Non-covalent Small-Molecule Kelch-like ECH-Associated Protein 1

May 11, 2018 - After employment as a scientific assistant, he began his Ph.D. studies ... Kim T. Tran obtained his Bachelor's degree within pharmacy i...
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Perspective Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

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Non-covalent Small-Molecule Kelch-like ECH-Associated Protein 1− Nuclear Factor Erythroid 2‑Related Factor 2 (Keap1−Nrf2) Inhibitors and Their Potential for Targeting Central Nervous System Diseases Jakob S. Pallesen, Kim T. Tran, and Anders Bach* Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark

ABSTRACT: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has a protective effect against oxidative stress and plays a major role in inflammation and central nervous system (CNS) diseases. Inhibition of the protein−protein interaction (PPI) between Nrf2 and its repressor protein, Kelch-like ECH-associated protein 1 (Keap1), leads to translocation of Nrf2 from the cytosol to the nucleus and expression of detoxifying antioxidant enzymes. To date, several non-covalent smallmolecule Keap1−Nrf2 inhibitors have been identified; however, many of them contain carboxylic acids and are rather large in size, which likely prevents or decreases CNS permeability. This Perspective describes current small-molecule Keap1−Nrf2 inhibitors with experimental evidence for the ability to inhibit the Keap1−Nrf2 interaction by binding to Keap1 in a non-covalent manner. Binding data, biostructural studies, and biological activity are summarized for the inhibitors, and their potential as CNS tool compounds is discussed by analyzing physicochemical properties, including CNS multiparameter optimization (MPO) scoring algorithms. Finally, several strategies for identifying CNS-targeting Keap1 inhibitors are described.

1. INTRODUCTION Oxygen, O2, is essential for cellular energy metabolism and living systems, but as a side effect of these metabolic processes, reactive oxygen species (ROS) are produced. Also, ROS play a major role in cancers, inflammatory diseases, and neurodegenerative diseases.1,2 The damaging effect of ROS is attenuated by the antioxidant defense system, consisting of detoxification and antioxidant enzymes such as superoxide dismutase (SOD), catalase, GPx, thioredoxin, HO-1, ferritin, glutathione reductase, NAD(P)H dehydrogenase (quinone) 1 (NQO1), and glutathione S-transferase (GST).3,4 The antioxidant enzymes are regulated through the antioxidant response element (ARE), and nuclear factor erythroid 2-related factor 2 (Nrf2) is one of the major ARE-binding transcription factors.5,6 Studies have confirmed the protective effect of Nrf2 against oxidative stress, which makes upregulation of Nrf2 activity an interesting therapeutic strategy especially against inflammation and central nervous system (CNS) diseases.7−9 Under unstressed conditions, the cellular concentration of Nrf2 remains low because it is negatively regulated by the substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1), © XXXX American Chemical Society

which binds to Nrf2 in the cytosol and targets it for ubiquitination and proteasomal degradation.6 Upon oxidative stress, Keap1 acts as a redox sensor and regulator, as the reactive oxidants modify the sulfhydryl groups on specific Keap1 cysteine residues (i.e., Cys151, Cys257, Cys273, Cys288, and Cys297).8,10,11 This results in a conformational change in Keap1 that perturbs the Keap1−Nrf2 protein−protein interaction (PPI) and prevents ubiquitination. As a result thereof, Nrf2 accumulates and translocates into the nucleus, where it forms a transcription factor complex that binds to the ARE promoter region and induces gene expression of antioxidant enzymes.8,12 Thus, inhibition of the PPI between Keap1 and Nrf2 has been suggested as a potential strategy for therapeutic agents as a means to further boost the antioxidant response against oxidative stress. Nrf2 modulators can be divided into two categories: covalent Keap1 modifiers and non-covalent (peptide or small-molecule) Keap1−Nrf2 PPI inhibitors.13 Dimethyl fumarate (DMF, Received: March 12, 2018 Published: May 11, 2018 A

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Figure 1. Molecular mechanisms of the Keap1−Nrf2−ARE pathway and the hypothesized effect of reversible non-covalent inhibitors targeting Keap1 and thus the Keap1−Nrf2 PPI.

failure;19 however, CDDO-Me is still being investigated in clinical trials of kidney and pulmonary hypertension disorders. In general, it can be difficult to control the reactivity of covalent modifiers, and it is a general concern that such compounds bind unspecifically and thus lead to side effects and reduced modulation of the intended target. Thus, non-covalent inhibitors might provide a more attractive strategy.4,8,20,21 To date, several peptide-based Keap1−Nrf2 inhibitors have been designed and serve as useful research tool compounds,22,23 but low bioavailability and cell permeability limit their use. To overcome the lack of cell permeability, a Tat peptide and a

Tecfidera) is a covalent Keap1 modifier and an approved drug for multiple sclerosis. During absorption, DMF is converted to monomethyl fumarate (MMF), which covalently reacts with Cys151 of Keap1 and results in Nrf2-mediated gene transactivation in brain neurons and glial cells.14,15 Bardoxolone (CDDO) is another example of a potent inducer of the Nrf2 pathway that also covalently, yet reversibly, reacts with Cys151 of Keap1.16−18 The methyl ester of bardoxolone (CDDO-Me) failed a phase 3 clinical trial for the treatment of kidney disease in diabetic patients because it increased the risk of heart B

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ETGE and DLG from the amino acids they contain.9,37,39 Although ETGE binds the Kelch domain significantly more strongly than does DLG (Kd = 19 nM vs 1 μM, respectively), both motifs have been shown to be indispensable for the Keap1-dependent repression of Nrf2. Thus, it is proposed that the two-site binding of Neh2 provides a fine-tuned spatial orientation of the lysine-rich α-helix flanked by the ETGE and DLG motifs that facilitates CRL3-mediated polyubiquitination.37,39,40 In particular, three models have been proposed for the molecular events initiated in the induced state (i.e., the presence of oxidants or electrophiles) that eventually lead to Nrf2 stabilization (Figure 1). The “hinge-and-latch model” proposes that covalent modification of cysteine residues within the Keap1 IVR domain (especially Cys273 and Cys288) perturbs the oligomeric complex conformation in such a way that the low-affinity DLG motif dissociates from the Keap1 Kelch domain, thus disrupting the fixed arrangement of Nrf2 needed for ubiquitination.8,37 The model is weakened by the lack of empirical evidence for DLG dissociation under oxidative assault and by observations that inducers (exemplified by two well-known chemopreventive Nrf2 activators, 5,6dihydrocyclopenta[c]-1,2-dithiole-3(4H)-thione (CPDT) and sulforaphane) have in fact been shown to increase the association between Nrf2 and Keap1 in human and rat bladder cancer cell lines.41,42 The “Cul3-dissociation model” proposes that the ability of Keap1 to assemble into a fully functional CRL3 complex is crucial for regulation of Nrf2 levels in response to inducers; here, covalent modification of cysteine residues in the BTB domain (especially Cys151) has been shown to decrease the association between Keap1 and Cul3 and thus encumber ubiquitination.43 The model is undermined by recent conflicting experimental results showing that Cul3−Keap1 dissociation is not required for immediate Nrf2 stabilization.12,42 The employment of Förster resonance energy transfer (FRET)-based methods has provided new insights into the dynamic nature of Keap1−Nrf2 complexation.12 Under basal conditions, the Keap1−Nrf2 complex appears to alternate in a cyclic fashion between an “open state”, in which Keap1 interacts with only the ETGE motif, and a “closed state”, in which the Keap1 homodimer interacts with both Neh2 motifs. Only the closed state allows polyubiquitination to take place, and the presence of the open state has been suggested to allow further regulatory modes to exist. The “conformation cycling model” proposes that cysteine modifications on Keap1 produce a dysfunctional closed state of the Keap1−Nrf2 complex in which both Neh2 domains are still bound to Keap1 but ubiquitination cannot take place (Figure 1).12,41 Although distinctive, all three models agree that Nrf2 does not dissociate (fully) from Keap1 in the induced state and that increased Nrf2 levels are due to trapping of Keap1 in some kind of unproductive state combined with de novo synthesis of Nrf2. As it is believed that the Keap1−Nrf2 system operates close to saturation, even a slight decrease in functional Keap1 would allow Nrf2 to accumulate.12,41 The precise mechanism(s) of a non-covalent inhibitor of the Keap1−Nrf2 PPI has to our knowledge not yet been elucidated. On the basis of the three models following cysteine modifications on Keap1, we hypothesize that in the context of oxidative stress, such an inhibitor could (1) affect a subpopulation of Keap1−Nrf2 complexes that have not entered

calpain (Cal) cleavage sequence were fused to the Nrf2-derived peptide LDEETGELFLP, resulting in the peptide inhibitor TatCal-DEETGE, which demonstrates Nrf2 activation and antioxidant gene induction in brain-injured mice.24 Nrf2 has been shown to play an important role in CNS diseases. Activation of Nrf2 is found to protect neurons against ischemic and hemorrhagic stroke, traumatic brain injury, and neurodegenerative disorders in animals.8,25−27 These data have been elucidated by genetic knockout studies and inhibition of Keap1−Nrf2 by either covalent modifiers or peptides.4 For example, the covalent modifier acetyl-11-keto-β-boswellic acid reduced infarct volume by 34% when administrated at reperfusion after 2 h of middle cerebral artery occlusion in rats.28 Also, Tat-Cal-DEETGE reduced traumatic brain injuryassociated disruption of the blood−brain barrier (BBB) after intracerebroventricular injection 2 h before injury and when administered directly into the cortex 10 min after injury.29 Additionally, this peptide reduced oxidative stress and neuronal cell death in the hippocampus in a global cerebral ischemia rat model.24 Small-molecule Keap1−Nrf2 PPI inhibitors that enter the brain after peripheral administration have not yet been identified but would be an ideal tool to further investigate the potential of Keap1−Nrf2 inhibition as a drug discovery strategy for modulating CNS diseases. The focus of this Perspective is non-covalent small-molecule Keap1−Nrf2 inhibitors and their potential for targeting CNS diseases. We have included those compounds where experimental evidence for their ability to inhibit the Keap1−Nrf2 interaction by binding to Keap1 in a non-covalent manner has been presented in the literature or patent applications. Binding data, biostructural studies, biological activity, and physicochemical properties will be discussed to elucidate the potential of the published small-molecule inhibitors as tool compounds or lead molecules for studying the Keap1−Nrf2 system in relation to CNS diseases. The CNS multiparameter optimization (CNS MPO) desirability tool combined with a second version (CNS MPO.v2) will guide this discussion,30−33 and various strategies for targeting Keap1 for CNS diseases with small molecules will be described. Also, structural similarity to pan-assay interference compounds (PAINS) 34,35 or molecular properties indicating toxicity or reactivity issues will be analyzed and discussed using the FAF-Drugs4 filtering tool.36

2. MOLECULAR MECHANISMS OF KEAP1−NRF2 INHIBITION Nrf2 consists of six domains, designated Nrf2−ECH homology (Neh) 1−6, of which the N-terminally located Neh2 domain constitutes a negative regulatory domain, as it is responsible for binding to Keap1.6 Keap1 consists of three functional domains: (1) A broad complex, tramtrack, and bric-à-brac (BTB) domain, (2) an intervening region (IVR), and (3) a Kelch domain, also known as the double glycine repeat (DGR) or DC domain (Figure 1).37,38 Under basal conditions, Keap1 self-associates to form a homodimer via its BTB domain, which also constitutes the adaptor site for the assembly of cullin 3 (Cul3), RING-box protein 1 (Rbx1), and ubiquitin-charged E2 enzyme to form the fully functional cullin−RING ligase 3 (CRL3) complex that ubiquitinates Nrf2 (Figure 1).38,39 It is well-established that Keap1 associates with Nrf2 in a two-site molecular recognition fashion: via its two Kelch domains, the Keap1 homodimer interacts with the Neh2 domain of a single Nrf2 protein at two evolutionary conserved binding motifs within Neh2, called C

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Figure 2. Top row: X-ray structures of the Keap1 Kelch domain with truncated Neh2 peptides containing the ETGE (PDB ID 2FLU) or DLG (PDB ID 3WN7) motif. Rows 2−4: X-ray structures of the Kelch domain in complex with ligands (see Table 1 for PDB IDs). For compound 23, the cocrystallized and soaking forms of the ligand have been superimposed. The Keap1 Kelch domain is shown in the same orientation in all pictures.

weaker interaction to DLG would be sufficient to inhibit Nrf2 repression.

the induced state, e.g., by binding to free Keap1 or the openstate complex, in either case hampering the formation of the closed state and reducing the constitutive Nrf2 degradation, or (2) aid in liberating Nrf2 from the induced-state complex, thus directly increasing Nrf2 levels (Figure 1). It is unknown whether such an inhibitor would disrupt the Keap1−Nrf2 ETGE interaction or the Keap1−Nrf2 DLG interaction, or both; however, on the basis of the above models, breaking the

3. THE KEAP1−NRF2 PPI INTERFACE PPIs are generally considered difficult or even “undruggable” as drug targets, as the interaction surface can be very large, shallow, and dispersed. However, certain PPI classes have recently been shown to be suitable for small-molecule D

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Table 1. Published Non-covalent Small-Molecule Keap1−Nrf2 PPI Inhibitors

E

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Table 1. continued

F

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Table 1. continued

a

All data were obtained from ChemAxon or calculated as described by Ertl et al.71 bCNS MPO algorithm.30 cCNS MPO.v2 algorithm.33

pocket to range from difficult to druggable (Dscore = 0.7−1.0 depending on the X-ray structure used). The main challenge appears to lie in the fact that many known ligands rely on interactions of carboxylic acid residues with the arginine hot spot residues to get affinity, which results in polar compounds with low cell permeability and CNS availability. Taking advantage of all of the features of the binding pocket, especially the nonpolar and aromatic residues, is likely key to balancing affinity and pharmacokinetic properties.

disruption, including more globular protein−peptide interactions such as the Keap1−Nrf2 PPI.44 The binding between Keap1 and Nrf2 is confined to the interactions between the six-bladed β-propeller Keap1 Kelch domain and the two Nrf2 motifs, ETGE and DLG, of the intrinsically disordered peptide Neh2 domain (Figure 2, top row).37 X-ray crystallography of truncated Neh2 peptides containing either the ETGE or DLG motif has provided detailed structural information on these interaction surfaces.45−47 Although the buried surface area of the Kelch−DLG interface (∼780 Å2) is significantly larger than that of the Kelch−ETGE interface (∼550 Å2), both are relatively small compared with those of classical PPIs (1000−6000 Å2) and are characterized by the Kelch protein partner adopting a concave binding surface similar to small-molecule binding pockets of traditional targets.44,47 The binding pocket has classically been subdivided into five interconnected epitopes, designated P1− P5, where P1 and P2 mainly contain polar residues (Arg483, Ser508, Ser363, Arg380, Asn382, and Arg415), P4 and P5 contain mostly nonpolar residues (Tyr525, Gln530, Tyr572, Tyr334, and Phe577), and P3 contains small polar and nonpolar residues (Gly509, Ala556, Gly571, Ser602, Ser555, and Gly603) (Figure 2, top row).48 Recently, a hitherto unexplored P6 subpocket on the Kelch surface induced by an extended DLG-containing peptide (DLGex) was observed.47 Although interesting, this subpocket is fairly superficial and disconnected from the other epitopes, which might make it difficult to target with small molecules. Overall, the Kelch binding pocket is quite polar and basic because of the presence of multiple charged arginine residues (i.e., Arg483, Arg415, and Arg380), which make key salt bridges with carboxylic acid residues in both the ETGE and DLG motifs. Importantly, mutation studies, in silico binding analysis, and medicinal chemistry efforts have indicated the crucial importance of occupying P1 and P2, which can thus be considered “hot spots” of the Kelch binding pocket.46,48,49 Taking the size, depth, and polarity of the Keap1 Kelch pocket into account, in silico analysis by SiteMap50 predicts the

4. KEAP1−NRF2 PPI INHIBITORS To date, several non-covalent small-molecule Keap1−Nrf2 inhibitors have been identified, as summarized in Table 1. 4.1. Inhibitors Containing a 1,2,3,4-Tetrahydroisoquinoline Core. Hu et al.51 were the first to report a reversible small-molecule Keap1−Nrf2 inhibitor. Compound 1 (Table 1) was identified by high-throughput screening (HTS) of the commercially available MLPCN library using a homogeneous fluorescence polarization (FP) competition assay measuring the interaction between the Keap1 Kelch domain and a fluorescently labeled Nrf2-derived peptide. The initial HTS hit was a mix of four stereoisomers, and the most promising stereoisomer had an IC50 of 3 μM (FP). After resynthesis and separation of the stereoisomers, 1, also known as LH601A, was shown to be at least 100-fold more potent than the other stereoisomers, with a Kd of 1.0 μM in a surface plasmon resonance (SPR) competition assay. Compound 1 was also active in functional cell assays, where it could induce downstream ARE activation (EC50 of 18 μM) and promote translocation of Nrf2 in the nuclei of human bone osteosarcoma epithelial cells (EC50 of 12 μM).51 A later X-ray structure of the Keap1 Kelch domain cocrystallized with 1 revealed that 1 occupies the polar subpocket P2, the nonpolar subpocket P5, and P3 (Figure 2).52 This information was used to guide further structure− activity relationship (SAR) studies in which 12 analogues were synthesized and used to demonstrate that the phthalimide, the aryl ring of the tetrahydroisoquinoline, and the cycloG

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Figure 3. Structure-based progression of the initial X-ray crystallography fragment hits 14, 15, and 16 (PDB IDs 5FNQ, 5FZJ, and 5FZN, respectively) into 17 (PDB ID 5FNT) and finally to 13.

improved the pKa and cLogD profiles compared with 6 (Table 1). Jain et al.49 generated the diacetamide-substituted analogue of 5, i.e., compound 9, which was more promising than a series of asymmetric analogues with different substituents on the sulfonamide. In an FP assay, compound 9 showed activity in the low nanomolar range (IC50 of 63 nM) without having to resort to carboxylic acid groups (Table 1). The binding mode of 9 was confirmed by X-ray analysis, which showed the location of the amide groups in the two hydrophilic subpockets P1 and P2 (Figure 2).49 The first asymmetric compound of the 1,4-diaminonaphthalene series to be cocrystallized with Keap1 was compound 10, also called RA839 (Figure 2 and Table 1). Compound 10 was identified by screening followed by structural modifications and exhibited an IC50 of 0.14 μM in an FP assay (Table 1).57 The binding mode is similar to that of 5, but instead of occupying the hydrophobic subpocket P4, the carboxylic acid forms a charge-assisted interaction with Arg483 in P1. The cellular activity of 10 was confirmed in various assays, and it significantly increased hepatic mRNA levels of the Nrf2 target genes in mice.57 Saito et al.58 identified compound 11, which is similar to 5, by FP-based HTS of the Drug Discovery Initiative Library (Table 1). Compound 11 inhibits the interaction between Keap1 and a phosphorylated peptide of p62 (p-p62), which is found to bind in the same pocket of the Keap1 Kelch domain as Nrf2 does.58 The inhibitory activity was confirmed by a SAR study of 11 where the compound demonstrated inhibition of Keap1−p-p62 and Keap1−Nrf2 PPIs (IC50 values of 1.5 and 6.2 μM, respectively; Table 1).59 Also, the X-ray structure of the Keap1 Kelch domain cocrystallized with 11 disclosed a similar binding mode as with 5 (Figure 2).58 Compound 11 contains an aliphatic ketone, which is categorized as a proteinreactive electrophile (FAF-Drugs4 analysis), which could give rise to undesirable off-target effects.72 Compound 12 has a unique benzoindole core substituted with a hydroxylamine group and was discovered by HTS of the Drug Discovery Initiative Library using an FP assay.60 Currently, 12 is the most rigid analogue of 5 and also demonstrates an improved Keap1−Nrf2 PPI inhibitory activity (IC50 of 0.20 μM; Table 1). The metabolic stability of 12 in human liver microsomes is more than 8-fold greater than that of 5. A series of N-substituted hydrazide compounds based on 12 was developed and demonstrated similar inhibitory activity and metabolic stability as 12.60 There is a risk that the hydroxamic acid of 12 could undergo acylation and Lossen

hexanecarboxylic acid were all important for the potency of 1. However, a few modifications were allowed, as bioisosteric replacement of the carboxylic acid with a tetrazole led to a 3fold increase in the IC50 of 2 relative to 1 in the FP assay. Interestingly, X-ray structures show that the carboxylic acid of 1 and the tetrazole of 2 are both placed in subpocket P2 and interact with Asn414 (Figure 2). Removal of the phthalimide carbonyl of 1, as in 3, resulted in a 2-fold reduction in the IC50 relative to 1, and further methylation at the 5-position (4) resulted in a similar IC50 value as for 3 (Table 1).52 4.2. Inhibitors Containing a 1,4-Diaminonaphthalene Core. In recent years, Keap1−Nrf2 PPI inhibitors with a 1,4diaminonaphthalene core have attracted a lot of interest. Compound 5 was identified by Marcotte et al.53 using a homogeneous confocal fluorescence anisotropy assay (twodimensional fluorescence intensity distribution analysis (2DFIDA)) to screen the Evotec Lead Discovery Library (267 551 compounds). The symmetric compound 5 showed an IC50 of 2.7 μM in the 2D-FIDA assay and was able to increase Nrf2dependent luciferase reporter activity in cell-based assays (Table 1). The X-ray structure of the Keap1 Kelch domain cocrystallized with 5 (Figure 2) showed that the naphthalene group of 5 occupied subpocket P3 and the anisole groups occupied the two hydrophobic subpockets P4 and P5.53 Crucially, the two important hydrophilic subpockets P1 and P2 were unoccupied. Jiang et al.54 used this information for structure-based optimization and introduced N-acetic acid groups on both sulfonamides of 5 to furnish compound 6. At that time, 6 was the most potent Keap1−Nrf2 inhibitor, with more than a 200fold improvement in potency relative to 5; the Kd of 6 was determined to be 3.59 nM in a biolayer interferometry (BLI) assay and its IC50 to be 28.6 nM by FP (Table 1). This improvement in activity indicates interactions between the carboxylic acids of compound 6 and the important arginine residues in subpockets P1 and P2 (Arg415 and Arg483, respectively), as also suggested by in silico docking.54 In order to improve the physicochemical and drug-like properties of compound 6, further SAR studies were performed. Compound 7, with p-acetamido substituents instead of p-methoxy groups, demonstrated a good balance of activity, physiochemical properties (solubility and cLogD), and cellular Nrf2 activity slightly better than 6.55 Lu et al.56 did a carboxylic acid bioisosteric replacement study of 7 to further optimize the drug-like properties and membrane permeability. Replacing the carboxylic acid moieties with tetrazoles led to compound 8, which retained the activity in the low nanomolar range and H

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18, with an IC50 in the range of 10−100 nM in the FP assay and IC50 < 10 nM in the TR-FRET assay (Table 1).62 In the second phenylpyrazole patent application, approximately 25 compounds with a cycloalkylarylpyrazole core were presented, with compound 19 being one of the most promising compounds, showing high Keap1−Nrf2 inhibitory activity (IC50 in the range of 10−100 nM in both the FP and TRFRET assays) and potency in cells (EC50 of 79 nM) (Table 1).63 4.5. Inhibitors Containing a 1,4-Diphenyl-1,2,3-triazole Core. An in silico screen of 178 000 fragments from the ZINC database formed the starting point for the discovery of the 1,4-diphenyl-1,2,3-triazole-containing compound 20.64 On the basis of their docking scores, 364 molecules were selected for further structural analysis, such as common features of the molecules and key amino acid interaction partners in the binding pocket. After pharmacophore modeling, an initial hit was found containing two carboxylic acids. Further SAR studies were conducted to identify 20 with improved Keap1−Nrf2 inhibitory activity (IC50 of 7.1 μM, FP) and promising gene induction in cells (Table 1).64 The nitroarene scaffold, as in 20, is flagged as a “low risk” problem in the FAF-Drugs4 analysis because of its potential toxicity. The primary pathways of biotransformation to toxic metabolites involve reduction of the nitro group to stabilized nitro radicals. Under anaerobic conditions, the nitro radicals can be reduced further to hydroxylamine and amine metabolites. In a more aerobic environment, the radicals can reduce oxygen to form toxic oxygen species.75 4.6. Inhibitors Containing a 4-Phenyl-1,2,4-triazole Core. Compounds 21 and 22, containing a 4-phenyl-1,2,4triazole core, were identified by screening and a follow-up SAR study.65 They are both able to activate the Nrf2−ARE pathway in cells. On the basis of docking results, the nitro group in the two compounds interacts with the key amino acid Arg483. Binding to the Keap1 Kelch domains of 21 and 22 was confirmed by SPR, suggesting Kd values of 22.8 and 16.5 μM, respectively (Table 1).65 Compounds 21 and 22 contain a triazole-3-thiol core, which can be categorized as an activated aryl thioether. The heterocycle is electron-withdrawing and can in some cases activate the thioether, leading to nonspecific reactivity with biological nucleophiles.76,77 4.7. Inhibitors Containing a 1-(1,3,4-Oxadiazol-2yl)urea Core. Satoh et al.66 selected 65 compounds on the basis of in silico screening of both in-house and commercially available compounds tested against the Keap1 Kelch domain using an existing crystal structure (PDB ID 2FLU). By SPR, 27 compounds were found to be active, and medicinal chemistry performed on the most promising hit led to the discovery of compound 23. Interestingly, two different crystal structures of the human Keap1 Kelch domain with 23 have been determined by soaking (PDB ID 3VNH) and cocrystallization (PDB ID 3VNG) (Figure 2). In the soaking form, compound 23 binds deeper within the binding pocket of the Kelch domain compared with the cocrystallization form and interacts with amino acids in subpockets P1−P3. This tighter binding was confirmed by molecular dynamics simulations.66 The structurally related compound 24, called NK-252, was presented as a potential Keap1−Nrf2 PPI inhibitor in a study focusing on nonalcoholic fatty liver disease.67 Compound 24 caused dose-dependent Nrf2 activation in a luciferase NQO1− ARE reporter gene assay, and SPR analysis revealed dosedependent binding to the Keap1 Kelch domain at 12.5−100

rearrangement to form a reactive isocyanate intermediate, which could interact with different biomolecules (FAF-Drugs4 analysis).73 4.3. Inhibitors Containing a 3-Phenylpropanoic Acid Core. In a collaboration between Astex Pharmaceuticals and GlaxoSmithKline Pharmaceuticals, Davies et al.61 identified compound 13 using fragment-based drug discovery (FBDD) (Table 1). Approximately 330 fragments were screened using X-ray crystallography, which identified subpockets P1, P4, and P5 as hot spots for fragment binding. The X-ray structures revealed that three different fragments, 14−16, occupied each of their hotspots and interacted with key amino acids such as Arg483 (subpocket P1), Tyr525 (P4), and Ser602 (P5), respectively (Figure 3). Despite a low potency (IC50 > 1 mM, FP), phenylacetic acid fragment 14 was identified as a promising “anchor fragment” for hit elaboration because of its multiple exit vectors as shown by the X-ray structure (Figure 3). Fragment 14 was modified in a stepwise manner to occupy all three hotspots in the binding pocket. First, a benzotriazole moiety was attached to the benzylic position of fragment 14 in order to reach hotspot P4 and form a π−π stacking interaction with Tyr525 (similar to fragment 15) (Figure 3). This modification led to an improved activity (IC50 of 61 μM). Growth from the 3-position of the phenyl ring of fragment 14 with a sulfonamide moiety (corresponding to fragment 16) led to an interaction with Ser525 in hotspot P5 and a further improved activity (IC50 of 3.4 μM) (compound 17) (Figure 3). Subsequent modifications, including replacement of the p-chlorine with a methyl group, introduction of the electron-donating methoxy group on the benzotriazole, and cyclization of the phenylsulfonamide, resulted in compound 13 with very high affinity as measured by FP (IC50 of 15 nM) and isothermal titration calorimetry (ITC) (Kd of 1.3 nM) (Table 1 and Figures 2 and 3). Compound 13 was found to be selective toward the Keap1 Kelch domain across a panel of 49 targets with potential toxicity liabilities. Furthermore, 13 activates the Nrf2 pathway in human bronchial epithelial cells (both normal cells and cells derived from chronic obstructive pulmonary disease (COPD) patients) and thereby induces target gene expression and downstream antioxidant activities. Also, assessment of 13 in in vivo models related to COPD was performed. After intravenous infusion over 6 h in rats, animals treated with 13 showed induced pulmonary expression of Nrf2-regulated genes, and the compound attenuated ozone-induced pulmonary inflammation and depletion of lung glutathione levels.61 Further development and biological characterization of 270 analogues of 13 containing the same 3-phenylpropanoic acid core have been published in a patent application.74 The inhibitory activity of the compounds was determined in a Keap1−Nrf2 competition FP assay and a BEAS-2B cell assay of NQO1 activity. Eleven of the analogues demonstrated a strong inhibitory activity with IC50 < 10 nM. 4.4. Inhibitors Containing a 1-Phenylpyrazole Core. Recently, two new compound series, both containing a 1phenylpyrazole core, were presented in patent applications by Astex Pharmaceuticals and GlaxoSmithKline.62,63 In the first, about 65 compounds with a biarylpyrazole core were presented and tested for inhibitory activity in the Keap1−Nrf2 competition FP assay and the NQO1-activity cell assay. Also, the ability to inhibit the Keap1−Nrf2 PPI was assessed in a time-resolved FRET (TR-FRET) assay. One of the most promising biarylpyrazole-containing compounds is compound I

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μM (Table 1); however, a Kd value was not determined, likely because saturation was not achieved at the tested concentrations.67 4.8. Inhibitor Containing a Hydrazinecarbohydrazide Core. Structure-based virtual screening of the Specs database was performed by Sun et al.68 to discover the hydrazinecarbohydrazide-containing compound 25. First, a focused Keap1− Nrf2 library of 21 199 compounds was prepared from the 251 774 compounds from the Specs database. After a pharmacophore search, docking, and visual inspection, 25 was identified as a promising inhibitor of the Keap1−Nrf2 interaction. Compound 25 was shown to have an EC50 of 9.8 μM in an FP assay and Nrf2 transcriptional activity in a cellular ARE−luciferase reporter assay (Table 1).68 However, compound 25 contains acylhydrazone groups, which have been shown to be electrophilic under acidic conditions and hydrolyze to hydrazides, which can undergo metabolic oxidation to reactive nitrogen species (FAF-Drugs4).76 4.9. Inhibitor Containing a Pyrimidin-4(3H)-one Core. Besides compound 5, Marcotte et al.53 also identified 26 with a pyrimidin-4(3H)-one core. Compound 26 demonstrated a lower activity than 5, with an IC50 of 118 μM in the FP assay and no activity in a luciferase cell reporter assay (Table 1). The X-ray structure of the Keap1 Kelch domain cocrystallized with 26 demonstrated that two molecules were bound side by side in the binding pocket (Figure 2).53 Compound 26 contains a pyrimidine-2-thiol core, which can be categorized as an activated aryl thioether, similar to compounds 21 and 22, that can show nonspecific reactivity with biological nucleophiles.76,77 4.10. Inhibitors Containing a Pyrazolidin-3-one Core, a 4-Aminophenol Core, or a Thiazolidine-2,4-dione Core. Three structurally different series of Keap1−Nrf2 PPI inhibitors were identified by Zhuang et al.69 using structurebased virtual screening of 153 611 compounds from the Specs database, resulting in 65 initial hits. Nine compounds demonstrated inhibitory activity in the low micromolar range in an FP assay, leading to the identification of the three scaffolds. SAR studies on the pyrazolidin-3-one compounds led to the identification of compound 27, which exhibited good inhibitory activity with a Kd of 15.2 μM in the FP assay (Table 1).69 However, FAF-Drugs4 identified 27 as a PAINS because of the α,β-unsaturated carbonyl, which can react with protein thiol groups and thus give rise to off-target activity.76 The other pyrazolidin-3-one-containing compounds described in this study also contain an α,β-unsaturated carbonyl; however, they are not all active in the FP assay, indicating that 27 could be a true Keap1−Nrf2 PPI inhibitor. The second lead scaffold consists of a 4-aminophenol core, which was used for SAR analysis. The initial hit from the screening, compound 28, was actually the most potent one among the compounds from the SAR study, with a Kd of 2.9 μM (Table 1).69 Compound 28 is also categorized as a PAINS because of the p-hydroxysulfonamide motif, which has redox cycling activity that could potentially lead to generation of reactive metabolites and irreversible alkylation of endogenous proteins.73,76,77 Also, similar to 21, 22, and 26, compound 28 contains an activated aryl thioether. Again, however not all of the compounds from this SAR analysis containing a phydroxysulfonamide motif were active in the FP assay, indicating that 28 could be a true Keap1−Nrf2 PPI inhibitor. Compound 28 was later used as a lead scaffold for a SAR study where the introduction of N-acetic acid groups on the

sulfonamide and replacement of the p-isopropyl group with an ethyl gave a 6-fold-improved affinity (Kd of 1.14 μM compared with 7.24 μM for 28) (Table 1).70 The last of the three new inhibitors identified by Zhuang et al. contained a thiazolidine-2,4-dione core. SAR analysis did not improve the inhibitory activity, as the initial hit, compound 29, remained the most potent derivative, with Kd of 10.4 μM in an FP assay (Table 1).69 Similar to 27, compound 29 also contains an α,β-unsaturated carbonyl and is categorized as a PAINS.76

5. CENTRAL NERVOUS SYSTEM PERMEABILITY Targeting the CNS is challenging because the BBB provides a unique membrane that segregates the CNS and the circulation system and prevents unwanted chemicals from entering the brain. The BBB is formed by capillary endothelial cells kept together by tight junctions that efficiently avoid penetration of polar compounds.78 As the binding pocket of the Keap1 Kelch domain is polar, development of a potent Keap1 inhibitor with high CNS permeability is likely even more challenging.78 Many of the published Keap1−Nrf2 inhibitors contain carboxylic acids (1, 3, 4, 6, 7, 10, 13, 18, 19, 23, 25, 27, and 29; Table 1), the charge of which at physiological pH is likely to prevent or decrease CNS permeability.33 Also, similar to many other PPIengaging proteins, Keap1’s binding pocket is relatively large. These challenges are confirmed by the only two studies where CNS exposure has been experimentally investigated, as described in the following section together with a discussion of CNS MPO scores. To further analyze the promise of current Keap1 inhibitors as CNS agents, we calculated their CNS MPO30 and modified CNS MPO.v233 scores. Through study of a set of marketed CNS drugs and an internal set of Pfizer CNS candidates, the CNS MPO algorithm was defined to provide guidance in drug design to accelerate the identification of CNS-permeable and drug-like compounds. The CNS MPO score is based on a set of six fundamental physiochemical parameters: (1) lipophilicity, as expressed in the partition coefficient (cLogP); (2) distribution coefficient at pH 7.4 (cLogD); (3) molecular weight (MW); (4) topological polar surface area (tPSA); (5) number of hydrogen-bond donors (HBD); and (6) most basic center (pKa).30,32 Each parameter is scored with a value between 0 and 1 based on a simple linear function, and the six values are added to obtain the final CNS MPO score, which can vary from 0−6. Higher CNS MPO scores are desirable for CNS drug candidates, as they would suggest BBB permeability and absorption, distribution, metabolism, and excretion (ADME) properties in ranges similar to those for the CNS drug test set. An optimal CNS candidate with a CNS MPO score of 6 would have the following property values: cLogP ≤ 3, cLogD ≤ 2, MW ≤ 360 Da, tPSA = 40−90 Å2, HBD = 0, and pKa ≤ 8. The algorithm was applied to the two data sets and showed that 74% of the marketed CNS drugs were characterized with CNS MPO scores greater than 4, in comparison with 60% of the Pfizer CNS candidates. Additionally, approximately 95% of the compounds with CNS MPO scores greater than 5 showed high passive permeability, low P-glycoprotein (P-gp) liability, favorable metabolic stability, and high cellular viability.32 Rankovic33 developed a modified version of the CNS MPO algorithm, termed CNS MPO.v2, describing the physicochemical properties for brain-penetrant compounds versus peripherally restricted molecules. In this analysis, the test sets included not only marketed drugs but also research molecules with available mouse brain exposure data. The results suggested that J

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Table 2. CNS Permeability of Inhibitors Containing a 1,2,3,4-Tetrahydroisoquinoline Core52

a

MDCK-MDR1 cells. bAlso see Table 1.

fold and in vivo brain exposure is significantly increased, with an unbound brain-to-plasma ratio of 0.9 (Table 2). Unfortunately, the inhibitory activity does not follow the improved CNS profile, as an IC50 of >100 μM was determined for 32.52 The CNS MPO analysis suggests that the reason for the improved CNS profile could be not only the reduced tPSA but also the reduction of the molecular weight. Noticeably, the calculated CNS MPO scores for 1, 2, and 3 correlate with the experimentally determined ERs (Table 2). Kazantsev at al.65 used HPLC to quantify compound 21 in wild-type mouse cortices (N = 3). After daily intraperitoneal injections of 21 escalating from 50 to 275 mg/kg, the concentration of 21 was determined to ∼0.5 μM in homogenized brain tissue. The concentration of 21 in the blood was not specified, which makes it impossible to evaluate the brain-to-plasma ratio. Also, the duration of administration of the compound is uncertain and a very high dose was injected. Overall, this study indicates that only small amounts of 21 can enter the brain, which correlates with an intermediate CNS MPO.v2 score of 4.0 (Table 1). Most of the compounds in Table 1 have rather low MPO scores ( 120 Å2, and HBD ≥ 2, which combined make them unlikely to penetrate the BBB. Furthermore, the essential physicochemical properties of compound 25, 26, 28, and 29 do not comply with CNS permeability, as can be seen from the very low MPO scores (Table 1). Compound 10 is the only compound from the 1,4diaminonaphthalene series with a reasonable MPO score of

the most critical physicochemical properties in regard to CNS permeability are molecular size and hydrogen-bond capacity and not lipophilicity as previously stated. In general, brainpenetrant compounds were found to have MW ≤ 470 Da, tPSA ≤ 90 Å2, and HBD ≤ 2.33 However, it should be noted that both data sets contained a low number (