Discovery and Development of Kelch-like ECH-Associated Protein 1

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Perspective

Discovery and Development of Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction Inhibitors: Achievements, Challenges and Future Directions Zhengyu Jiang, Mengchen Lu, and Qi-Dong You J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00586 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 4, 2016

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Journal of Medicinal Chemistry

Discovery and Development of Kelch-like ECHAssociated Protein 1: Nuclear Factor Erythroid 2Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction Inhibitors: Achievements, Challenges and Future Directions Zheng-Yu Jiang a,b, ‡, Meng-Chen Lu a, ‡, Qi-Dong You*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 The transcription factor Nrf2 is the primary regulator of the cellular defense system, and enhancing Nrf2 activity has potential usages in various diseases, especially chronic age-related and inflammatory diseases. Recently, directly targeting Keap1-Nrf2 protein-protein interaction (PPI) has been an emerging strategy to selectively and effectively activate Nrf2. This Perspective summarizes the progress in the discovery and development of Keap1-Nrf2 PPI inhibitors, including the Keap1-Nrf2 regulatory mechanisms, biochemical techniques for

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inhibitor identification, and approaches for identifying peptide and small-molecule inhibitors, as well as discusses privileged structures and future directions for further development of Keap1Nrf2 PPI inhibitors.

1. Introduction Transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is the primary regulator of cytoprotective genes in cells,1, 2 and it activates transcription in response to electrophiles and reactive oxygen species (ROS) under cellular stress3. Nrf2 binds DNA as a heterodimer with small musculoaponeurotic fibrosarcoma (Maf) proteins, MafF, MafG, and MafK.4 The binding site of Nrf2 in the DNA sequence is the antioxidant/electrophile response element (ARE/EpRE, 5’-TGACNNNGC-3’)5. Previous studies have determined that Nrf2 controls the expression of numerous cytoprotective proteins, including phase I and phase II drug-metabolizing enzymes, drug transporters and antioxidants.6 However, there has been increasing evidence that the target genes of Nrf2 are involved in a much wider range of activities, including lipid and glucose metabolism, heme and iron metabolism and gene transcription.7-11 Thus, Nrf2 not only plays an important role in cellular defense, but also influences energy metabolism, inflammation and cell growth.12 These important biological functions of Nrf2 have attracted interest from a broad range of researchers in various fields, especially the drug discovery community.13, 14 Many studies have demonstrated that Nrf2 plays important roles in protection against various diseases, especially chronic age-related and inflammatory diseases, including cancer15-17, neurodegenerative diseases18, cardiovascular diseases, acute lung injury19, chronic obstructive pulmonary disease20,


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kidney diseases21 and inflammation.22 To date, numerous Nrf2 activators have been reported, and some of these have been used in clinical trials to explore clinical applications. Among them, Tecfidera™ (dimethylfumarate, DMF) has been the most successful Nrf2 activator that has been approved by the FDA to treat patients with relapsing forms of multiple sclerosis.23 Another potent Nrf2 activator, the synthetic triterpenoid [2-cyano-3, 12-dioxo-oleana-1, 9(11)-dien-28oic acid, methyl ester] (CDDO-Me), is now being assessed in a phase 3 study of patients with connective tissue disease-associated pulmonary arterial hypertension. Sulforaphane, an isothiocyanate compound extracted from broccoli sprouts, has also been evaluated in several clinic studies.24, 25 This progress has inspired further development of therapeutic agents around the cytoprotective role of Nrf2. In this Perspective, we focused on the discovery and development of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors as a selective and effective way to explore Nrf2-targeting therapies.

2. Regulatory PPIs that modulate Nrf2 activity Given the central role of Nrf2 in the cellular defense system, Nrf2 activity is tightly regulated. Clearly understanding the regulatory mechanism of Nrf2 activity is the basis for developing targeting modulators. Despite the identification of an increasing number of regulatory mechanisms, Keap1-involved PPIs play a central role in the precise regulation of Nrf2 in accordance with the cellular oxidative state (as shown in Figure 1).26, 27 2.1 General Model of Keap1-Cul3 E3 ligase-mediated Nrf2 ubiquitination First, Keap1 (Kelch-like ECH-associated protein 1) is an adaptor in the Cul3 (cullin3)-based ubiquitin E3 ligase that is responsible for Nrf2 ubiquitination. E3 ligases take part in ubiquitin transfer to a target protein and mediate substrate specificity in the highly heterogeneous


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ubiquitination system (there are > 600 E3s in humans).28 The cullin-based E3 ligase is composed of multiple subunits that are assembled on a cullin scaffold, binding a RING-box protein at the N terminus and an adaptor protein as a substrate receptor (responsible for substrate specificity) at the C terminus.29 In the Keap1-Nrf2-Cul3 complex, Keap1 acts as the substrate receptor and selectively recognizes Nrf2, linking Nrf2 to the scaffold protein Cul3. The recognition of the substrate by Keap1 is the key step for the selectivity. Therefore, Keap1-Nrf2 PPI ensures ubiquitination substrate selectivity. Cul3 is the scaffold protein in the assembly of the E3 ligase complex, and Keap1-Cul3 PPI ensures that specific lysines of Nrf2 are located in the proper position at the catalytic center. This PPI-mediated substrate ubiquitination is the typical mechanism in the ubiquitin system. In addition, Keap1 also acts as the sensor and switch for the Nrf2 ubiquitination machine.30 Keap1 is a cysteine-rich protein that contains 25 (mouse) or 27 (human) cysteine residues, some of which have been identified as sensors of electrophilic and/or oxidative assault.31-35 Under stress conditions, these sensitive cysteine residues are oxidized to disulfides or conjugated to electrophiles.36 These covalent modifications affect the precise assembly of the E3 ligase complex and suppress the ubiquitination of Nrf2, which finally leads to the activation of Nrf2.37, 38

Activation of the Nrf2-regulated cytoprotective system promotes cytosolic reductase activities,

elevates the reduced glutathione level and accelerates the metabolism and export of xenobiotics. These findings have also inspired the development of electrophilic Nrf2 activators that mimic the endogenous process of Nrf2 activation.39 These Nrf2 activators enhance the transcriptional activity of Nrf2 via S-alkylation of reactive cysteines in Keap1.5,

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The mechanism of

electrophilic Nrf2 activation has been further confirmed by the crystal structure of a well-known electrophilic Nrf2 activator, CDDO with Keap1.41 Issues regarding this type of Nrf2 activator


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have been summarized by another excellent Perspective.42 The results mentioned above provide the Keap1-Cul3 E3 ligase-mediated Nrf2 ubiquitination model with a unique sensor-switch regulatory mechanism mediated by the Keap1 cysteine residues.

Figure 1. Schematic diagram of the regulation of Nrf2 activity. 2.2 Unique Mechanism underlying Keap1-Nrf2 PPI The case of Nrf2 is much more complex. Existing achievements have proved that Nrf2 contains two Keap1 binding motifs, called ETGE and DLG. Two Keap1 protein molecules form a homodimer and interact with these two motifs in one molecule of Nrf2 protein. The early experimental proof for this PPI model was from an ITC (isothermal titration calorimetry) experiment of the Keap1 DC domain and the Nrf2 Neh2 domain.43 The obtained titration curve exhibits a biphasic curve, which is fitted best with a two-site binding model. After that


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experiment, the co-crystal structures of the Keap1-Nrf2 ETGE peptide and the Keap1-Nrf2 DLG peptide44 were reported, which confirmed that Nrf2 contains two Keap1 binding sites. The available crystal structures of Keap1 and Keap1 with ligands are listed in Table 3. In addition, the experimental results showed that the Keap1 protein exists as a stable dimer rather than a monomer in mammalian cells, and the dimerization of Keap1 is indispensable for its interaction with Cul3.45 The direct structural evidence for the Keap1 homodimer is the result of single particle electron microscopy that clearly showed two large spheres attached by short linker arms to the sides of a small forked-stem structure, resembling a cherry-bob.46 These research results clearly supported the two-site molecular recognition model of Keap1-Nrf2 and were also in line with the ‘hinge and latch’ model that previously obtained wide acceptance.44 In that model, the high binding affinity ETGE motif acts as the hinge to tightly link Nrf2 with the Keap1 dimer, and the weak binding affinity DLG motif acts as the latch to switch ubiquitination. Only when both of the two motifs bind to the Keap1 dimer will the ubiquitination of Nrf2 be carried out. The intracellular oxidative stress signals mainly induce the disassociation of the Keap1-Nrf2 DLG motif and block the ubiquitination of Nrf2. However, recent studies have provided new insights into the Keap1-Nrf2-Cul3 regulation pattern. Dinkova-Kostova’s group developed a quantitative Förster resonance energy transferbased system using multiphoton fluorescence lifetime imaging microscopy to investigate the interaction between Nrf2 and Keap1 in single live cells.47 Their research results showed that the complex of the Keap1 dimer with Nrf2 adopts the following two distinct conformations under homeostatic conditions: the “open” conformation, in which Nrf2 binds to the Keap1 dimer through the high binding affinity ETGE motif and the “closed” conformation, in which both of the two motifs in Nrf2 bind with the Keap1 dimer. The conformation of the Keap1-Nrf2 complex


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switches between the two states, and ubiquitination of Nrf2 only occurs when the “closed” conformation is formed, which is termed the “conformation cycling model” (as shown in Figure 2).48 Under induced conditions, the accumulation of the complex in the closed conformation, not the open conformation, has been observed. This accumulation most likely occurs because the inducers have disrupted the ubiquitination step, and the closed conformation of the complex cannot begin cycling. Thus, intracellular Keap1 is anchored to Nrf2, and newly translated Nrf2 accumulates and translocates into the nucleus, which finally turns on the expression of cytoprotective genes. Then, inducers activate Nrf2 by inhibiting the regeneration of free Keap1 rather than releasing Nrf2 from the Keap1 dimer. Recent studies on the Nrf2 DLG motif have further confirmed the conformation cycling model. The previously obtained minimal DLG motif-derived peptide has shown weaker binding affinity and is poorly tolerated with further optimization.49 This ambiguity was revealed by the further investigation of Masayuki Yamamoto’s group of the minimum Keap1-binding sequence of the Nrf2 DLG motif. The authors defined a new DLGex motif that covers a sequence much longer than what was previously defined.50 The binding affinity of the DLGex motif-derived peptide, rather than the DLG motif peptide, is consistent with the second binding constant of the Nrf2 Neh2 domain. Moreover, in kinetic analyses, Keap1-DLGex binding follows a fast association and fast dissociation model, whereas Keap1-ETGE binding contains a slow-reaction step that leads to a stable conformation. This research suggested that the dissociation of Keap1-DLGex may occur spontaneously, which indicates that the ‘hinge and latch’ behavior is a dynamic process.


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Figure 2. Conformation cycling model of Keap1-Nrf2 regulation. These studies together confirmed PPIs’ decisive role in modulating the activation of Nrf2. Keap1 bridges Nrf2 with Cul3 through PPIs, which enables the Keap1-mediated selective ubiquitination of Nrf2. Besides, the Keap1 dimer interacts with Nrf2 through a unique pattern, which thereby benefits the precise modulation of Nrf2. Among these PPIs, Keap1-Nrf2 PPI is closely associated with Nrf2 activity, which provides a great way to discover Nrf2-targeting modulators.

3. Characteristics of Keap1-Nrf2 PPI


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PPIs are involved in almost every biological function. Hundreds of thousands of PPIs51, 52 together constitute the huge interactome that affects cellular behaviors.53,

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Thus, it is not

surprising that extraordinary diversity and complexity exist in PPIs. Although PPIs may adopt quite different sizes, shapes and electrostatics,55 PPIs also share some similar characteristics, such as a large interface area, shallow interaction sites and multiple binding interactions. These characteristics together present a challenge for agents targeting PPIs. Thus, it is imperative to obtain a thorough understanding of the mechanism of action of a given PPI target before discovery of modulators. The Keap1-Nrf2 interaction is an especially unique PPI. The binding pocket in Keap1 is open, which is similar to most PPIs, but it is well defined. The size of the binding site is relatively small. The interface area of the Keap1-Nrf2 ETGE motif is approximately 529 Å2 (calculated from PDB code: 1X2R), and that of the Keap1-Nrf2 DLGex motif is a bit larger, approximately 820 Å2 (calculated from PDB code: 3WN7). Previously, we divided the Keap1 substrate binding cavity into five sub-pockets (P1-P5) based on the co-crystal structure of the Keap1-Nrf2 ETGE motif (Figure 3A).56 However, given the Keap1-Nrf2 DLGex interaction,50 a P6 sub-pocket should be included in Keap1 (as shown in Figure 3B). The contact surfaces involved in Keap1Nrf2 PPI are more typical of those involved in protein-small-molecule interactions (300-1000 Å2) than of PPIs (1500-3000 Å2).57,

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It is reasonable that the two regions in Nrf2 that are

responsible for Keap1 dimer binding are adjacent in the primary sequence, which does not allow for a large binding surface. However, the binding strength between Keap1 and Nrf2 is quite potent. The 9-mer Nrf2 ETGE peptide can reach a Kd of approximately 20 nM.59 This information indicates that the substrate binding cavity of Keap1 has more well-characterized hot-


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spots on the one hand and is precisely specific with ligands on the other hand. This feature ensures substrate selectivity but raises difficult issues for developing modulators.

Figure 3. Sub-pocket analysis of Keap1 substrate binding cavity. Based on the Keap1-Nrf2 crystal structure, the Keap1 substrate binding cavity can be divided into six sub-pockets. (A) Sub-pocket analysis of Keap1 substrate binding cavity based on the Keap1-Nrf2 ETGE complex (PDB code: 1X2R); (B) Sub-pocket analysis of Keap1 substrate binding cavity based on the Keap1-Nrf2 DLGex complex (PDB code: 3WN7); (C) Summary of key residues in each subpocket. In general, PPIs are classified into the following two categories: a domain-domain pattern that is mediated by the interaction between two protein domains and a peptide-domain pattern that is mediated by the interaction between a linear sequence of residues of one of the partners and a domain of the other one.60, 61 The Keap1-Nrf2 ETGE interaction and the Keap1-Nrf2 DLGex interaction belong to the two types, respectively (as shown in Figure 4A & 4C). The Keap1-Nrf2 ETGE interaction is a typical example of peptide-domain-mediated PPI. The Nrf2 ETGE peptide possesses a tight four-residue β-hairpin conformation that comprises the conservative DXETGE motif, specifically the residues Asp77, Glu78, Glu79, Thr80, Gly81, and Glu82. This conformation is stabilized by intramolecular hydrogen bonds involving the Asp77 and Thr80


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side chains and the peptide backbone. Glu79 and Glu82 insert into the P1 and P2 sub-pockets and form multiple electrostatic interactions with key arginine residues in Keap1, including Arg483, Arg415 and Arg380 (Figure 4D). The Keap1-Nrf2 DLGex interaction belongs more to the domain-domain pattern. The cocrystal structure shows that the DLGex region constitutes three helices, helix 1 (Leu19 to Arg25), helix 2 (Ile28 to Leu30), and helix 3 (Arg34 to Phe37), which form a U shape that is distinctly different from the β-hairpin conformation of the ETGE region.50 Helix 1 and helix 3 cover the outer surface of the P1 and P2 sub-pockets in Keap1, and helix 2 and the surrounding linking residues are located on the central part of the cavity. The overall binding pattern of DLGex is significantly different from that of the previously reported short DLG peptide (as shown in Figure 4). In the case of the DLGex motif, the side chain of Gln27 moves from the inside to the outside of the P1 sub-pocket and occupies a shallow groove composed of Ser431, Gly433, Cys434, His436 and Arg380. The side-chain amide group of Gln26 supplies hydrogen bonds to the imidazole group of His436 and the main-chain carbonyl group of Gly433 in Keap1, which is not found among other Keap1 ligands. Moreover, the side-chain carbonyl oxygen atom of Gln26 can form hydrogen bonds with Arg380 in Keap1. Asp29 is inserted into the P1 subpocket and forms multiple hydrogen bonds with Arg415 and Arg483 simultaneously, while the short DLG peptide occupies this cavity with Gln27. Because of its negative charge, Asp29 can make a firm salt bridge with Arg415 in Keap1. Closer examination reveals that the positions of Asp27 also adopt a displacement, which allows the side-chain carboxyl group to form multiple interactions with Arg415, Arg380, Asn382 and Asn414. Only the interaction with Arg415 can be observed in the Keap1-Nrf2 DLG complex. This result indicates that the DLGex peptide can form more polar interactions in the P2 sub-pocket than the DLG peptide. Overall, the


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incorporation of two helixes on both sides of the DLG peptide significantly changes the positions of the polar residues, especially Asp27 and Asp29, which form more polar interactions in the P1 and P2 sub-pockets. Compared with the Nrf2 ETGE peptide, the DLGex peptide can form similar polar interactions in the P1 and P2 sub-pockets with a completely different sequence (the DLD and ETGE sequence). Not only does this result stress the importance of polar interactions in Keap1 substrate recognition but also provides new ideas for both novel natural ligand discovery and the design of potent modulators.

Figure 4. Co-crystal structure of Keap1-Nrf2 PPI. (A) & (D) Binding surface and key polar residue interactions of the Keap1-Nrf2 ETGE motif (PDB code: 1X2R); (B) & (E) Binding surface and key polar residue interactions of the Keap1-Nrf2 DLG motif (PDB code: 2DYH); (C) & (F) Binding surface and key polar residue interactions of the Keap1-Nrf2 DLGex motif (PDB code: 3WN7). Hydrogen bonds are represented by green dashed lines, and electrostatic


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interactions are represented by yellow dashed lines. The carbon atoms of Nrf2 residues and Keap1 residues are colored cyan and purple, respectively. Moreover, the binding thermodynamics and kinetics profiling of the two regions also show significant differences. Several studies have elucidated that Keap1-ETGE binding is a single enthalpy-driven process,43, 62 which is consistent with the key role of polar interactions in Keap1ETGE binding. Our computational studies also showed that the polar sub-pockets, P1 and P2, contribute the major part of the total binding energy.63 The ITC results of Keap1-DC with a deletion mutant of Nrf2 Neh2, in which the residues of ETGE were removed, showed that the total entropy effects were beneficial for binding.43 These data indicated that the second binding site may have distinct binding thermodynamic behavior. The identification of the DLGex motif further confirmed this suggestion. The ITC experiment with the DLGex peptide and Keap1 clearly showed a synergistic enthalpy/entropy-driven process.50 This finding shows that even though the binding cavity of Keap1 has many polar residues, the overall entropy effects can benefit binding and stresses that both polar and hydrophobic interactions contribute to anchoring small molecules and peptides. The binding kinetics of Keap1-ETGE and Keap1-DLGex also have great differences. The kon and koff determined from both SPR (Surface Plasmon Resonance)59 and the BLI (Bio-Layer Interferometry)62 assays show that the Nrf2 ETGE peptide has a very slow dissociation rate, confirming the recognition and anchoring effects of this region in the conformation cycling model. By using quantitative Förster resonance energy transferbased methods, Baird et al. investigated Keap1-Nrf2 interactions in a single live cell and proposed a ‘conformational cycling’ model for the Keap1-mediated degradation of Nrf2.47 The authors found that the Keap1-Nrf2 complex sequentially takes on the following two distinct conformations: an ‘open’ conformation in which the high-affinity ETGE motif of Nrf2 interacts


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with a single molecule of Keap1, followed by a ‘closed’ conformation in which Nrf2 binds to both members of the Keap1 dimer.48 Subsequently, Masayuki Yamamoto group’s recent study proposed a two-state binding model of Keap1-ETGE in which Nrf2-ETGE binds to Keap1 by two steps. In the first step, Keap1 DC recognizes Nrf2 ETGE by a fast association and dissociation step, and then the complex adopts the fully optimized conformation by a slow conformational adjustment reaction in the second step.64 The current findings are reasonable but are far from a thorough confirmation. In contrast, the kinetic analysis results from an SPR experiment showed that Keap1-DLGex binding follows a fast association and fast dissociation model50, which allows other signaling pathways to regulate Nrf2 activity. Without a doubt, the in-depth investigation of Keap1-Nrf2 PPI, coupled with the co-crystal structure, can facilitate the discovery of Keap1-Nrf2 PPI antagonists. 4. Assays for evaluating Keap1-Nrf2 PPI Inhibitors To modulate a PPI of interest, well-established agents must be identified first. However, the identification of the PPI inhibitors is another hurdle in PPI drug discovery. Unlike traditional drug targets, especially enzymes, PPIs do not involve enzymatic activity in most cases. Currently, various interdisciplinary techniques, especially biochemical and biophysical techniques, can be used to develop screening assays to find active hits from small arrays and large compound libraries. Cell-based high content screening, cellular downstream phenotype examination and in vivo imaging to monitor PPIs have also been used to further evaluate the biological effects of PPI modulators. For the Keap1-Nrf2 PPI, various methods have been developed to meet the needs of drug discovery, including high-throughput screening (HTS) of hits, improvement of PPI inhibition activity and optimization for drug-like properties.


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Hereafter, we will describe currently available assays for the identification and evaluation of Keap1-Nrf2 inhibitors (Table 1). Basic principles and advantages as well as strengths and weaknesses of these different assay types will be included. 4.1 In vitro assays Accordingly, depending on the general propose of in vitro methods, they can be divided into the following two categories: Keap1 binding evaluation and Keap1-Nrf2 interaction inhibition. 4.1.1 Keap1 binding assay The most straightforward parameter reflecting binding strength is the equilibrium dissociation constant Kd. Therefore, assays that can determine Kd and precisely measure direct binding are valuable in evaluating Keap1-Nrf2 PPI inhibitors. Because the estimation of Kd also plays an important role in PPI biology, these methods are often available before the discovery of PPI modulators. The Kd can be obtained via two distinct ways, the binding thermodynamic method and the binding kinetic method. The most commonly used technique for the binding thermodynamic method is ITC assay, which has been used in the evaluation of Keap1-Nrf2 interactions. The most attractive advantage of ITC is that it can give the quantitative contribution of enthalpy and entropy simultaneously, which can benefit further drug-like optimization of the inhibitors.65, 66 Values for both ∆G and ∆H can be obtained from the ITC titration profiles, and entropic contribution (T∆S) can be calculated using the fundamental equation ∆G = ∆H − T∆S. Our group has successfully used this method to optimize Keap1-Nrf2 peptide inhibitors and obtained the most potent peptide inhibitor with a Kd value of approximately 10 nM.62 For the binding kinetic method, SPR and BLI are the commonly used techniques. In addition to the resulting Kd value, a binding kinetic assay can monitor the association and dissociation process in real-time and obtain the association rate constant kon and the dissociation rate constant koff. As


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described above, these kinetic results further enhance the understanding of the Keap1-Nrf2 interaction mechanism. In the optimization of inhibitors, the kinetic profile is also an important drug-like index, which directly impacts drug efficacy and safety.67 Longqin Hu’s group used SPR to carry out kinetic analyses of the Keap1-Nrf2 interaction and determine the minimal Nrf2 ETGE peptide sequence required for Keap1 binding.59 By using the BLI technique, we assessed the kinetic profile of the potent small-molecule Keap1-Nrf2 inhibitor and found that the inhibitor followed the binding behavior of the ETGE peptide, which has quite a slow dissociation rate.56 Generally, the binding kinetic and thermodynamic profile together ensure the precise assessment of Keap1-ligand binding and provide useful tips for further optimization of inhibitors. The advantages of these methods are obvious, such as label-free detection, real-time monitoring of binding interactions and quantitative evaluation of binding strength. The shortcomings are also conspicuous. All of these assays need a separate operation for each test sample, which restricts the throughput of the assay. These assays can only be used for validation and step-by-step optimization of the inhibitors, rather than high-throughput screening to find novel hits. In recent years, differential scanning fluorimetry (DSF) has been developed to detect ligand-receptor binding interactions that promote protein stability.68 The main advantage of DSF is that it can be performed with higher throughput without requiring large amounts of protein. Chunlin Zhuang et al. used this method to confirm the binding interaction of lead compounds with Keap1.69 4.1.2 Keap1-Nrf2 inhibition assay Compared to Keap1 binding strength, Keap1-Nrf2 interaction inhibition activity is a more direct index reflecting the potency of the compounds. Because the PPI itself cannot induce biological effects directly in most cases, extra reporter groups, especially fluorescent groups, must be


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introduced. Among these fluorescent-based techniques, fluorescence polarization (FP) is the most widely used. The basic principle of FP is that moving fluorescent molecules emit light on a different plane when excited with linear polarized light.70 This effect is induced by the movement of the molecules in solution between excitation and emission, which can be significantly affected by the molecular size. The difference in polarization can be observed for fluorescently labeled species of different sizes, which essentially relies on a mass difference (preferably ∼10×). Thus, the FP assay is often used in a competition format, i.e., a short peptide derived from the binding motif of one protein will be fluorescently labeled, and the binding partner will be treated as the receptor. Only in this way can we obtain a sufficient difference in the mass of the unbound and bound state of the fluorophore-bearing peptide, which can induce a strong difference in FP. For this reason, the main disadvantage of the FP assay is that one protein in a PPI must be minimized to a short peptide size to meet the need for the FP difference. It is more suitable for the peptide-domain PPI, to which the Keap1-Nrf2 ETGE PPI belongs. Longqin Hu’s group first reported using the FP assay for the discovery of Keap1-Nrf2 PPI inhibitors by an optimized fluorescently labeled Nrf2 ETGE peptide.71 The authors also used this assay to conduct HTS and discovered a first-in-class small-molecule Keap1-Nrf2 inhibitor.72 Since those reports, the FP assay has been widely used in the discovery and optimization of Keap1-Nrf2 inhibitors.56, 69, 73-75 The main advantage of the FP assay is that it can quantitatively evaluate the activity of inhibitors with a biological reagent and compound saving system, simple operation process (“mix-and-read assay”) and high-throughput screening. The FP assay also has some weaknesses. In addition to the size-dependent nature and the need for the fluorescent tracer peptide mentioned above, the FP assay cannot be conducted in a time-resolved manner and lacks time stability. A further improvement of the FP method, the two-dimensional fluorescence


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intensity distribution analysis (2D-FIDA),76 has also been used to perform HTS of Keap1-Nrf2 PPI inhibitors, and it has also identified active hits.77 To overcome the drawbacks of FP assays, some proximity-based fluorescent methods have also been established. Among them, a fluorescence resonance energy transfer (FRET)-based assay is the most widely used. FRET is the radiationless transmission of energy from a donor molecule that initially absorbs the energy to an acceptor molecule to which the energy is subsequently transferred. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Thus, it is a proximity-based method, which can be adapted to detect the distance between two proteins and avoids the process of minimizing one binding partner to a short peptide, which facilitates the setup of the screening assay. Moreover, the time stability of FRET over FP can facilitate HTS. This type of assay also works in the Keap1-Nrf2 PPI system, and Geoff Wells’ group has successfully developed a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 PPI.78 In addition to evaluating PPI inhibition, the fluorescent tracer displacement assay can also be used to rapidly assess thermodynamic parameters for targetligand binding.79 The main advantage of this type of thermodynamic binding assay is that it can reach medium throughput, which can be used in hit-to-lead programs.80 A research group from Evotec AG reported a protocol for the determination of the thermodynamic parameters of Keap1-Nrf2 PPI inhibitors and tested the small-molecule inhibitors obtained from HTS.81 The Keap1-Nrf2 inhibition assays mentioned above are all fluorescence related, which may not work on compounds that fluoresce. The SPR-based solution competition assay can be used to examine the Keap1-Nrf2 PPI inhibition activity of fluorescent compounds.59 In this assay, a biotin-labeled Nrf2 ETGE peptide is captured on a streptavidin chip and competitively binds


18

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Keap1 with the inhibitors in the solution. Because of the nature of the SPR technique, it is not disturbed by the compound’s fluorescence, and throughput is also limited. 4.2 Cellular and in vivo assays 4.2.1

Assays for the Keap1-Nrf2 PPI inhibition

Compared with the determination of the Keap1-Nrf2 PPI inhibition activity in vitro, direct evaluation of Keap1-Nrf2 PPI inhibition activity in live cells or in vivo is hard to achieve. Recently, a cell-based luciferase enzyme fragment complementation (EFC) assay has been reported to measure the dissociation of the Keap1-Nrf2 complex.82 The authors generated HEK293T cells that stably express the Nrf2 protein with the N-terminal luciferase fragment (CLuc-Nrf2) and the Keap1 protein with the C-terminal luciferase fragment (NLuc-Keap1). When Keap1 interacts with Nrf2, the complemented luciferase enzymes achieve their active form. Once the Keap1-Nrf2 PPI is disrupted, the complemented luciferase enzymes are disassembled, leading to a decrease in luciferase activity. Thus, the disruption of the Keap1-Nrf2 PPI can be associated with the decrease in luciferase activity, which can be easily detected and used in a high throughput intact cell-imaging platform. Moreover, with the help of a noninvasive molecular imaging approach, this assay can also be used to evaluate compounds in small animal models. The FRET-based assay system is an alternative to evaluate Keap1-Nrf2 inhibition activity in live cells. As mentioned above, Geoff Wells’ group has successfully established and used this type of assay to investigate the Keap1-Nrf2 PPI regulatory mechanism.47, 48 They also applied it to the evaluation of small-molecule inhibitors.75 4.2.2

Assays for Nrf2 activation

Because of the transcriptional activation activity of Nrf2, it is much easier to construct an assay to estimate the potency of Nrf2 activation qualitatively and quantitatively. Among the available


19


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methods, the most straightforward way is the Nrf2-ARE-luciferase reporter gene assay. Nrf2 induces a battery of cytoprotective genes by binding to ARE located in the promoter regions; thus, a luciferase gene under the control of ARE promoters can reflect Nrf2 activation. Moreover, the luminescence signal affected by the luciferase activity can be easily detected, which provides a rapid and convenient quantification of Nrf2-ARE induction. Therefore, a cellular AREluciferase reporter gene assay can be used in HTS, and several HTS studies using this assay have been reported.83, 84 Apart from using luciferase as the reporter gene, some other ARE-controlled reporter systems are also commercially available. For example, Invitrogen provides the Cell Sensor™ ARE-bla HepG2 Cell-based Assay, which uses β-lactamase as the reporter. The main disadvantage of ARE-luciferase reporter gene assays is the frequent occurrence of false positives. The cell-based luciferase enzyme fragment complementation (EFC) assay, to a certain extent, can overcome this shortcoming. This method depends on the reconstitution of a ‘split’ luciferase. Based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2), a research group from GlaxoSmithKline developed such an approach in which firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively.85 Thus, a non-transcriptional readout is needed to minimize false positives, and the luminescence signal can provide information directly about the level of Nrf2 protein and its function, which is a clear advantage over the classical ARE-luciferase reporter assay. The commercially available PathHunter® U2OS Keap1-Nrf2 Nuclear Translocation Cell Line from DiscoverX uses a very similar mechanism. Nrf2 also controls the transcription of several enzymes, and thus, the enzyme activity of these downstream enzymes can be used as reporters directly. The examination of NQO1 enzyme activity is widely used in both classic Nrf2


20

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activators86 and Keap1-Nrf2 PPI inhibitors75. This type of assay is a good complement to the ARE-luciferase reporter gene assay for eliminating false positives. Compared with these medium- to high-throughput assays for evaluating the activation of Nrf2, there are a number of low-throughput but direct methods to validate the activation of Nrf2 and its function at the cellular level and in vivo, including western blot and qRT-PCR analysis of Nrf2 and its downstream genes. As the prototypical Nrf2-target genes, NQO-1 (NAD(P)H:quinone oxidoreductase-1)87-89 and HO-1 (heme-oxygenase-1)90-92 are the most widely evaluated. However, it has been noticed that the basal expression of NQO-1 is considerably high in certain cell lines, especially tumor cell lines such as HCT116 cells56,

74, 92, 93

. Thus, a variety of

downstream genes should be used to accurately evaluate the compound’s effects on Nrf2regulated genes. The current suite of available assay technologies, some of which are available as commercial kits, allow scientists to efficiently identify activators, confirm mechanism of action in vitro, and demonstrate pathway engagement in vivo in a tissue of interest. As such, the tools are available for robust drug-discovery efforts.

Table 1. Summary of Assays for Evaluating Keap1-Nrf2 PPI Inhibitors Assay type

Assay principle

Biological effect

Notes

ref

In vitro system Binding Kinetic Analysis

Biolayer Biophysics-Optics

56, 62

Keap1 binding

Interferometry (BLI)

Quantitative methods


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Medium throughput Label free; Quantitative methods; Isothermal

Titration Physical ChemistryKeap1 binding

Calorimetry (ITC)

Binding thermodynamics analysis

62

Calorimetry Low throughput

Competitive

Molecular size-based Keap1-Nrf2 ETGE High throughput

Fluorescence

luminescence

peptide disruption

71


Polarization (FP) Wide

applicability

for

compounds； Keap1-Nrf2 ETGE

SPR-based competition

59

Biophysics-Optics peptide disruption

assay

Competition assay； Medium throughput

Fluorescence Proximity-based resonance

Keap1-Nrf2 ETGE High throughput 78

energy luminescence

peptide disruption

transfer assay Fluorescence Tracer

displacement


Keap1-Nrf2 ETGE Medium throughput 94

anisotropy and Van’t peptide disruption

assay Hoff analysis Differential

Scanning

High throughput

Melting temperatures Fluorimetry

Binding thermodynamics analysis

69

Keap1 binding

(DSF) of Keap1 protein

Assay


22

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Cellular and In vivo system Luciferase enzymatic Nrf2

ARE-Luciferase

transcription High

throughputHigh

false 83, 84

Reporter Gene Assay

activity

activity

ARE-β-lactamase

β-lactamase

Nrf2

Reporter Gene Assay

enzymatic activity

activity

positives

transcription High throughput Provided by Invitrogen

Nrf2-MafK or Keap1Nrf2 translocation RunX2

luciferase Luciferase enzymatic

High throughput 85

and Nrf2-MafK or enzyme

fragment activity Nrf2-RunX binding

Low false positives

complementation Nrf2−Keap1 Luciferase

enzyme Luciferase enzymatic Keap1-Nrf2 82

Works in vivo

fragment activity

Disruption

complementation (EFC) assay FRET

and Keap1-Nrf2

multiphoton

Direct detection of Keap1-Nrf2 47, 75

fluorescence lifetime fluorescence

Disruption

lifetime

interaction

imaging microscopy

Enzymatic

assay

Protein level

of enzymatic

Nrf2

75

dependent (NQO1, HO-1, etc.)

protein

High throughput

activity Nrf2-regulated genes


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High throughput β-gal enzyme fragment β-gal

enzymatic Nrf2 translocation

complementation

Low false positives

activity Provided by DiscoverX mRNA level Cells and in vivo tissue;

qRT-PCR Detection

87

Gene expression

Nrf2-regulated

of mRNA

Low throughput genes

antigen-antibody

Protein level Cells and in vivo tissue;

Protein expression

reaction

93

Nrf2-regulated Low throughput

Detection of protein

genes

5. Medicinal chemistry strategies in the discovery of Keap1-Nrf2 PPI Inhibitors Generally, Keap1-Nrf2 PPI antagonists often represent a challenging target for drug development approaches95. That is especially true for the Keap1-Nrf2 PPI, as there is a high surface area and open binding site at the interface of the Keap1-Nrf2 PPI. Despite this challenge, with the remarkable progress that has been made in discovery techniques and the characterization and development of PPI inhibitors, several different types of Keap1-Nrf2 PPI inhibitors have been recently reported.96, 97 5.1 Peptide Keap1-Nrf2 antagonists


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The high surface area and lack of characterized interactions are the main hurdles for targeting PPIs. Peptides are the ideal candidates to tackle this limitation and match the PPI target as they are able to closely mimic the principle features and proper conformation of a protein but can be easily synthesized and modified for functional fine-tuning.60 In addition, the sequence of the natural protein can directly guide the design of peptides. It is feasible to discover first-in-class PPI inhibitors from peptides and to explore the mechanisms of action of PPIs using peptides,61 as has been the process of discovering the Keap1-Nrf2 PPI inhibitors98. Representative Keap1-Nrf2 PPI peptide inhibitors are listed in Table 2. 5.1.1 Exploration of the minimal Nrf2 peptide sequence required for Keap1 binding As mentioned previously, the strong binding site (Nrf2 ETGE region), which is a typical peptide-domain PPI pattern, provides the primary sequence for discovering peptide antagonists. Searching for the minimal peptide sequence from the primary sequence is a general approach for peptide PPI inhibitors in early stages. The process for identification of the minimal sequence can help researchers to understand the hot-spots of PPI. Moreover, the minimal peptide can lead to useful tools to investigate the PPI chemical biology and set up the screening assays previously mentioned in 4.1.2. The reported co-crystal structure of Keap1-Nrf2 ETGE provides direct guidance for this research. In the two reported structures, the Nrf2 ETGE regions are the 9-mer and 16-mer ETGE-containing peptides, respectively. The structure indicated that the 9-mer peptide (LDEETGEFL) may be a good starting point for the search for the minimal peptide. Two different groups carried out similar research using different evaluation methods. Using a competitive SPR assay, Lonqin Hu’s group found that the 10-mer to 14-mer Nrf2 peptides had similar binding affinities to the Keap1 Kelch domain, whereas the 9-mer Nrf2 peptide had a moderate binding affinity of 352 nM, and Nrf2 peptides < 9 amino acid residues failed to bind to


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the Keap1 Kelch domain.59 In addition, acetylation of the N-terminus could significantly improve the binding affinity to approximately 20 nM (peptides 4 and 5). The binding affinity of fluorescein isothiocyanate (FITC)-labeled Nrf2 peptides also gave similar results, with the FITC9-mer Nrf2 peptide maintaining binding affinity (peptide 1, FITC-LDEETGEFL-NH2), and the FITC-8-mer Nrf2 peptide only showing moderate binding affinity (835 nM). Thus, peptide 1 was chosen as the tracer to construct the FP assay. Using this assay, the authors found that the 8-mer Nrf2 peptide (H-DEETGEFL-OH) also had moderate inhibition activity, which indicated that the 8-mer peptide can interact with Keap1. Geoff Wells’ group used the FP method to obtain an apparent Kd value to determine the minimal peptide. Their results showed that the FITC-labeled 7-mer ETGE-containing peptide (peptide 2, FITC-βAla-DEETGEF-OH) is the shortest peptide that can maintain the binding affinity, which showed an apparent Kd of 95.7 nM. The results seem different from those of Lonqin Hu’s group. However, the FITC-labeled 7-mer peptide contains a β-Ala to act as the linker between the FITC and the N-terminus. This β-Ala has effects similar to that of the leucine at the N-terminus of the 9-mer peptide, and thus, the FITC-labeled 7-mer peptide should be treated as an 8-mer peptide. Therefore, the main difference occurs at the leucine of the C-terminus. In the co-crystal structure, this residue has an obvious interaction with the Keap1 protein, and only its terminal carboxyl acid can form an intramolecular hydrogen bond with the amine at the N-terminus, which may have slight effects on maintaining the peptide conformation. It is reasonable that it can be simplified to obtain the minimal fluorescent peptide tracer for Keap1 binding. Using this tracer, Hancock et al. found that another 7-mer Nrf2 peptide (peptide 6, Ac-DEETGEF-OH) also has moderate peptide inhibition activity, which indicates that this type of 7-mer Nrf2 peptide is the minimal sequence for Keap1 binding.49 Taken together, we can conclude that the 7-mer Nrf2 peptide 6 is the shortest Keap1 binding peptide


26

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that can retain the proper conformation. Moreover, for most Keap1 binding interactions, the FITC-labeled ‘8-mer’ ETGE containing peptide 2 is the simplest fluorescent peptide tracer for the FP assay, and the 9-mer peptide 5 (Ac-LDEETGEFL-OH) is the optimal peptide inhibitor with potent Keap1-Nrf2 inhibition activity. 5.1.2 Structure-based optimization of the Nrf2 ETGE peptide Although the 9-mer peptide showed fair Keap1 binding affinity and Keap1-Nrf2 inhibition activity, its Keap1 binding affinity is still lower than that of the Nrf2 protein. Moreover, the four acidic residues strongly disfavour cellular penetration. Further optimization is necessary to improve the activity, shorten the length of the peptide and replace the unnecessary acidic residues. An SAR study of the Ac-DEETGEF-OH sequence was carried out. The first glutamic acid in the peptide can be optimized, and changing it to alanine improves the IC50 to 0.73 µM. Moreover, inspired by the primary sequence of another Keap1 substrate protein, p62, replacement of glutamic acid with proline further increased the activity to an IC50 of 0.248 µM. The core ETGE motif is highly conserved for the activity, and changing the glutamic acid to glutamine or asparagine is not conducive to activity. Even changing the second glutamic acid in ETGE to aspartic acid can abolish activity, and if only the first glutamic acid in ETGE is replaced by aspartic acid, there is a 3-fold loss in activity. The threonine that can form a hydrogen bond with Keap1 is also sensitive to residue-alterations, and only the serine derivative retains some activity. The phenylalanine on the C-terminal side of ETGE has a high tolerance for residue changes. Replacement with tryptophan, leucine or tyrosine can maintain activity, and leucine is the optimal and can result in the most potent peptide (peptide 7, Ac-DPETGEL-OH) with an IC50 of 0.115 µM in this study.49 In a subsequent study, Wells’ group found that transforming the first aspartic acid to asparagine is also acceptable, though it will induce an 8-


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fold activity loss (peptide 9). Moreover, they found that terminal modifications have dramatic effects on activity. To promote the lipophilicity of the peptide, the C18 fatty acid stearic acid was introduced at the N-terminal. This conjugate (peptide 10) showed 5-fold greater activity (0.022 µM vs. 0.115 µM), whereas the introduction of a benzoyl group had almost no effect on activity. Their research also showed that the C-terminal carboxyl group is important for Keap1-Nrf2 inhibition activity, and changing the carboxyl group to carboxamide caused a 6- to 10-fold decrease in activity (peptides 7 & 8 as an example).99 Our group further investigated this activity loss by molecular dynamics (MD) simulation, and found that the C-terminal carboxyl group can form hydrogen bonds with Arg483, whereas the terminal carboxyl oxygen atom of the peptide only forms much weaker interactions with Asn382. The terminal carboxyl group at this site can form favorable electrostatic interactions with the Arg483 in the P2 sub-pocket, which can strengthen the multiple polar interaction network in the P2 sub-pocket.100 This result indicated that the C-terminal carboxyl group should be retained when this 7-mer residue scaffold is used, but when the growth of the residue occurs at the C-terminal, the terminal carboxyl will be far away from the key arginine. On the basis of Hu’s study,59 a terminal amide analog would be optimal. In addition to explaining this activity change, we also used the systematic MD approach to set up a computational workflow to investigate the Keap1-Nrf2 PPI peptide inhibitors. We found that the terminal leucines residues in the 9-mer Nrf2 peptide contribute to Keap1 binding through favourable hydrophobic interactions in an additive manner, which is also consistent with the positive results obtained by introducing the C18 fatty acid stearic acid at the N-terminal. Based on this result, the Leu residues were added to the optimal 7-mer peptide inhibitor, resulting in a more potent inhibitor (Ac-LDPETGEFL-OH) with an IC50 of 42.6 nM. Further MD simulation of this peptide


28

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indicated that replacing the Phe with Tyr may be beneficial for activity, and the experimental results also proved that this replacement (peptide 11) can further enhance the activity. 5.1.3 Conformational Restriction. During the optimization of ETGE-containing peptide inhibitors, the most dramatic finding was that the Pro replacement significantly improved the activity of the peptide described above. Although Pro has limited effects on the formation of interactions, it has proven helpful for mimicking the β-hairpin secondary conformation for long periods of time.101,

102

Our

computational results stressed this concept. MD simulation results have shown that Pro replacement can significantly stabilize the β-hairpin conformation and enhance the hydrogen bond network formed by the key glutamates in the ETGE motif.100 The dramatic effect of Pro in stabilizing the peptide conformation further indicates conformational restriction may be a valuable strategy in PPI inhibitor design. Terminal cyclization is another conformational restriction method. In addition to the remarkable effects on conformation locking, cyclic peptides also have enhanced stability against both exo- and endo-proteases, which significantly strengthens the therapeutic potential of this class of peptides. The secondary structure of the ETGE region is a typical β-hairpin, which consists of two antiparallel β-strands connected by a turn sequence. Head-to-tail cyclizing or disulfide bridges can be used to develop conformationally stable cyclic peptides.103 Previously, based on optimized Keap1-Nrf2 peptide inhibitors, we introduced a terminal disulfide linkage to stabilize the peptide, resulting in the most potent peptide inhibitor (peptide 12, Acc[CLDPETGEYLC]-OH) with a Kd value of 10.4 nM for binding to Keap1 in the ITC assay and an IC50 value of 9.4 nM in the FP assay.62 This result proved that terminal cyclization is quite useful in the design of Keap1-Nrf2 PPI inhibitors. Another head-to-tail cyclized Nrf2-derived


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peptide (cycloGDEETGE) has been reported, and its structure in complex with the Keap1 Kelch domain is also available in the PDB.104 Although the activity of this peptide is still unknown, it provides valuable insight for further development of peptide inhibitors. 5.1.4 Cellular activity of peptide inhibitors Multiple acidic residues are one of the most distinguishing features of the Nrf2 ETGE peptide. Though these acidic residues have important roles in both conformation stabilization and Keap1 binding, they strongly disfavor cell membrane permeability. Thus, reducing the number of acid residues and enhancing lipophilicity are the main goals in improving the cellular activity of peptide inhibitors. Hancock et al. demonstrated that the introduction of a lipophilic stearic acid group can significantly improve cellular activity. However, restricted by the key roles of the Glu residues in Keap1 binding, the net charge of the peptide can hardly be higher than -2. Thus, the cellular activity is still limited. Directly conjugating a cell-penetrating peptide (CPP) to the primary sequence is a more direct way to develop a cell-penetrating probe to investigate PPI inhibition effects. Steel et al. used a trans-activating transcriptional activator (TAT) peptide that was derived from HIV as the CPP to construct cell-penetrating Nrf2 ETGE peptides.105 The resulting activated peptide (peptide 13) can elevate the protein level of Nrf2 and up-regulate its downstream target gene HO-1 at both the mRNA and protein levels in a dose-dependent manner in intact human THP-1 monocytes. Moreover, this research also proved that this Keap1-Nrf2 peptide inhibitor can also inhibit production of the pro-inflammatory cytokine TNF, which indicates the potential antiinflammatory uses of Keap1-Nrf2 PPI inhibitors. Jing et al. also used the same strategy to construct a similar peptide but found that the peptide did not work in brain-injured mice. The authors further introduced a calpain cleavage sequence


30

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(PLFAER) between the TAT sequence and the Nrf2 ETGE motif, which makes the peptide sensitive to Ca2+ increases and allows injury-specific activation of Nrf2. This new peptide (peptide 14) significantly increased the mRNA levels of Nrf2-regulated genes. Moreover, it attenuated blood–brain barrier (BBB) compromise following traumatic brain injury (TBI), which is beneficial for reducing neurovascular dysfunction in the injured brain.106 This research not only indicated the potential therapeutic application of the Keap1-Nrf2 PPI inhibitor but also provided an enlightening thought for the design of target-activated Nrf2 activators. A very recent study further confirmed that this peptide induced Nrf2-regulated cytoprotective genes, reduced oxidative stress, and induced strong neuroprotection and marked preservation of hippocampaldependent cognitive function after global cerebral ischemia (GCI) in an injury-specific manner.107 This study further confirmed Keap1-Nrf2 PPI inhibitors as a potentially promising new therapeutic modality for the treatment of GCI. 5.1.5 Searching for novel peptide sequences Although these peptide antagonists are based on the primary sequences of the natural binding partner, display methods, in particular the phage display method, are good sources for the discovery of antagonists with novel binding sequences.108 Brian Kuhlman et al. used a phage display combined with a computational loop grafting protocol to discover a engineered monobody that is a potent competitive inhibitor of the Keap1-Nrf2 PPI and binds to Keap1 with a Kd of 300 pM.109 However, this study could not find a novel Keap1 binding sequence, and the core binding sequence was RDEETGEFHWP, which also included the primary Nrf2 sequence DEETGEF. Geoff Wells’ group also set up a phage display library approach to discover peptide ligands with non-native sequence motifs, but only weak peptides were discovered.49 The hurdles


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in discovering novel Keap1-Nrf2 peptide inhibitors further confirmed the conservative nature of the core ETGE motif. Taken together, the development of Keap1-Nrf2 peptide inhibitors has made considerable progress. This research has confirmed that this PPI can be disrupted by artificial compounds and that directly disrupting Keap1-Nrf2 PPI could be an effective way to activate Nrf2. Moreover, the potential uses of Keap1-Nrf2 inhibitors as anti-inflammatory and organ protective agents have also been validated. The SAR study of the peptides also noted that the P1 and P2 subpockets occupied by the two Glu residues are hot-spots, which can give direct guidance to the design of small molecules.

Table 2. Representative Keap1-Nrf2 PPI peptide inhibitors. Keap1-Nrf2 Keap1 Inhibition No.

binding

Sequence

ref

activity (FP affinity/Kd assay) /IC50 Peptide 1

FITC-LDEETGEFL-NH2

Peptide 2

FITC-βAla-DEETGEF-OH

25.6 nM Fluorescent

LQLDEETGEFLPIQGK(MR121)-OH

Peptide 4

LDEETGEFL-NH2

95.7 nM 49

tracer Peptide 3

71

3570 nMa

(Kdapparent) Not reported

77

355 nM

59, 71


32

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Peptide 5

Ac-LDEETGEFL-NH2

194 nMa

Peptide 6

Ac-DEETGEF-OH

5390 nMb

21.4 nM

49

Peptide 7

Ac-DPETGEL-OH

115 nMb

Peptide 8

Ac-DPETGEL-NH2

634 nMb

Peptide 9

Ac-NPETGEL-OH

NA

875 nMb

Peptide 10

St-DPETGEL-OH

22 nMb

Peptide 11

Ac-LDPETGEYL-OH

29.6 nMa

99

46.5 nM 62

9.45 nMa

Peptide 12

Ac-c[CLDPETGEYLC]-OH

10.4 nM

Peptide 13

YGRKKRRQRRRLQLDEETGEFLPIQ

Cell penetrating

RKKRRQRRR-PLFAER-LDEETGEFLP-

Cell penetrating and target

105

107

Peptide 14 NH2

activation

Fluorescent tracers used in FP assays: aFITC-LDEETGEFL-NH2; bFITC-βAla-DEETGEFOH. 5.2 Small-molecule Keap1-Nrf2 inhibitors Compared with the discovery of peptide inhibitors, finding small-molecule Keap1-Nrf2 PPI inhibitors is more challenging. With the help of progress in both screening methods and peptide inhibitors, a number of small-molecule inhibitors have been identified.96,

97

Representative

small-molecule Keap1-Nrf2 PPI inhibitors are listed in Table 4.


33


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Figure

5.

Discovery

and

optimization

of

Keap1-Nrf2

Page 34 of 88

inhibitors

with

a

THIQ

(tetrahydroisoquinoline) scaffold. (A) The development process of the Keap1-Nrf2 inhibitors with THIQ scaffold. (B) The binding mode differences between the hit and optimal compound. Key modifications are labeled with yellow dashed lines. Hydrogen bonds are represented by green dashed lines, and electrostatic interactions are represented by yellow dashed lines. The carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively. Key water molecules are represented by red dots. 5.2.1 Experimental HTS of hit compounds


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The first hits targeting Keap1-Nrf2 PPI were obtained from HTS. Hu et al. used the FP assay to screen the NIH MLPCN library for small molecules (containing approximately 330,000 compounds) and identified a small molecule hit, 1, as a first-in-class direct small-molecule inhibitor of Keap1-Nrf2 PPI.72 Because this compound contains three chiral centers, the authors further investigated the effects of chirality on activity through a combination of flash and chiral chromatographic separation. They finally obtained one stereoisomer, which is predominantly responsible for Keap1 binding activity (Kd = 1 µM), and the stereochemistry of this stereoisomer 2 was determined by the X-ray crystallography. This compound also showed fair activity in a cellular ARE gene reporter assay and Nrf2 nuclear translocation assay, with an EC50 of 18 µM and 12 µM, respectively. Another group from Biogen Idec conducted HTS of the Evotec Lead Discovery library (Evotec, Hamburg, Germany) using a 2D-FIDA assay. They obtained 18 active hit compounds, which can be divided into two subclasses based on the chemical structures, specifically the N-phenylbenzenesulfonamide class and the benzenesulfonyl-pyrimidone class. The representative compounds 4 and 5, with an IC50 of 118 µM and 2.7 µM, respectively, were chosen to carry out the following hits validation process. The NMR and native mass spectrometry results together confirmed the direct interaction between the Keap1 DC domain and the small molecules. Moreover, this group determined the co-crystal structures of the two inhibitors with the Keap1 DC domain, providing useful information for structure-based design. The cellular AREluciferase reporter assay was also used to measure the action of Nrf2, and only compound 5 upregulated Nrf2 response genes. Western blot measuring Nrf2 stabilization also provided the similar results which could be due to the extremely weak Keap1-Nrf2 PPI inhibition activity of 4.


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5.2.2 Virtual screening of Keap1-Nrf2 PPI inhibitors In addition to HTS, virtual screening (VS) is another commonly used hit screening method with high efficiency and low costs. In a previous study, we reported the first hierarchical structure-based virtual screening utilizing a receptor-ligand binding model of Keap1-Nrf2.73 In that study, considering that the ETGE motif displays multiple acidic residues that form strong electrostatic interactions with Keap1, the database was pre-filtered using the criteria that the molecules should have a calculated formal charge ≤ 1. The co-crystal structure of the Keap1Nrf2 ETGE peptide was used to construct the pharmacophore. Using this pharmacophore together with the molecular docking method, we screened the SPECS database (SPECS, Zoetermeer, Netherlands) and obtained an active hit with an EC50 of 9.80 µM in the FP assay. This compound also elevated Nrf2 transcription activity in the cell-based ARE-luciferase reporter assays in a concentration-dependent manner. Zhuang et al. also carried out a VS of the SPECS database using the cascade docking method integrated in Schrodinger’s Glide module (Schrodinger, New York, NY). The previously reported active hit 5 was chosen as the control, and only those compounds that performed better than 5 were retained. Maximum chemical diversity was used as the criteria to select compounds, and clustering based on the calculated Tanimoto coefficient using the 2D fingerprint110 was carried out to obtain the final purchasing list. The authors obtained nine compounds representing three chemotypes of Keap1-Nrf2 PPI inhibitors, which possessed good inhibitory activity with KD2 values (the equilibrium dissociation constants of test compounds)69 ranging from 2.9 to 75.48 µM (Figure 6). Then, the authors used the hit-based substructure search method to investigate the preliminary SAR of the new scaffold. Among these compounds, compound 14 is the most potent, and it also contains a naphthalene sulfonamide scaffold.


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Figure 6. Active hits from virtual screening. 5.2.3 Structure-based SAR and optimization of hit compounds The emergence of the pioneer small-molecule inhibitors and co-crystal structures of Keap1small molecule ligands (as shown in Table 3) inspired further SAR and optimization work around these hits. Currently, the activity of inhibitors can reach the nanomolar range, and the SARs of certain scaffolds have been clarified. Tetrahydroisoquinolines After the discovery of hit compound 1, Hu’s group conducted a preliminary SAR study. Their research indicated that the acidic functionality of the cycloalkane ring is important for Keap1 binding, and the amide or ester analog can induce significant loss of or even abolish activity. The aryl ring on the tetrahydroisoquinoline (THIQ) scaffold is also indispensable, and the one-carbon linker between THIQ and the phthalimido group is naturally optimal. A further optimization may be a phthalimido group that can tolerate simplification.


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This compound has also attracted the interests of UCB Pharma. The company determined the co-crystal structure of the Keap1-compound 2 complex, which clarified the binding mode.111 Based on this co-crystal structure, they first conducted a detailed SAR study of the cyclohexane carboxylic acid. Both the cyclohexane and cyclopentane were optimal, and further reducing the size of the ring harmed the activity. Removal of the carboxylic acid or its replacement by carboxamide or nitrile totally abolished the activity, and only the tetrazole analog was active with a small decrease in potency. Introducing a polar nitrogen atom in the cyclohexane significantly reduced the activity. These experiments together indicate that the cyclohexane carboxylic acid is a privileged fragment at this site. The phthalimido group was also investigated by this group, and the research results were consistent with the previous study. One of the carbonyls can be moved, but the amide group is necessary. The removal of the phenyl ring in the phthalimido group was also feasible, and had no significant effect on potency. There were also some positive findings. Methylation of the 5-position of THIQ further improved activity, leading to the most potent compound 3 in this study (IC50 = 0.75 µM). This research group also investigated the physicochemical and DMPK properties, and they found that compound 2 is a Pgp substrate and restricted to the peripheral compartment in mice, whereas the isoindole analog 3, with reducing PSA, exhibited a trend toward decreased efflux. These results provide guidance for improving drug-like properties and brain availability. Naphthalene sulfonamides Another active hit 5, which was reported by Biogen Idec, attracted our research interest. The co-crystal structure of this compound shows that it adopts a unique binding mode that is quite different from the Nrf2 ETGE motif (Figure 7). The naphthalene ring is inserted into the central cavity, and the two side-chain aryl rings occupy the P4 and P5 sub-pockets. These aromatic rings


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can form multiple cation-pi and pi-pi interactions with the side-chains of Tyr334, Tyr572, Tyr525 and Arg415, which cannot be observed in the Keap1-Nrf2 ETGE structures. With the help of these hydrophobic interactions, compound 5 can effectively compete with the Nrf2 ETGE peptide, with an EC50 of 2.7 µM, without the tight polar interactions in the P1 and P2 polar sub-pockets. However, in our computational analysis, the electrostatic interactions with the polar residues in the P1 and P2 sub-pockets, especially the key arginines, are pivotal for Keap1 binding. Accordingly, we introduced a polar recognition group, a di-acetic moiety, to mimic the binding mode of the ETGE motif. Based on the crystal structure of the Keap1-5 complex, the nitrogen atom of the sulfonamide was chosen as the linking site, resulting in a potent Keap1-Nrf2 PPI inhibitor 6, with a Kd of 9.91 nM to Keap1 and an IC50 of 28.6 nM in the FP assay. This is the first nanomolar small-molecule Keap1-Nrf2 PPI inhibitor, which confirms that Keap1-Nrf2 PPI can be effectively regulated by small molecules. Functional affinity-based small-molecule probes of Keap1 have been developed based on compound 6.112 We then further optimized the substituents on the side-chain aryl rings and found that p-acetamido substituents (compound 8) were the best choice for balancing PPI inhibition activity, physicochemical properties, and cellular Nrf2 activity.74 In addition, in this study, we also found that the core naphthalene ring plays an important role in Keap1 binding, and simplifying the naphthalene to a phenyl ring causes an approximately 50-fold decrease in activity. Recently, exploration of bioisosteric replacements of the diacetic moiety afforded the ditetrazole analog, which maintains the potent PPI inhibition activity and improves the cellular potency.113 Moore et al. also investigated the structural requirements of naphthalene sulfonamide-based Keap1-Nrf2 inhibitors.114 Their study showed that the 1, 4-disubstitution pattern of the naphthalene scaffold is important for Keap1Nrf2 PPI inhibition, and a singly substituted acetic acid analog can maintain most of the activity.


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Moreover, the di-amide analog 9 also showed good potency, and its X-ray structure with Keap1 fully confirmed the binding pattern, especially the water displacement effects. In this study, they also found that ethyl ester 7, which showed moderate in vitro potency in the FP and SPR assays, is more potent in cellular assays, exhibiting ex vivo efficacy similar to the well-known electrophilic activator, sulforaphane. These results provide valuable insight into the development of prodrugs for improving drug-like properties.

Figure 7. The development process for the naphthalene sulfonamide class of Keap1-Nrf2 PPI inhibitors. Hit structure 5 was discovered from HTS.77 Hotspot-based design provided the first nanomolar small-molecule inhibitor 6.56 SAR and optimization further enhanced the cell activity and solubility.74, 114


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Very recently, Schmoll et al. also reported a novel Keap1-Nrf2 PPI inhibitor (10, RA839)115, which contains a naphthalene sulfonamide scaffold. This compound also has a 1, 4-disubstitution pattern for the naphthalene scaffold but just one sulfonamide group. The other substituent is the pyrrolidine-3-carboxylic acid, which can form key polar interactions with Arg483 in the crystal structure. This compound exhibited a Kd of 6 µM to the Keap1 kelch domain in the ITC experiment, partly because it cannot fully occupy the Keap1 binding cavity.

Figure 8. Binding mode of the naphthalene sulfonamide class of the Keap1-Nrf2 PPI inhibitors. (A) The binding mode of 5 (PDB code: 4IQK); (B) The binding mode of 9 (PDB code: 4XMB); (C) The binding mode of 10 (PDB code: 5CGJ). Hydrogen bonds are represented by green dashed lines, the electrostatic interactions are represented by yellow dashed lines and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively. Key water molecules are represented by red dots.

Other structure classes Geoff Wells’ group reported a series of 1,4-diphenyl-1,2,3-triazole compounds that inhibit Keap1-Nrf2 PPI (Figure 9A).75 The design of this series of compounds was inspired by docking-


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based design. The two functionalized phenyl rings can mimic the side-chain of the key Glu residues, while the triazole ring acts as the scaffold, which can be easily synthesized. The SAR study showed that the benzoic acid moiety was optimal in the in vitro FP assay but not preferable in the cell-based NQO1 assays (compound 18). The nitro-substituted compounds showed more potent cell-based activity, and a meta-nitro group on the 4-phenyl ring and a meta-nitro, methyl, or halogen on the 1-phenyl unit are the best combinations (compound 15-17).75 The nitro group provided an alternative choice for the polar recognition group that can be further investigated. Recently, our group reported a series of Nrf2 activators with similar overall shape but with a 1,2,4-oxadiazole core (e.g. 19B).116 However, these compounds did not show Keap1-Nrf2 PPI inhibition activity, which further indicates that the Keap1 cavity is strictly specific with substrate structures.

Figure 9. The discovery of 1, 4-diphenyl-1, 2, 3-triazole compounds as Keap1-Nrf2 PPI inhibitor.


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In another study, Mochizuki et al. reported another Nrf2 activator 20 (Figure 10), NK-252, which can interact with the Keap1 DC domain.117 This compound may be a Keap1-Nrf2 PPI inhibitor, but a detailed study was absent. This scaffold has also recently been reported by Kunishima’s group. However, their ligands contain an acidic group that can interact with arginine. In addition, their study found that the small molecule could adopt two different binding modes with the Keap1 DC domain in the soaking and cocrystallization forms (Figure 10 B & C). In the cocrystallization form, the 2-phenoxyacetic acid moiety is deeply inserted into the P2 subpocket and forms multiple hydrogen bonds with Ser363, Arg380 and Asn382. In this binding, the phenyl ring can form two pi-pi interactions with Tyr572 and Tyr334 simultaneously, while the biaryl moiety is located on the outside of the central cavity and interacts with Arg336. The soaking experiment revealed a distinct binding mode whereby the molecule is approximately located on the central hole of Keap1. The phenyl ring occupies the P3 sub-pocket in a manner similar to the naphthalene ring of compound 5. The acetic acid group extends to the P1 subpocket and forms multiple polar interactions with Arg483, Arg415 and Ser508. The carbamide linker and the biaryl moiety occupy the P2 sub-pocket by a novel pattern. The authors also used MD simulation to further investigate the differences between the two forms, and the binding pattern from the cocrystallization form tended to dissociate more easily compared to the structure of the soaking form. Although the Keap1-Nrf2 inhibition activity of this compound was unknown, this study provides a fresh binding pattern with Keap1 that can facilitate the design of novel Keap1-Nrf2 PPI inhibitors.


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Figure 10. Binding mode investigation of the Keap1 ligands with urea fragment. (A) Chemical structures of the Keap1 ligands with urea fragment; (B) Binding mode of 21 with Keap1 in the co-crystallization form (PDB code: 3VNG); (C) Binding mode of 21 with Keap1 in the soaking form (PDB code: 3VNH). Hydrogen bonds are represented by green dashed lines, the electrostatic interactions are represented by yellow dashed lines and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively. Key water molecules are represented by red dots.


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Very recently, researchers from Astex Pharmaceuticals and GlaxoSmithKline Pharmaceuticals identified a series of novel Keap1-Nrf2 PPI inhibitors with high cell potency by a fragmentbased approach.118 At the beginning of their research, a crystallographic screen of a fragment library was carried out, and three fragments were identified that occupied three discrete hot-spots within the Keap1 cavity (as shown in Figure 11). Fragment 1 showed a binding pattern similar to compound 21 in the soaking form. The aliphatic acid of Fragment 1 inserts into sub-pocket P1, forming electrostatic interactions with Arg483 and Arg415, and the core phenyl ring is located in the P3 pocket. Fragment 2 forms a pi-pi interaction with Tyr525 and several hydrogen bonds with Gln530 and Ser555. On the basis of the crystal structure, fragment 1 was chosen as the template, and a benzotriazole moiety similar to fragment 2 was attached directly to the benzylic carbon of the phenyl acetic acid. This modification gave the first hit structure 22, which showed inhibition activity in the FP assay. The crystal structure also proved that 22 retained the Keap1binding interactions found in fragments 1 and 2. Moreover, the chlorophenyl ring of 22 provided the site for the introduction of privileged fragments. The benzenesulfonamide was linked to the 3-position of the chlorophenyl ring of 22 by using the methylene group as the linker, which gave a good hit 23, with an IC50 of 0.27 µM in the FP assay. This hit structure showed some outstanding characteristics, including the following two anchoring characteristics: it forms hydrogen bonds with Ser555 and Ser602 in the same manner as the Nrf2 ETGE peptide, and the core phenyl ring inserts into the P3 sub-pocket in a manner similar to the naphthalene sulfonamide inhibitors. Further structure-based optimization gave the nanomolar inhibitor 24, which contains a quite ingenious seven-member heterocyclic structure. This inhibitor further locks the binding conformation of the benzsulfamide, and the extra methyl group in the ring can


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form favourable hydrophobic interactions with Tyr572. This compound also showed good potency in activating the Nrf2 antioxidant response in cellular and in vivo models.

Figure 11. Fragment-based discovery of novel Keap1-Nrf2 PPI inhibitors with high cell potency. Three distinct fragments were identified through a crystallographic screen of a fragment library (Fragment 1: PDB code 5FNQ; Fragment 2: PDB code 5FZJ and Fragment 3: PDB code 5FZN). The first hit in the fragment-to-hit process, 22, was designed based on the binding mode of fragments 1 and 2 (PDB code: 5FNR). The structure of fragment 3 was integrated into 22, resulting in a good hit 23 (PDB code: 5FNT). Further structure-based optimization gave the


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nanomolar inhibitor 24, with a quite ingenious seven-member heterocyclic structure (PDB code: 5FNU). Hydrogen bonds are represented by green dashed lines, the electrostatic interactions are represented by yellow dashed lines and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively. Key water molecules are represented by red dots.

Table 3. Summary of the available crystal structures of Keap1 and Keap1 with ligands. Keap1

Ligand

PDB code

ref

1U6D

119

1ZGK

120

1X2J

121

mKeap1-DC domain; residues 309-624 mKeap1-DC domain; residues 309-624 mKeap1-DC domain; residues 309-624 hKeap1 DC domain; residues 4IFJ 321-609 hKeap1 BTB domain; residues 4CXI 48-180 41

hKeap1 BTB domain C151W 4CXJ mutant; residues 48-180


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Page 48 of 88

Keap1 BTB domain C151W CDDO

4CXJ

Nrf2/Neh2 DLGex; residues 17-51

3WN7

50

Nrf2/Neh2 ETGE; residues 76-84

1X2R

121


4IFL


2FLU

122

Nrf2/Neh2 DLG; residues 24-29

2DYH

44

Prothymosin α Residues 39-54

2Z32

123

p62 peptide residues 346-359

3ADE

124

3WDZ

125

3VNG

64

mutant; residues 48-180 hKeap1 DC domain; residues 321-609 mKeap1-DC domain; residues 309-624 hKeap1 DC domain; residues 321-609 hKeap1 DC domain; residues 325-609 mKeap1-DC domain mKeap1-DC domain; residues 309-607 mKeap1-DC domain; residues 309-624 mKeap1-DC domain; residues phosphorylated p62 residues 346309-624

359

hKeap1 DC domain; residues 21 321-609


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hKeap1 DC domain; residues 3VNH 321-609 mKeap1 DC domain; residues 10 (RA389)

5CGJ

115

9

4XMB

114

309-624 hKeap1 DC domain; residues 321-609 hKeap1 DC domain; residues Keap1-Nrf2 inhibitors with THIQ 4L7B, 4L7C, 111

321-611

scaffold

4L7D, 4N1B

2

4IFN

hKeap1 DC domain; residues 321-609 hKeap1 DC domain; residues 321-609;

Mutation:

E540A, cycloGDEETGE

3ZGC

E542A 104

hKeap1 DC domain; residues 321-609;

Mutation:

E540A,

3ZGD

E542A hKeap1 DC domain; residues 4

4IN4

321-609 77

hKeap1 DC domain; residues 5

4IQK

321-609


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5FNQ, 5FNR, 5FNS, mKeap1-DC domain; residues Fragments and hits used in the 118

5FNT, 309-607

design of 24 5FNU, 5FZJ, 5FZN

Table 4 Representative small-molecule Keap1-Nrf2 PPI inhibitors. Scaffold

Compound

Tetrahydroisoquinolines

1

(THIQ)

Keap1-Nrf2 inhibition activity

Method identification

of

ref

Kd = 1900 nM

HTS

72

111

(FP assay)

Structure-based SAR and optimization

IC50 = 1460 nM

HTS

126

Structure-based SAR and optimization

74

(competitive SPR assay) 3

Naphthalene sulfonamides

Chemical structure

5

IC50 = 750 nM

(FP assay)

8

IC50 = 14.4 nM (FP assay)


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14

KD2 = 2900 nM

Virtual screening

110

118

(FP assay)

Other structure classes

21

unknown

HTS and Structure-based SAR and optimization

24

IC50 = 15 nM

Fragment-based approach and structure-based optimization

(FP assay)

6. Privileged structures and key interactions for the Keap1-Nrf2 PPI inhibitors Both the experimental mutation experiments and the computational binding energy results together confirmed the central role of the P1 and P2 sub-pockets in Keap1 binding; thus, these two cavities can be regarded as the ‘hot-spots’ of Keap1-Nrf2 PPI. The Nrf2 ETGE motif used two glutamic acid residues to occupy the hot-spots, whereas in the Nrf2 DLGex motif, two aspartic acid residues replaced the glutamic acid residues. This information indicated that the aliphatic carboxylic acid was the natively privileged structure for the hot-spots, and it has been successfully used in the optimization of naphthalene sulfonamide inhibitors by our group. Moreover, many other Keap1-Nrf2 PPI inhibitors also contains aliphatic carboxylic acid fragments, including cyclopentanecarboxylic acid, cyclohexanecarboxylic acid, 2-oxyacetic acid and 2-thioacetic acid. Another point that should be mentioned is that the carboxyl acid group is quite beneficial for in vitro Keap1-Nrf2 inhibition activity but may be disadvantageous for the pharmacokinetic properties, especially for brain exposure. Recent studies have proven that monoacidic inhibitors also show good potency,118 which may benefit further development.


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Furthermore, both bioisosteric replacements and prodrug forms of carboxyl acid can work at the cellular level, which may provide alternative ways to improve ADME behaviors. In contrast, the high polarity and acidity of the carboxyl group may also have benefits in the development of gastrointestinal drugs, especially drugs targeting the colon. The carboxy group can prevent the absorption of drugs in the stomach and small intestine and thereby make drugs reach the colon. Olsalazine is an example of this type of colon-targeting drug. The P3 sub-pocket is located in the central part of the PPI interface and provides the anchoring site for the scaffold. The peptide backbone is located in this cavity and forms several key hydrogen bonds with key serine residues (Ser555 and Ser602) to stabilize binding.63 However, established potent small-molecule inhibitors have a unique way of utilizing this site. Most of the inhibitors have an aromatic ring in this site that can form cation-pi interactions with the guanidine group of Arg415 and hydrophobic interactions with the side carbon chain of Arg415 and the side chain of Ala556. In addition to the favorable interactions, the aromatic ring can provide multiple sites for the introduction of functional fragments that occupy the other four sub-pockets. The next step in optimizing the scaffold structure may be the combination of the currently used aromatic ring and the key hydrogen bonds utilized by the Nrf2 ETGE motif, which has been explored in a very recent study118. The P4 and P5 sub-pockets show a high tolerance for chemical groups. The residues that naturally occupy these two pockets are Leu and Phe. In the development of small-molecule inhibitors, diverse aromatic rings have been identified as the optimal pharmacophore. Aromatic rings can form pi-pi interactions with Tyr525, Tyr334, Phe577 and Tyr572, and the aromatic rings can be appropriate sites for diverse substituents that can further tighten the binding. Moreover, the outer surface of the P4 and P5 sub-pockets has not yet been explored with small


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molecules and may be useful in subsequent inhibitor design studies. The P6 sub-pocket, induced by the DLGex motif, also provides new opportunities for small molecules, but whether it can be used by small molecules is still unknown. 7. Future Directions and Conclusions To date, considerable progress has been made in the discovery and development of Keap1Nrf2 PPI inhibitors. The potency of inhibitors now reaches single-digit nanomolar values in vitro, and the in vivo Nrf2 activation effects have been proven. Several different types of potent Keap1Nrf2 PPI inhibitors, including cell-penetrating peptides and several potent small molecules with different chemotypes, are available for further exploration of the therapeutic potential of Keap1Nrf2 PPI inhibition. However, there are some open questions for Keap1-Nrf2 PPI inhibitors. The first is whether the inhibitors disrupt the Keap1-Nrf2 ETGE and/or Keap1-Nrf2 DLGex interaction. The existing results have proven that Nrf2 ubiquitination only occurs only after twosite binding has been achieved. These results indicate that the inhibition of the Keap1-Nrf2 DLGex interaction is enough to disturb the Nrf2 ubiquitination process. This result has been further confirmed by the conformation cycling model. Moreover, disrupting the Keap1-Nrf2 DLGex interaction has also been utilized by the protein competition regulation of Nrf2 in cells. Among these Keap1 substrates, p62 is well studied, and it binds to Keap1 by the PSTGE motif. The phosphorylation of serine can remarkably enhance the binding affinity to Keap1, but even that affinity is much weaker than that of the Keap1-Nrf2 ETGE motif. However, excess p62, especially phosphorylated p62 can significantly activate Nrf2.124,

125, 128

In addition, p21 can

directly bind the Nrf2 DLG motif and inhibit two-site binding, resulting in Nrf2 activation.129 Current studies of Keap1-Nrf2 inhibitors are mainly focused on the Keap1-Nrf2 ETGE motif, especially the tracer peptides used in in vitro screening assays. However, this interaction is very


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tight and difficult to disturb, which may hinder the identification of hits with low in vitro activity. Moreover, to enhance activity and mimic the ETGE binding mode, acidic and polar groups have been introduced into molecules, which may harm the drug-like properties and ADME behavior. However, the Keap1-Nrf2 DLGex interaction is much weaker and exhibits a fast association and fast dissociation behavior. Moreover, hydrophobic interactions play an important role in Keap1Nrf2 DLGex binding. These characteristics together benefit inhibitor design. Thus, efficiently inhibiting the Keap1-Nrf2 DLGex motif interaction seems to be a possible way to further explore Keap1-Nrf2 inhibitors. Another question is how to make the Keap1-Nrf2 inhibitors selectively target a specific organ. In particular, developing brain-targeting Keap1-Nrf2 inhibitors has attracted the most interest. The brain is very sensitive to changes in redox status; thus, maintaining redox homeostasis in the brain is critical for the prevention of oxidative damage. The neuroprotective effects of Nrf2 have been identified, and the therapeutic potential of Nrf2 activation in neurological diseases has been revealed.130,

131

However, the chemical characteristics of Keap1-Nrf2 inhibitors are not

conducive to brain availability. Reducing the number of polar groups, decreasing PSA and removing unnecessary acidic groups are quite necessary for further optimization of inhibitors. If inhibiting the Keap1-Nrf2 DLGex motif as a Keap1-Nrf2 inhibition strategy works as expected, it would simplify this hurdle to some degree. Using active transport by conjugating small molecules with biologics is an alternative choice, and it has been used in the development of peptide inhibitors targeting brain injury and global cerebral ischemia.106, 107 Targeting activation of Nrf2 is another key point in the development of clinical therapeutics around Nrf2. The doubleedged sword of Nrf2 in cancer has been clarified.15, 132, 133 Elevated Nrf2 activity can accelerate the evolution of cancer, protect cancer cells from stress conditions and strengthen resistance to


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multiple therapies. Thus, over- and unselective activation of Nrf2 may contribute to the initiation and progression of cancer. Positive and negative effects associated with Nrf2 activation in diabetes have also been observed.134 Thus, targeting activation is an urgent need in the further development of Keap1-Nrf2 inhibitors. The key pharmacophore, the carboxyl group, provides benefits in this situation. It can be used to design a targeted prodrug that not only improves the ADME properties but also selectively activates Nrf2. The specific oxidative stress microenvironment, such as the presence of ROS and specific enzymes, can be used to design the prodrug. The huge therapeutic potential of Nrf2 activation has led the drug community to focus on Nrf2 activation. Keap1-Nrf2 PPI inhibition as a selective Nrf2 activation mechanism is now the research hot-spot in this field. To date, potent inhibitors have been developed, but the drug-like properties need to be optimized. Furthermore, the Nrf2 activation effects of the inhibitors have been identified, but the therapeutic applications have not been clarified. It remains to be seen whether the PPI target is druggable.

AUTHOR INFORMATION Corresponding Author * Prof. Qi-Dong You E-mail address: [email protected]; Telephone number: +86-02583271414 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.


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. ACKNOWLEDGMENT This work is 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), Specialized Research Fund for the Doctoral Program of Higher Education, 20130096110002, the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Fundamental Research Funds for the Central Universities, 2016ZPY016 and the Natural Science Foundation of Jiangsu Province of China (No. BK20160746). ABBREVIATIONS PPIs, protein-protein interactions; Nrf2, nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein; DC domain, DGR (double glycine repeat) and CTR (Cterminal region) domain; Maf, musculoaponeurotic fibrosarcoma; ITC, Isothermal Titration Calorimetry; FP, fluorescence polarization; SPR, Surface Plasmon Resonance; BLI, Bio-Layer Interferometry; DSF, differential scanning fluorimetry; HTS, high throughput screening; ARE, antioxidant

response element; NQO1, NAD(P)H:quinone oxidoreductase-1; HO-1, heme-

oxygenase-1; EFC, enzyme fragment complementation; FITC, fluorescein isothiocyanate; MD, molecular dynamics; BBB, blood–brain barrier; 2D-FIDA, the two-dimentional fluorescence intensity distribution analysis; FRET, fluorescence resonance energy transfer; RunX2, runtrelated transcription factor 2; CPP, cell penetrating peptide; TAT, trans-activating transcriptional activator; GCI, globalcerebral ischemia; THIQ, tetrahydroisoquinoline.


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BIOGRAPHIES Zhengyu Jiang received his Bachelor’s degree from China Pharmaceutical University, where he also obtained his Ph.D. in Medicinal Chemistry under the supervision of Professor Qidong You. He is currently a member of the Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University. He works as a medicinal chemist and his main research topics focus on the following: (i) rational design of protein-protein interaction inhibitors, (ii) the study of Keap1 related interactome and (iii) chemical biology study of oxidative stress.

Mengchen Lu earned her Bachelor of Science degree in medicinal chemistry from China Pharmaceutical University. Since 2014 she has been Ph.D. student in medicinal chemistry at China Pharmaceutical University under the supervision of Professor Qidong You. Her research interests lie in the discovery and chemical biology study of the Keap1-Nrf2 protein–protein interactions inhibitors.

Qidong You obtained his Bachelor’s degree from the Nanjing College of Pharmacy, China in 1982 and his Ph.D. in Medicinal Chemistry from Shanghai Institute of Pharmaceutical Industry in 1989. He did his research in medicinal chemistry as a visiting-scholar at Department of Pharmaceutical Sciences, Strathclyde University, Glasgow, UK between 1994 and 1995. Currently, he is a Professor of Medicinal Chemistry in the Department of Pharmacy, China Pharmaceutical University and the director of Jiang Su Key Laboratory of Drug Design and Optimization. His research interests mainly focus on small-molecule inhibitors of protein– protein interactions, anti-tumor natural products and targeted anticancer drugs.


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Figure 1. Schematic diagram of the regulation of Nrf2 activity. 177x98mm (300 x 300 DPI)



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Figure 2. Conformation cycling model of Keap1-Nrf2 regulation. 177x145mm (300 x 300 DPI)


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Figure 3. Sub-pocket analysis of Keap1 substrate binding cavity. Based on the Keap1-Nrf2 crystal structure, the Keap1 substrate binding cavity can be divided into six sub-pockets. (A) Sub-pocket analysis of Keap1 substrate binding cavity based on the Keap1-Nrf2 ETGE complex (PDB code: 1X2R); (B) Sub-pocket analysis of Keap1 substrate binding cavity based on the Keap1-Nrf2 DLGex complex (PDB code: 3WN7); (C) Summary of key residues in each sub-pocket. 177x49mm (300 x 300 DPI)



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Figure 4. Co-crystal structure of Keap1-Nrf2 PPI. (A) & (D) Binding surface and key polar residue interactions of the Keap1-Nrf2 ETGE motif (PDB code: 1X2R); (B) & (E) Binding surface and key polar residue interactions of the Keap1-Nrf2 DLG motif (PDB code: 2DYH); (C) & (F) Binding surface and key polar residue interactions of the Keap1-Nrf2 DLGex motif (PDB code: 3WN7). Hydrogen bonds are represented by green dashed lines, and electrostatic interactions are represented by yellow dashed lines. The carbon atoms of Nrf2 residues and Keap1 residues are colored cyan and purple, respectively. 177x105mm (300 x 300 DPI)


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Figure 5. Discovery and optimization of Keap1-Nrf2 inhibitors with a THIQ (tetrahydroisoquinoline) scaffold. (A) The development process of the Keap1-Nrf2 inhibitors with THIQ scaffold. (B) The binding mode differences between the hit and optimal compound. Key modifications are labeled with yellow dashed lines. Hydrogen bonds are represented by green dashed lines, and electrostatic interactions are represented by yellow dashed lines. The carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively. Key water molecules are represented by red dots. 177x143mm (300 x 300 DPI)



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Figure 6. Active hits from virtual screening. 177x70mm (300 x 300 DPI)


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Figure 7. The development process for the naphthalene sulfonamide class of Keap1-Nrf2 PPI inhibitors. Hit structure 5 was discovered from HTS.77 Hotspot-based design provided the first nanomolar small-molecule inhibitor 6.56 SAR and optimization further enhanced the cell activity and solubility.74, 114 177x111mm (300 x 300 DPI)



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Figure 8. Binding mode of the naphthalene sulfonamide class of the Keap1-Nrf2 PPI inhibitors. (A) The binding mode of 5 (PDB code: 4IQK); (B) The binding mode of 9 (PDB code: 4XMB); (C) The binding mode of 10 (PDB code: 5CGJ). Hydrogen bonds are represented by green dashed lines, the electrostatic interactions are represented by yellow dashed lines and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively. Key water molecules are represented by red dots. 177x48mm (300 x 300 DPI)


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Figure 9. The discovery of 1, 4-diphenyl-1, 2, 3-triazole compounds as Keap1-Nrf2 PPI inhibitor. 84x101mm (600 x 600 DPI)



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Figure 10. Binding mode investigation of the Keap1 ligands with urea fragment. (A) Chemical structures of the Keap1 ligands with urea fragment; (B) Binding mode of 21 with Keap1 in the co-crystallization form (PDB code: 3VNG); (C) Binding mode of 21 with Keap1 in the soaking form (PDB code: 3VNH). Hydrogen bonds are represented by green dashed lines, the electrostatic interactions are represented by yellow dashed lines and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively. Key water molecules are represented by red dots. 177x160mm (300 x 300 DPI)


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Figure 11. Fragment-based discovery of novel Keap1-Nrf2 PPI inhibitors with high cell potency. Three distinct fragments were identified through a crystallographic screen of a fragment library (Fragment 1: PDB code 5FNQ; Fragment 2: PDB code 5FZJ and Fragment 3: PDB code 5FZN). The first hit in the fragment-tohit process, 22, was designed based on the binding mode of fragments 1 and 2 (PDB code: 5FNR). The structure of fragment 3 was integrated into 22, resulting in a good hit 23 (PDB code: 5FNT). Further structure-based optimization gave the nanomolar inhibitor 24, with a quite ingenious seven-member heterocyclic structure (PDB code: 5FNU). Hydrogen bonds are represented by green dashed lines, the electrostatic interactions are represented by yellow dashed lines and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively. Key water molecules are represented by red dots. 177x153mm (300 x 300 DPI)



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TOC 82x43mm (300 x 300 DPI)


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Discovery and Development of Kelch-like ECH-Associated Protein 1

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