kon = 6.24E+04 M-1S-1 and koff = 1.35E-03 S-1 resulted in a Kd value of 21.6 nM. The kinetic determinants showed very slow dissociation kinetics, which indicated a long residence

The above results suggested that the introduction of the FITC group had nearly no effect on the Keap1 binding affinity, which ensured that probe 2 could be a potent tool to fluorescently visualize cellular Keap1. To highlight this potential, immunofluorescence microscopy was achieved on NCM460 colonic cells. As shown in Figure 5, probe 2 enabled the labelling of Keap1 at 10 µM concentration. Probe 2 was found to co-localize with Keap1 in both nuclear and cytoplasm. These results indicated that probe 2 could exhibit fluorescence response to Keap1 in living cells, and it can be further developed as the biological sensor of Keap1.



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Figure 5. Confocal microscope image of NCM460 colonic cells treated with probe 2. NCM460 colonic cells were treated with probe 2 (10 µM) for 10 h prior to fixation, then incubated at 4 °C overnight with Keap1 primary antibody. After that, cells were incubated with Alexa Fluor-labeled secondary antibody. Nuclears were stained with fluorochrome dye DAPI prior to observation. The bars indicate the magnification (10 µM). All images are representative of images obtained in three separate experiments.

CONCLUSION In this study, we produced the high-affinity Keap1 functional ligands and showed their potential to be used in Keap1 studies in vitro and in cells. Using the potent small-molecule Keap1 ligand as the structure template, the functional groups were attached to the linker substituted on the benzene sulfonamide ring, which afforded probe 1 and 2 with biotin and FITC group, respectively. The two probes exhibit high potency, selectivity and on-target action which provide great promise for the in situ profiling of Keap1, visualization of the dynamic changes in live cells and specific and real-time detection of the Keap1 protein. We envision future applications of these probes in studying the role of Keap1 in its interactome as well as in the progression of some diseases such as autophagy, neurodegenerative disorders, cardiovascular disease, ageing and cancer.

EXPERIMENTAL SECTION General Chemistry. All reagents were purchased from commercial sources and, unless otherwise noted, were used without further purification. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates with fluorescent indicator (GF254) and visualized under UV light. Melting points were determined with a Melt-Temp II apparatus. The 1H NMR and 13C NMR spectra were measured on a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane (TMS) as an internal standard. ESI-mass and high resolution mass spectra (HRMS) were recorded on a Water Q-Tof micro mass spectrometer. Purity (> 95%) of target compounds was determined by the HPLC study performed on Agilent C18 (4.6 mm×150 mm, 3.5 µm) column using a mixture of solvent methanol/water 70:30 methanol:water with 1‰ TFA) at the flow rate of 0.5 mL/min and peak detection at 254

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nm under UV. The detailed experimental procedures can be found in the Supporting Information. 2-(N-(4-(N-(carboxymethyl)-4-(5-oxo-5-((2-(5-((3aS,4S,6aR)2-oxohexahydro-1H-thieno[3,4-d]imidazol-4yl)pentanamido)ethyl) amino)pentanamido)phenylsulfonamido)naphthalen-1-yl)-4methoxyphenylsulfonamido)acetic acid (probe 1): To a solution of compound 10 (20 mg, 0.026 mmol) in THF was added N-succinimidyl D-biotinate (10 mg, 0.029 mmol). The reaction mixture was stirred at room temperature for 8 h and then extracted with ethyl acetate. The reaction mixture was partitioned between ethyl acetate (10 mL) and water (15 mL). The water layer was extracted with ethyl acetate (5 mL × 3). The organic layer was combined, washed with saturated NaCl solution (5 mL × 2), dried over sodium sulfate, filtered and concentrated. The filtrate was concentrated under reduced pressure to give the crude product. Purification of the crude product by column chromatography on silica gel (eluent:10% CH3OH in CH2Cl2) gave probe 1 as the light yellow solid, yield 33%; m.p.: 181.4-182.3 °C; 1H-NMR (300 MHz, MEOD-d4) δ 9.81-9.92 (d, 1H), 8.31-8.3 (m, 2H), 7.87 (s, 1 H),7.74-7.69 (m, 3H), 7.68-7.56 (m, 5H), 7.51-7.43 (m, 2H), 7.19-7.13 (m,1H), 6.90-6.93 (m, 2H), 6.83-6.86 (m, 2H), 4.554.75 (m, 2H), 4.20-4.45 (m, 2H), 4.11-4.23 (m, 2H), 3.84-3.85 (d, 3H), 3.62-3.75 (m, 2H), 3.38-3.41 (m, 2H), 3.16 (s, 1H), 2.84 (m, 1H), 2.65 (m, 1H), 2.31-2.41 (m, 2H), 2.12-2.26 (m, 4H), 1.95-2.01 (m, 2H), 1.52-1.73 (m, 3H), 1.41-1.58 (m, 3H); 13 C-NMR (75 MHz, MEOD-d4): δ 172.20, 169.11, 136.92, 132.53, 129.49, 128.54, 128.34, 127.66, 126.99, 126.76, 126.16, 123.78, 118.19, 113.37, 66.96, 61.43, 59.89, 59.66, 55.03, 54.59, 54.39, 52.29, 39.17, 38.27, 38.11, 35.29, 34.99, 34.33, 29.00, 27.88, 27.57, 24.91, 24.60, 20.84, 18.99; HRMS (ESI): found 1004.2598 (C44H51N7NaO13S3, [M+Na]+, requires 1004.2599); HPLC (85 : 15 methanol : water with 1‰ TFA ): tR = 3.281 min, 97.78%. 2-(N-(4-(N-(carboxymethyl)-4-(5-((2-(3-(3',6'-dihydroxy-3oxo -3H-spiro[isobenzofuran-1,9'-xanthen]-5yl)thioureido)ethyl) amino)-5oxopentanamido)phenylsulfonamido)naphthalen-1-yl)-4methoxyphenyl-sulfonamido)acetic acid (probe 2): To a solution of compound 10 (20 mg, 0.026 mmol) in THF was added FITC (13.7 mg, 0.29 mmol). The reaction mixture was stirred at 60 °C for 8 h under nitrogen. After cooling to the room temperature, the solution was concentrated under reduced pressure. The crude product was then diluted in 20 mL of diethyl ether and stirred for 30 min. Then the solution was filtered and the residue was purified by column chromatography on silica gel (eluent:15% CH3OH in CH2Cl2), resulting in probe 2 as a pink solid, yield 43%; m.p.: 226.2-227.1 °C; 1HNMR (75 MHz, MEOD-d4) δ 8.31-8.3 (m, 2H), 7.87 (s, 1 H), 7.74-7.69 (m, 3H, 7.68-7.56 (m, 5H), 7.51-7.43 (s, 1H), 7.24 (d, 1H), 7.19-7.13 (m, 2H), 7.11-6.92 (m, 2H), 6.66-6.89 (m, 6H), 6.50-6.56 (m, 2H), 4.49-4.40 (m, 2H), 4.20-4.25 (m, 2H), 3.77-3.83 (d, 3H), 3.45 (m, 4H), 2.39-2.48 (m, 2H), 2.32-2.36 (m, 2H), 1.95-2.01 (m, 2H); 13C-NMR (MEOD-d4, δ) 172.20, 169.51, 161.46, 161.00, 160.06, 152.64, 152.45, 136.96, 132.60, 129.33, 128.60, 128.42, 127.47, 125.88, 123.89, 121.13, 118.18, 114.40, 113.23, 112.665, 112.12, 109.74, 109.60, 109.35, 101.72, 54.34, 53.84, 37.97, 34.40, 29.00, 20.69; HRMS (ESI): found 1145.2346 (C23H22N3O5S2, [M+H]+, requires 1145.2362); HPLC (90 : 10 methanol : water with 1‰ TFA ): tR = 4.281 min, 95.18%.


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Affinity Pull-down Experiments. The streptavidin coupled magnetic beads (Pierce™ Pull-Down Biotinylated Protein: Protein Interaction Kit 21115, Thermo Scientific) were loaded with probe 1 (50 µg/100 µL) and the mixture was incubated for 30 min at 4 °C with gentle rotation. Afterwards, beads were washed with 250 µL of Biotin Blocking Solution to block available streptavidin sites with free Biotin. The mixture was incubated for 5 min at room temperature with gentle rotation and washed with TBS again. Beads were suspended in 300 µL of the NCM460 colonic cell lysate. After incubation (2h, 4 °C, gentle rotation), beads were washed with TBS and suspended in 24 µL assay buffer containing 2% (w/v) SDS. The mixture was incubated at 95 °C for 5 min. Finally, beads were removed to yield the samples, which were further investigated by SDS-Page and western blot analysis. Biolayer Interferometry. The interaction between the ligand and the Keap1 Kelch domain was determined by biolayer interferometry using an Octet Red 96 instrument (FortéBio Inc.). Super Streptavidin Biosensors tips (FortéBio, Inc., Menlo Park, CA) were prewetted with buffer (FortéBio) to establish a baseline before immobilization. Then probe 1 was immobilized onto Super Streptavidin Biosensors. All of the binding data were collected at 30 °C. The experiments comprised five steps: (1) baseline acquisition, (2) probe 1 loading onto the sensor, (3) second baseline acquisition, (4) association of Keap1 for the measurement of kon, and (5) dissociation of Keap1 for the measurement of koff. Six concentrations of Keap1 were used for detection. The association and dissociation plot and kinetic constants were obtained with FortéBio data analysis software. Equilibrium dissociation constants (Kd) were calculated by the ratio of koff to kon. Isothermal Titration Calorimetry. Isothermal titration calorimetry was performed at 25 °C with the ITC200 system (MicroCal). The Keap1 Kelch domain protein was lyophilized. Both the protein sample and the probe 2 were dissolved in 10 mM HEPES buffer (pH 7.4). 2 µL aliquots of 0.05 mM probe 2 were injected 19 times at 2.5 min intervals from a stirring syringe (750 rpm) into the sample cell containing 220 µL of 0.005 mM Keap1 Kelch domain. Data were analyzed with the computer program Origin, version 7.0, supplied by MicroCal. Immunofluorescence. NCM460 colonic cells were treated with probe 2 (10 µM) for 10 h, then incubated at 4 °C overnight with Keap1 primary antibody (abcam, UK). After washing with PBS, cells were incubated at 37 °C for 1 h with Alexa Fluor-labeled secondary goat anti-rabbit IgG antibody (KeyGEN BioTECH). Cells were then stained with fluorochrome dye DAPI (Santa Cruz Biotechnology, Santa Cruz, CA) to visualize the nuclei and observed under a laser scanning confocal microscope (Olympus Fluoview FV1000, Japan) with a peak excitation wave length of 570 nm and 340 nm.

ASSOCIATED CONTENT Supporting Information. Detailed experimental procedures of the synthesis, the expression and purification of Keap1 Kelch Domain and the detailed procedure of the fluorescence polarization competition assay. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Code The coordinate and structure factor files for Keap1 in complex with compound 1 is 4IQK.

Corresponding Author *Q. D.Y. E-mail: [email protected]; Telephone number: +86-02583271414 *Z.Y. J. E-mail: [email protected]; Telephone number: +86-025-83271414

Author Contributions The manuscript was written through contributions of all authors.

ACKNOWLEDGMENT The present work was supported by the project 81230078 (key program) and 81573346 of National Natural Science Foundation of China, 2014ZX09507002-005-015 and 2013ZX09402102-001005 of the National Major Science and Technology Project of China (Innovation and Development of New Drugs), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. SKLNMZZCX201611) and the Fundamental Research Funds for the Central Universities (2016ZPY016).

ABBREVIATIONS Keap1, Kelch-like ECH-associated protein-1; Cul3, Cullin 3; Nrf2, NF-E2-related factor-2; ARE, antioxidant response element; Isothermal titration calorimetry, ITC; heat shock protein-90, Hsp90; PD, Parkinson’s disease; IKKβ, IκB kinase β; NF-κB, nuclear factor-κB; FP, fluorescence polarization; BLI, biolayer interferometry assay; FITC, fluorescein isothiocyanate; ITC, isothermal titration calorimetry; TLC, thin layer chromatography; TMS, tetramethylsilane; HRMS, high resolution mass spectra.

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J.-P.; Yonchuk, J. G.; Feldser, H. G.; Sanchez, Y.; Foley, J. P.; Bolognese, B. J.; Logan, G.; Podolin, P. L.; Yan, H.; Callahan, J. F.; Heightman, T. D.; Kerns, J. K. Monoacidic inhibitors of the Kelchlike ECH-associated protein 1: nuclear factor erythroid 2-related factor 2 (KEAP1:NRF2) protein–protein interaction with high cell potency identified by fragment-based discovery. J. Med. Chem. 2016, 59, 3991-4006. (24) Jiang, Z. Y.; Xu, L. L.; Lu, M. C.; Chen, Z. Y.; Yuan, Z. W.; Xu, X. L.; Guo, X. K.; Zhang, X. J.; Sun, H. P.; You, Q. D. Structureactivity and structure-property relationship and exploratory in vivo evaluation of the nanomolar Keap1-Nrf2 protein-protein interaction inhibitor. J. Med. Chem. 2015, 58, 6410-6421. (25) Bertrand, H. C.; Schaap, M.; Baird, L.; Georgakopoulos, N. D.; Fowkes, A.; Thiollier, C.; Kachi, H.; Dinkova-Kostova, A. T.; Wells, G. Design, synthesis, and evaluation of triazole derivatives that induce Nrf2 dependent gene products and inhibit the Keap1-Nrf2 protein-protein interaction. J. Med. Chem. 2015, 58, 7186-7194. (26) Winkel, A. F.; Engel, C. K.; Margerie, D.; Kannt, A.; Szillat, H.; Glombik, H.; Kallus, C.; Ruf, S.; Gussregen, S.; Riedel, J.; Herling, A. W.; von Knethen, A.; Weigert, A.; Brune, B.; Schmoll, D. Characterization of RA839, a noncovalent small molecule binder to Keap1 and selective activator of Nrf2 signaling. J. Biol. Chem. 2015, 290, 28446-28455. (27) Wen, X.; Thorne, G.; Hu, L.; Joy, M. S.; Aleksunes, L. M. Activation of NRF2 signaling in HEK293 cells by a first-in-class direct KEAP1-NRF2 inhibitor. J. Biochem. Mol. Toxicol. 2015, 29, 261-266. (28) Lu, M.-C.; Yuan, Z.-W.; Jiang, Y.-L.; Chen, Z.-Y.; You, Q.D.; Jiang, Z.-Y. A systematic molecular dynamics approach to the study of peptide Keap1-Nrf2 protein-protein interaction inhibitors and its application to p62 peptides. Mol. Biosyst. 2016, 12, 1378-1387. (29) Lu, M.-C.; Chen, Z.-Y.; Wang, Y.-L.; Jiang, Y.-L.; Yuan, Z.W.; You, Q.-D.; Jiang, Z.-Y. Binding thermodynamics and kinetics guided optimization of potent Keap1-Nrf2 peptide inhibitors. RSC Advances 2015, 5, 85983-85987. (30) Lo, S.-C.; Li, X.; Henzl, M. T.; Beamer, L. J.; Hannink, M. Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling. EMBO J. 2006, 25, 3605-3617. (31) Padmanabhan, B.; Tong, K. I.; Ohta, T.; Nakamura, Y.; Scharlock, M.; Ohtsuji, M.; Kang, M.-I.; Kobayashi, A.; Yokoyama, S.; Yamamoto, M. Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer. Mol. Cell 2006, 21, 689-700. (32) Fukutomi, T.; Takagi, K.; Mizushima, T.; Ohuchi, N.; Yamamoto, M. Kinetic, thermodynamic, and structural characterizations of the association between Nrf2-DLGex degron and Keap1. Mol. Cell. Biol. 2014, 34, 832-846. (33) Marcotte, D.; Zeng, W.; Hus, J.-C.; McKenzie, A.; Hession, C.; Jin, P.; Bergeron, C.; Lugovskoy, A.; Enyedy, I.; Cuervo, H.; Wang, D.; Atmanene, C.; Roecklin, D.; Vecchi, M.; Vivat, V.; Kraemer, J.; Winkler, D.; Hong, V.; Chao, J.; Lukashev, M.; Silvian, L. Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism. Bioorg. Med. Chem. 2013, 21, 4011-4019. (34) Goldhahn, H. Darstellung des 4-Nitronaphthylamin-1. J. Prakt. Chem. 1940, 156, 315-316. (35) Stanley, M.; Martin, S. R.; Birge, M.; Carbain, B.; Streicher, H. Biotin-, fluorescein- and 'clickable' conjugates of phosphaoseltamivir as probes for the influenza virus which utilize selective binding to the neuraminidase. Org. Biomol. Chem. 2011, 9, 5625-9. (36) Rich, R. L.; Myszka, D. G. Higher-throughput, label-free, real-time molecular interaction analysis. Anal. Biochem. 2007, 361, 16. (37) Wysocki, L. M.; Lavis, L. D. Advances in the chemistry of small molecule fluorescent probes. Curr. Opin. Chem. Biol. 2011, 15, 752-759. (38) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J. Fluorescent chemosensors based on spiroring-opening of xanthenes and related derivatives. Chem. Rev. 2012, 112, 1910-1956.


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Table of Contents



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Figure 1. Structure-based design of the affinity-based small molecule probe of Keap1. Chemical structures of 1(A) and 2 (B). Binding surface depiction of 1 (PDB code: 4IQK) and 2. Hydrogen bonds are represented as green dashed lines and electrostatic interactions are represented as yellow dashed lines. The carbon atoms in small molecules and the Keap1 residues are colored cyan and purple, respectively. 85x40mm (300 x 300 DPI)


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Figure 2. The validation of the Keap1 binding potency of probe 1. (A). The chemical structure of probe 1. (B). Dose-response inhibi-tion curve of probe 1 determined by the FP-based binding assay. (C). Western blot analysis of probe 1 bound to the streptavidin coupled magnetic beads after incubation with NCM460 colonic cell lysates. Keap1 was detected with an anti-Keap1 primary antibody. 83x48mm (300 x 300 DPI)



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Figure 3. Biolayer interferometry sensorgrams of the binding of varying concentrations of the Keap1 protein to the immobilized probe 1. 83x64mm (300 x 300 DPI)


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Figure 4. The Keap1 binding affinity of probe 2. (A). The chemical structure of probe 2. (B). The ITC titration profile of Keap1 Kelch Domain with probe 2. (C). Dose-response inhibition curve of probe 2 determined by the titration FP assay. 83x83mm (300 x 300 DPI)



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Figure 5. Confocal microscope image of NCM460 colonic cells treated with probe 2. NCM460 colonic cells were treated with probe 2 (10 µM) for 10 h prior to fixation, then incubated at 4 °C overnight with Keap1 primary antibody. After that, cells were incu-bated with Alexa Fluor-labeled secondary antibody. Nuclears were stained with fluorochrome dye DAPI prior to observation. The bars indicate the magnification (10 µM). All images are representative of images obtained in three separate experiments. 83x74mm (300 x 300 DPI)


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table of contents graphic 112x55mm (300 x 300 DPI)


Design, Synthesis, and Initial Evaluation of Affinity-Based Small

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