Keap1-Nrf2

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Perspective

Non-covalent Small-Molecule Kelch-like ECH-Associated Protein 1 - Nuclear Factor Erythroid 2-Related Factor 2 (Keap1Nrf2) Inhibitors and Their Potential for Targeting CNS Diseases Jakob S. Pallesen, Kim Tai Tran, and Anders Bach J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00358 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 12, 2018

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Journal of Medicinal Chemistry

Non-covalent Small-Molecule Kelch-like ECHAssociated Protein 1 - Nuclear Factor Erythroid 2Related Factor 2 (Keap1-Nrf2) Inhibitors and Their Potential for Targeting CNS Diseases Jakob S. Pallesen,† Kim T. Tran,† Anders Bach†*

†

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark

KEYWORDS: Kelch-like ECH-associated protein 1 (Keap1); nuclear factor erythroid 2-related factor 2 (Nrf2); CNS permeability; small-molecule inhibitors; protein-protein interactions (PPIs); fragment-based drug discovery (FBDD); bioisosteres; prodrugs.

ACS Paragon Plus Environment

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ABSTRACT

The transcription factor Nrf2 has a protective effect against oxidative stress and plays a major role in inflammation and CNS diseases. Inhibition of the protein-protein interaction (PPI) between Nrf2 and its repressor protein Keap1 leads to translocation of Nrf2 from the cytosol to the nucleus and expression of detoxifying antioxidant enzymes. To date, several non-covalent small-molecule 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 MPO scoring algorithms. Finally, several strategies are described for identifying CNS-targeting Keap1 inhibitors.


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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 Stransferase (GST).3-4 The antioxidant enzymes are regulated through the antioxidant response element (ARE), where nuclear factor erythroid 2-related factor 2 (Nrf2) is one of the major AREbinding 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 CNS diseases.7-9 At unstressed conditions, the cellular concentration of Nrf2 remains low as it is negatively regulated by the substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1), 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 interaction 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 interaction 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.


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Nrf2 modulators can be divided into two categories: Covalent Keap1 modifiers and noncovalent (peptides or small molecules) Keap1-Nrf2 protein-protein interaction (PPI) inhibitors.13 Dimethyl fumarate (DMF, 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 as it increased the risk of heart 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 less inhibition 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 calpain (Cal) cleavage sequence were fused to the Nrf2-derived peptide LDEETGELFLP, resulting in the peptide inhibitor Tat-CalDEETGE, 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 knock-


4

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out studies and inhibition of Keap1-Nrf2 by either covalent modifiers or peptides.4 For example, the covalent modifier, acetyl-11-keto-β-boswellic acid (AKBA), reduced infarct volume by 34% when administrated at reperfusion after 2 h of middle cerebral artery occlusion (MCAO) in rats.28 Also, Tat-Cal-DEETGE reduced traumatic brain injury-associated disruption of the bloodbrain 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 noncovalent manner has been presented in 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 are analyzed and discussed by using the FAF-Drug4 filtering tool.36


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2. MOLECULAR MECHANISMS OF KEAP1-NRF2 INHIBITION Nrf2 consists of six domains, designated Nrf2-ECH homology (Neh) 1-6, of which the Nterminally located Neh2 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-a-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


6

Basal conditions

DL G

ETGE ub

ub

Direct reversible inhibitor

R bx 1

E2

ub 1 Rbx

B BTB TB

Kelch

Kelch

Kelch

Liberated Keap1 R bx 1

ub 1 Rbx

B TB

BT B

Rbx

Kelch

ub

E2

E2

BT

Kelch

ETGE

ub 1 Rbx

E2

R bx 1

E2

Cu l3

CRL3

ub ub

ub

DL G

Nrf2 degradation

ETGE

Cu l3

Open state

26s proteasome

1

Kelch

DL G

Inhibited Keap1

R bx

B BTB

Kelch DL G

ETGE

DL G

Inhibited Keap1

ub 1

l3

l3

Kelch

R bx 1

Cu

E2

Cu

l3

B BTB TB

Kelch

ub

E2

Kelch

Cu l3

E2

Cu l3

ub

E2

Cu l3

ub

Rbx 1

E2

l3

l3

B BTB TB

1 Rbx

ub

E2

Cu

Cu

Cu l3

bx 1 Newly synthesized Nrf2 R E2

Cu

ub ub ub ub ub ub

ub

ub ub

Polyubiquitination

B BTB TB

Keap1 Kelch DL G

Nrf2

Kelch ETGE

Closed state

Induced conditions

Oxidative stress

l3

l3

Cu

Cu l3

ub

ub

E2

E2

ub

ub

R bx 1

1 Rbx

E2

E2

B TB

BT B

l3

BT B

1 Rbx

Cu

B TB

R bx 1

Cu

E2

B B TB T B

Kelch

Kelch ETGE

DL G

Hinge-and-latch model ub E2

ub E2

BT

Kelch

Kelch

DL G

ETGE

1

h Kelc D LG

h Kelc ETGE

Antioxidant enzymes

Cul3-dissociation model

Conformation cycling model

Rbx1

Cu l3

1 Rbx

Kelch

Rbx

Cu l3

ub

E2

Cu l3

ub 1 Rbx

l3 Cu

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cu l3

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B BTB

DL G

Kelch

Inhibited Keap1

ETGE

DL G

ETGE

Nr f2

Liberated Nrf2

S M ma af ll

ARE/EpRE

Newly synthesized Nrf2 Cytoplasm

Nucleus

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.


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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 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), both motifs have been shown to be indispensable for the Keap1dependent 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 will 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, CPDT and sulforaphane (SF)) have in fact been shown to increase the association between Nrf2 and Keap1 in human and rat bladder cancer cell lines.41-42


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The “Cul3-dissociation model” proposes that the ability of Keap1 to assemble into a fully functional CRL3 complex is crucial for regulating 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 the 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 produces a dysfunctional closed state of the Keap1-Nrf2 complex, i.e. where both Neh2domains are still bound to Keap1 but where 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, but 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 Keap1Nrf2 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. Based on the three models following cysteine modifications on Keap1, we hypothesize that in the context of oxidative stress such an inhibitor could: 1)


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Affect a subpopulation of Keap1-Nrf2 complexes that has not entered the induced state, e.g. by binding to free Keap1 or the open state complex; in both cases, the formation of the closed state would be hampered and the constitutive Nrf2-degradation reduced; and 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 or the Keap1-Nrf2 DLG interaction, or both; however, from the basis of the above models, breaking the weaker interaction to DLG would be sufficient for inhibiting Nrf2-repression.

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 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 βpropeller Keap1-Kelch domain and the two Nrf2 motifs, ETGE and DLG, of the intrinsically disordered peptide Neh2 domain (Figure 2, upper row).37 X-ray crystallography of truncated Neh2 peptides containing the ETGE and DLG motifs, respectively, have provided detailed structural information on these interaction surfaces.45-47 Although the buried surface area of the Kelch-DLG interface is significantly larger than that of the Kelch-ETGE (~780 Å2 vs. ~550 Å2), both are relatively small compared to classical PPIs (1,000-6,000 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


10

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(Arg483, Ser508, Ser363, Arg380, Asn382, and Arg415), P4 and P5 contain mostly non-polar residues (Tyr525, Gln530, Tyr572, Tyr334, and Phe577), and P3 contains small polar and nonpolar residues (Gly509, Ala556, Gly571, Ser602, Ser555, and Gly603) (Figure 2, upper row).48 Recently, a hitherto unexplored P6 sub-pocket was observed on the Kelch surface induced by an extended DLG-containing peptide (DLGex).47 Although interesting, this sub-pocket 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 due to the presence of multiple charged arginine residues (i.e. Arg483, Arg415, and Arg380), which make key saltbridges with carboxylic acid residues in both the ETGE and DLG motifs. Importantly, mutation studies, in silico binding analysis as well as 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 pocket to range from difficult to druggable (Dscore 0.7-1.0 dependent on X-ray structure used). The main challenge appears to lie in the fact that many known ligands rely on carboxylic acid residues interacting 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 the features of the binding pocket, especially the non-polar and aromatic residues, is likely key to balancing affinity and pharmacokinetic properties.


11


N O S O

O

O

O

N

N

N

N N

O O

OH

13

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O N H

O N H

O

OH

23

Figure 2. Upper row: X-ray structures of Keap1-Kelch domain with truncated Neh2 peptides containing ETGE (PDB ID: 2FLU) and DLG (PDB ID: 3WN7) motifs. Row 2-4: X-ray structures of the Kelch domain in complex with ligands (see Table 1 for PDB IDs). For


12

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compound 23 the co-crystallized and soaking forms of the ligand have been superimposed. The Keap1-Kelch domain is shown in the same orientation at all pictures.

4. KEAP1-NRF2 PPI INHIBITORS To date several non-covalent small-molecule Keap-Nrf2 inhibitors have been identified, as summarized in Table 1.


13


Table 1. Published non-covalent small-molecule Keap1-Nrf2 PPI inhibitors.

1,2,3,4-Tetrahydroisoquinoline core

Cmpd

Structure

1

2

3

4

1,4-Diaminonaphthalene core

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Physicochemical propertiesa MW: 446.5 cLogP: 3.3 cLogD: -0.2 HBD: 1 tPSA: 96.7 pKa: 4.0 MW: 470.5 cLogP: 3.4 cLogD: 1.7 HBD: 1 tPSA: 112.2 pKa: 4.8 MW: 432.5 cLogP: 3.4 cLogD: -0.1 HBD: 1 tPSA: 77.9 pKa: 4.2 MW: 446.6 cLogP: 3.9 cLogD: 0.4 HBD: 1 tPSA: 77.9 pKa: 4.1

Discovery strategy

Biological activity

MPOb/ MPO.v2c

Ref

4L7B

HTS of MLPCN library

Kd = 1.0 µM (SPR competition) EC50 = 12-18 µM (Functional cell assays) IC50 = 2.3 µM (FP assay)

4.8 / 4.7

51 52

4L7C

SAR on 1

IC50 = 7.4 µM (FP assay)

4.0 / 3.9

52

4N1B

SAR on 1


5.1 / 4.9

52

4L7D

SAR on 1


4.7 / 4.6

52

X-ray PDB ID

4IQK

5

MW: 498.6 cLogP: 3.6 cLogD: 3.5 HBD: 2 tPSA: 110.8 pKa: 8.2

2D-FIDA based HTS of Evotec Lead Discovery Library

-

SAR on 5

6

MW: 614.6 cLogP: 3.0 cLogD: -4.0 HBD: 2 tPSA: 167.2 pKa: 3.2

IC50 = 2.7 µM (2D-FIDA assay) Kd = 1.7 µM (BLI assay) IC50 = 1.46 µM (FP assay) Dose-dependent cell activity (ARE-luciferase reporter assay) Kd = 3.59 nM (BLI assay) IC50 = 28.6 nM (FP assay) Dose-dependent cell activity (ARE-luciferase reporter assay)

2.7 / 2.9

3.5 / 3.0

53 54

54


14

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7

MW: 668.7 cLogP: 1.8 cLogD: -5.2 HBD: 4 tPSA: 207.6 pKa: 13.8 MW: 716.8 cLogP: 1.0 cLogD: -0.6 HBD: 4 tPSA: 241.9 pKa: 13.8

-

-

SAR on 6

SAR on 7 (bioisostere)

IC50 = 14.4 nM (FP assay)

2.0 / 1.0

55

IC50 = 15.8 nM (FP assay)

2.0 / 1.0

56

2.5 / 2.0

49

8

9

10

MW: 612.7 cLogP: 1.4 cLogD: 1.4 HBD: 2 tPSA: 179.4 pKa: 14.5 MW: 452.6 cLogP: 3.8 cLogD: 2.0 HBD: 2 tPSA: 86.7 pKa: 8.2 MW: 494.6 cLogP: 4.3 cLogD: 4.1 HBD: 2 tPSA: 127.9 pKa: 14.9

IC50 = 63 nM (FP assay) Kd = 44 nM (SPR assay)

4XMB

SAR on 5

5CGJ

Screening, followed by medicinal chemistry

IC50 = 0.14 µM (FP assay) Kd = 6 µM (ITC)

4.4 / 3.9

57

4ZY3

HTS of Drug Discovery Initiative library

IC50 = 1.5 µM; Keap1-p-p62 IC50 = 6.2 µM; Keap1-Nrf2 (FP assay)

0.9 / 1.4

58 59

-

HTS of Drug Discovery Initiative library


1.8 / 2.1

60

5FNU

FBDD using X-ray crystallographic fragment screening

Kd = 1.3 nM (ITC assay) IC50 = 15 nM (FP assay) Increases NQO1gene expression and protein activity in cells

3.3 / 3.1

61

11 MW: 623.7 cLogP: 3.9 cLogD: 3.8 HBD: 2 tPSA: 151.3 pKa: 8.8 12 3-Phenylpropanoic acid

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MW: 550.6 cLogP: 4.2 cLogD: 0.6 HBD: 1 tPSA: 132.9 pKa: 3.8 13


15

18

MW: 511.6 cLogP: 6.0 cLogD: 2.4 HBD: 1 tPSA: 90.1 pKa: 3.4 MW: 463.6 cLogP: 5.6 cLogD: 2.0 HBD: 1 tPSA: 75.4 pKa: 3.4

1,4-Diphenyl1,2,3-triazole core 1-Phenyl-1,2,4-triazole core 1-(1,2,3-Oxadiazol-2-yl)urea core

20

21

22

23

24

25

MW: 392.2 cLogP: 4.6 cLogD: 4.6 HBD: 0 tPSA: 76.5 pKa: 0.5 MW: 455.5 cLogP: 5.4 cLogD: 5.4 HBD: 0 tPSA: 98.7 pKa: 0.8 MW: 405.4 cLogP: 4.6 cLogD: 4.6 HBD: 0 tPSA: 98.7 pKa: 0.2 MW: 358.3 cLogP: 0.9 cLogD: -3.1 HBD: 3 tPSA: 139.7 pKa: 7.1 MW: 285.3 cLogP: 0.4 cLogD: 0.0 HBD: 2 tPSA: 106.1 pKa: 7.1 MW: 486.4 cLogP: 3.5 cLogD: -3.6 HBD: 4 tPSA: 166.7 pKa: 15.0

Page 16 of 54

SAR

IC50 = 10-100 nM (FP assay) IC50 < 10 nM (TR-FRET assay)

3.6 / 3.7

62

-

SAR

EC50 = 10-100 nM (BEAS-2B NQO1 MTT assay) IC50 = 10-100 nM (FP assay) IC50 = 10-100 nM (TR-FRET assay)

4.1 / 3.9

63

-

Virtual screening of ZINC library followed by SAR

EC50 = 7.1 µM (FP assay) 2-fold increase of NQO1-activity in cells at 0.6 µM

4.0 / 5.0

64

HTS

Kd = 22.8 µM (SPR assay) Dose-dependently induces NQO1 and GCLM expression in cells

3.0 / 4.0

65

-

SAR on 21

Kd = 16.5 µM (SPR assay). Dose-dependently induces NQO1 and GCLM expression in cells

3.6 / 4.6

65

3VNH 3VNG

Virtual screening of in-house and ZINC database

Keap1 binding confirmed by SPR and AlphaScreen assays

4.2 / 3.3

66

5.0 / 4.5

67

1.9 / 0.9

68

-

19

Hydrazinecarb ohydrazide core

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1-Phenylpyrazole core


-

-

In-house synthesis

-

Virtual screening of Specs database

EC2 = 1.36 µM (ARE-luciferase reporter assay) Dose-dependent Keap1-binding (SPR assay) EC50 = 9.8 µM (FP assay) Dose-dependent cell-activity (ARE-driven luciferase cellbased assay)


16

Pyrazolidin-3one core 4-Aminophenol

26

27

MW: 497.5 cLogP: 4.4 cLogD: 4.1 HBD: 2 tPSA: 109.0 pKa: 12.0 MW: 443.2 cLogP: 3.9 cLogD: -0.2 HBD: 2 tPSA: 105.3 pKa: 6.7

28


29


Thiazolidine2,4-dione core

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pyrimidin4(3H)-one core

Page 17 of 54

IC50 = 118 µM (2D-FIDA assay) No cell activity (ARE-driven luciferase cellbased assay)

1.2 / 1.7

53

4IN4

2D-FIDA based HTS of Evotec Lead Discovery Library

-


Kd = 15.2 µM (FP assay)

3.9 / 3.4

69

-


Kd = 2.9-7.24 µM (FP assay)

2.0 / 1.9

69 70

-


Kd = 10.4 µM (FP assay)

2.6 / 2.1

69

a

All data are obtained from ChemAxon or Ertl 71 b 30 c (http://www.daylight.com/meetings/emug00/Ertl/); CNS MPO algorithm; CNS MPO.v2 algorithm.33

4.1. Inhibitors Containing a 1,2,3,4-Tetrahydroisoquinoline Core Hu et al. were the first to report a reversible small-molecule Keap1-Nrf2 inhibitor.51 Compound 1 (Table 1) was identified by high-throughput screening (HTS) of the commercially available MLPCN library using a homogenous fluorescence polarization (FP) competition assay measuring the interaction between the Keap1-Kelch domain and a fluorescently labeled Nrf2derived peptide. The initial HTS hit was a mix of four stereoisomers, and the most promising stereoisomer had an IC50 of 3 µM (FP). After re-synthesis 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


17


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activation (EC50 of 18 µM) and promote translocation of Nrf2 in the nuclei of human bone osteosarcoma epithelial (U2OS) cells (EC50 of 12 µM).51 A later X-ray structure of the Keap1-Kelch domain co-crystallized with 1 revealed that 1 occupies the polar sub-pocket P2, the non-polar sub-pocket P5, and P3 (Figure 2).52 This information was used to guide further SAR studies where 12 analogues were synthesized demonstrating that the phthalimide, the aryl ring of the tetrahydroisoquinoline, and the cyclohexane carboxylic 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 three-fold 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 sub-pocket P2 and interact with Asn414 (Figure 2). Removal of the phthalimide carbonyl of 1, as in 3, resulted in a two-fold reduction in the IC50 relative to 1, and further methylation on the 5-position (4) resulted in a similar IC50 value as 3 (Table 1).52

4.2. Inhibitors Containing a 1,4-Diaminonaphthalene Core In recent years Keap1-Nrf2 PPI inhibitors with a 1,4-diaminonaphthalene core have attracted a lot of interest. Compound 5 was identified by Marcotte et al. using a homogeneous confocal fluorescence anisotropy assay (two-dimensional fluorescence intensity distribution analysis, 2DFIDA) to screen the Evotec Lead Discovery Library (267,551 compounds).53 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 co-crystallized with 5 (Figure 2) showed that the naphthalene group of 5


18

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occupied the sub-pocket P3 and the anisole groups the two hydrophobic sub-pockets P4 and P5.53 Crucially, the two important hydrophilic sub-pockets P1 and P2 were unoccupied. Jiang et al. used this information for structure-based optimization and introduced N-acetic acid groups on both sulfonamides of 5 to furnish compound 6.54 At that time, 6 was the most potent Keap1-Nrf2 inhibitor with more than a 200-fold improvement in potency compared 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 of activity indicates interaction between the carboxylic acids of compound 6 and the important arginine residues in sub-pocket P1 and P2 (Arg415 and Arg483, respectively), as also suggested by in silico docking.54 In order to improve the physicochemical properties 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. 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 improved the pKa and cLogD profiles compared to 6 (Table 1).56 Jain et al. 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.49 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, showing the location of the amide groups in the two hydrophilic subpockets P1 and P2 (Figure 2).49


19


Page 20 of 54

The first asymmetric compound of the 1,4-diaminonaphthalene series to be co-crystallized with Keap1 was compound 10, also called RA839 (Figure 2 and Table 1). Compound 10 was identified by screening followed by structural modifications and exhibits an IC50 of 0.14 µM in an FP assay (Table 1).57 The binding mode is similar to 5, but instead of occupying the hydrophobic sub-pocket 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. identified compound 11, similar to 5, by FP-based HTS of the Drug Discovery Initiative Library (Table 1).58 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 Keap1Kelch 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 of 1.5 µM and 6.2 µM, respectively) (Table 1).59 Also, the X-ray structure of the Keap1-Kelch domain cocrystalized with 11 demonstrated a similar binding mode as 5 (Figure 2).58 Compound 11 contains an aliphatic ketone, which is categorized as a protein-reactive electrophile (FAF-Drug4 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 in human liver microsomes of 12 is increased more than eight-fold compared to 5. A series of N-substituted hydrazide compounds was developed based on 12, which demonstrated similar inhibitory activity and metabolic stability as 12.60 There is a risk that the hydroxamic acid of 12 can form a reactive isocyanate


20

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intermediate via acylation and a Lossen rearrangement, which can interact with different biomolecules (FAF-Drug4 analysis).73

4.3. Inhibitors Containing a 3-Phenylpropanoic Acid Core In a collaboration between Astex Pharmaceuticals and GlaxoSmithKline Pharmaceuticals, Davies et al. identified compound 13 using fragment-based drug discovery (FBDD) (Table 1).61 Approximately 330 fragments were screened using X-ray crystallography, which identified subpocket 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 (sub-pocket P1), Tyr525 (P4), and Ser602 (P5), respectively (Figure 3).

Figure 3. Structure-based progression of the initial X-ray crystallography fragment hits 14, 15, and 16 (PDB ID 5FNQ, 5FZJ and 5FZN, respectively) into 17 (PDB ID 5FNT) and finally to 13. Despite a low potency (IC50 > 1 mM, FP) the phenylacetic acid fragment 14 was identified as a promising “anchor fragment” for hit elaboration due to its multiple exit vectors as shown by the X-ray structure (Figure 3). Fragment 14 was modified in a stepwise manner to occupy all of the 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 π-π stacking interaction with Tyr525 (similar to fragment 15) (Figure 3). This modification led to an improved activity (IC50


21


Page 22 of 54

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 electrondonating methoxy group at the benzotriazole, and cyclization of the phenyl sulfonamide, resulted in 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 Figure 2-3). 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 IV 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 BEAS2B 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


22

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Recently, two new compound series, both containing a 1-phenylpyrazole 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 18 with an IC50 in the range of 10100 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 FP and FR-FRET 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 Based on the docking score, 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 in 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-Drug4 analysis due to 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


23


Page 24 of 54

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 1-Phenyl-1,2,4-triazole Core Compounds 21 and 22, containing a 1-phenyl-1,2,4-triazole core, were identified by screening and a follow-up SAR study.65 They are both able to activate the Nrf2-ARE pathway in cells. Based on 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 µM and 16.5 µM (Table 1), respectively.65 Compound 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 an 1-(1,2,3-Oxadiazol-2-yl)urea Core Satoh et al. selected 65 compounds based on in silico screening methods of both in-house and commercially-available compounds tested against the Keap1-Kelch domain using an existing crystal structure (PDB ID: 2FLU).66 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 either soaking (PDB ID: 3VNH) or co-crystallization (PDB ID: 3VNG) (Figure 2). In the soaking form, compound 23 binds deeper into the binding pocket of the Kelch domain compared to the co-crystallization form and interacts with amino acids in sub-pockets P1-3. This tighter binding was confirmed by molecular dynamics simulations.66


24

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A 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 dose-dependent binding to the Keap1-Kelch domain at 12.5-100 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. to discover the hydrazinecarbohydrazide-containing compound 25.68 First, a focused Keap1-Nrf2 library of 21,199 compounds was prepared based on 251,774 compounds from the Spec 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 shown to be electrophilic under acidic conditions and hydrolyze to hydrazides, which can undergo metabolic oxidation to reactive nitrogen species (FAF-Drug4).76

4.9. Inhibitor Containing a Pyrimidin-4(3H)-one Core Besides compound 5, Marcotte et al. also identified 26 with a pyrimidin-4(3H)-one core.53 Compound 26 demonstrated a lower activity than 5 with an IC50 of 118 µM in the FP assay and was inactive in a luciferase cell reporter assay (Table 1). The X-ray structure of the Keap1-Kelch domain co-crystallized with 26 demonstrated that two molecules were bound side by side in the


25


Page 26 of 54

binding pocket (Figure 2).53 Compound 26 contains a pyrimidine-2-thiol core, which can be categorized as an activated aryl thioether, similar to compound 21 and 22, that can show nonspecific reactivity with biological nucleophiles.76-77

4.10. Inhibitor 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. using structure-based virtual screening of 153,611 compounds from the Specs database, resulting in 65 initial hits.69 Nine compounds demonstrated inhibitory activity in the lowmicromolar 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 demonstrated good inhibitory activity with a Kd of 15.2 µM in the FP assay (Table 1).69 However, FAF-Drug4 identifies 27 as a PAINS, due to the α,β-unsaturated carbonyl, which can react with protein thiol groups and thus give rise to off-target activity.76 The other pyrazolidin-3one-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 with a Kd of 2.9 µM compared to the compounds from the SAR study (Table 1).69 Compound 28 is also categorized as a PAINS due the p-hydroxysulfonamide motif, which has redox cycling activity potentially leading 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


26

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aryl thioether. However, again not all the compounds from this SAR analysis containing a phydroxysulfonamide motif were active in the FP assay, indicating that 28 could be a true Keap1Nrf2 PPI inhibitor. Compound 28 was later used as lead scaffold for a SAR study where the introduction of N-acetic acid groups on the sulfonamide and introduction of p-ethyl instead of pisopropyl gave a 6-fold improved affinity (Kd of 1.14 µM compared to 7.24 µM for 28) (Table 1).70 The last of the three new inhibitors identified by Zhuang et al. contained a thiazolidine-2,4dione core. SAR analysis did not improve 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 as the BBB provides a unique membrane that segregates the CNS and the systemic circulation 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, 29; Table 1), which due to its charge at physiological pH is likely to prevent or decrease CNS permeability.33 Also, similar to many other PPI-engaging 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.


27


Page 28 of 54

To further analyze the promise of current Keap1 inhibitors as CNS agents we have calculated their CNS MPO score30 and modified CNS MPO.v2 score.33 By studying a set of marketed CNS drugs and an internal set of Pfizer CNS candidates, the CNS MPO algorithm was defined to serve as 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, 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); 6. Most basic center (pKa).30, 32 Each parameter is scored a value between 0 and 1, based on simple linear functions, 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 ADME properties in similar ranges to 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 from 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 score > 4 in comparison to 60% of the Pfizer CNS candidates. Additionally, approximately 95% of the compounds with CNS MPO scores > 5 showed high passive permeability, low P-gp liability, favorable metabolic stability, and high cellular viability.32 Rankovic et al. 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 the most critical physicochemical properties in regards to CNS permeability are molecular size


28

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and hydrogen bond capacity and not lipophilicity as previously stated.33 In general, brain penetrant 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 (< 5%) of carboxylic acids or related bioisosteres likely to be negatively charged at physiological pH. Several amino acid-like compounds are known to be actively transported across the BBB, but penetration by ionizable compounds and especially carboxylic acids is generally not well understood.33 Interestingly, the CNS MPOv.2 algorithm allows higher MW and a higher number of HBD compared to the CNS MPO. Whereas CNS MPO.v2 is predictive for brain penetration, CNS MPO is also taking metabolic stability and high cellular viability into account.

Jnoff et al. assessed the CNS permeability of the 1,2,3,4-tetrahydroisoquinoline-containing compounds (1-3 and 30-32) using an MDCK-MDR1 cell assay and measuring brain exposure in mice (Table 2).52 Compound 1 showed a very small unbound brain-to-plasma ratio (< 0.01) in mice and a high efflux ratio (ER = 20) in MDCK-MDR1 cells (Table 2). In Mdr1a/1b/Bcrp (Pgp) knock-out mice, the unbound brain-to-plasma ratio was improved more than 40-fold establishing that 1 is a P-gp substrate. The improved CNS profile of compound 30 indicates that the carboxylic acid of compound 1 is responsible for the poor CNS permeability, but it is unfortunately also important for affinity to Keap1 (Table 2). Replacement of the carboxylic acid of 1 with the tetrazole bioisostere (compound 2) or a zwitterionic amino acid (compound 31) did not improve the CNS profiles (Table 2). Interestingly, and in accordance with the analyses behind the CNS MPO algorithms,30,

32-33

it was seen that tPSA-reduction led to a threefold

improvement in ER for 3 compared to 1 (tPSA of 77.9 and 96.7 Å2, respectively). Furthermore, if the phthalimide group of compound 1 is replaced by a phenyl group (compound 32, tPSA of


29


Page 30 of 54

57.6 Å2) the ER is reduced 28-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 score for 1, 2, and 3 correlates with the experimentally determined ER (Table 2).

Table 2. CNS permeability of inhibitors containing a 1,2,3,4-tetrahydroisoquinoline core.52

Compound

Efflux ratioa

Unbound brain-to-plasma ratio (mice)

MPO / MPO.v2

IC50 / [µM]

20

< 0.01 (but 0.4 in Mdr1a/1b/Bcrp knock-out)

4.8 / 4.7b

2.3b

28

-

4.0 / 3.9b

7.4b

7.5

-

5.1 / 4.9b

1.1b

0.7

0.3

4.2 / 5.2

> 100

-

< 0.01

3.4 / 2.9

69.7

0.7

0.9

4.9 / 4.7

> 100

1

2

3

30

31

32 a

MDCK-MDR1 cells; bSee also Table 1


30

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Kazantsev at al. used HPLC to quantify compound 21 in wild-type mouse cortices (N=3).65 After daily intraperitoneal injections escalating from 50 mg/kg to 275 mg/kg of 21 the concentration of 21 was determined to ~0.5 µM in homogenized brain tissue. The concentration of 21 in the blood is 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 compounds of Table 1 have rather low MPO scores (< 3), such as 8, 11, and 26, indicating that it would likely be futile to use them in experiments directed toward CNS. Likewise, optimizing them into more promising CNS candidates will be difficult as the inhibitors are generally large and many contain carboxylic acids. For example, the CNS MPO and MPO.v2 scores for the 1,4-diaminonaphthalene-containing compounds (5-9, 11-12) are lower than 3.6 (Table 1) due to MW around 500 Da or higher, tPSA > 120 Å2, and at least two HBDs, 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 seen from the very low MPO scores (Table 1). Compound 10 is the only compound from the 1,4-diaminonaphthalene series with a reasonable MPO score of 3.9-4.4 (Table 1). A possible way to optimize 10 for BBB penetration could be to decrease the molecular weight, for example by removing chemical groups that may not contribute to affinity. As an example, if all four methyl groups were removed from the benzene ring of 10, the resulting compound would have an MPO.v2 score of 4.6, compared to 3.9 of 10, and thereby a higher theoretical chance of penetrating the BBB.


31


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Compound 19 is a very potent inhibitor of Keap1-Nrf2 (IC50 of 25 nM) and has a CNS MPO score of 4.1 and a CNS MPO.v2 score of 3.9 (Table 1). Without optimization it would probably be difficult for 19 to penetrate the BBB at relevant concentrations. However, further SAR studies focusing on maintaining high activity while improving CNS MPO scores slightly could lead to interesting CNS candidates. Another compound with promising MPO scores is 20 (Table 1). Further improvement of the MPO scores could be done by optimization of lipophilicity (cLogP), for example by exchanging the iodine atom with a methyl group. This modification reduces cLogP from 4.58 to 4.16, which will increase MPO scores with 0.2. Interestingly, this modification has been tested and show similar inhibitory activity as 20.64 Compound 24 has the most promising MPO scores among the compounds in Table 1, and a further reduction in HBDs from two to one, e.g. by replacing the urea with an amide, would increase the MPO.v2 score to 5.1, indicating very high chances of being CNS permeable. However, before making further SAR studies of 24, it is preferable to establish the Kd value to Keap1 and to test its ability to inhibit the Keap1-Nrf2 interaction in simple in vitro assays.

6. KEAP1-NRF2 SMALL-MOLECULE INHIBITORS TARGETING THE BRAIN As described, the binding pocket of the Keap1-Kelch domain has polar regions and is relatively large. Additionally, no experimental data suggest good BBB penetration of any published small-molecule Keap1-Nrf2 inhibitors. The fact that the majority of the compounds contain carboxylic acids or have MPO scores generally indicating poor CNS permeability underlines how challenging it will be to target Keap1 for CNS diseases. To circumvent this


32

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major drug discovery challenge we here suggest three strategies for targeting Keap1 in the CNS with small molecules.

6.1. Fragment-Based Drug Discovery FBDD has grown into a useful strategy for developing potent and drug-like PPI inhibitors, and has already been applied to discover the small-molecule Keap1-Nrf2 inhibitor 13 (Table 1).61-63 The principle of FBDD is to screen for fragments (i.e. small substructures of drug-like compounds with molecular weight of 100-300 Da) against the target followed by optimization by linking or merging fragment hits or growing a single fragment into larger and more potent molecules.44, 79-81 Fragment hits are often prioritized based on their ligand efficiency (LE),82 i.e. the molecule’s free energy of binding divided by its number of non-hydrogen atoms. A core principle of FBDD is to start from a small but ligand-efficient fragment hit, and by careful optimization improve the affinity while maintaining the high ligand efficiency and staying within the physicochemical drug-like space.79,

81

This mindset correlates well with the design and

optimization of compounds for CNS guided by CNS MPO scores. Thus, starting from a ligandefficient Keap1-binding fragment hit and ascertaining that size, lipophilicity, and number of HBDs are kept within recommended CNS MPO values during the optimization should enhance the chances of developing small molecules with high CNS permeability targeting the Keap1Nrf2 PPI.

6.2. Bioisosteric Replacement As seen from Table 1, many of the most potent Keap1 inhibitors contain carboxylic acids, which are often associated with poor CNS penetration due to high plasma protein binding, poor


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passive permeability, and P-gp recognition.31 A potential strategy to circumvent the CNS challenges of Keap1 inhibitors is to replace the carboxylic acid groups with a carboxylic acid bioisostere leading to analogues with more CNS-preferential physicochemical properties (e.g. related to lipophilicity, acidity, number of HBD, and molecular weight).83 Lu et al. performed a bioisosteric replacement study of 7 in order to improve drug-like properties and membrane permeability.56 Eight structurally different chemical groups were replacing the carboxylic acid group, including an ester, a tetrazole, and a hydroxamic acid, leading to diverse physicochemical properties of the compounds. The tetrazole (8, Table 1) was the only bioisoster that was able to maintain biological activity against the Keap1-Kelch domain. Furthermore, the higher pKa and cLogD compared to the carboxylic acid-containing 7 might be beneficial for the CNS permeability; however, brain exposure was not the focus of this study and was therefore not tested. Interestingly, replacing the carboxylic acids with amide groups led to an inactive compound.56 This is in contrast to the study by Jain et al., where the amide-substitution was able to retain the activity of the resulting compound (9) compared to the activity of 6 (Table 1).49 Overall, these studies show that tetrazoles can substitute the carboxylic acids with preserved affinity to Keap1, as was also shown for compound 1 and 2,52 and that amides in some cases can do the same. This could also indicate that carboxylic acid groups are not strictly required for obtaining high affinity to the Keap1-Kelch domain, and that the bioisosteric replacement strategy could be further explored in order to design CNS permeable Keap1-Nrf2 inhibitors.

6.3. The Prodrug Approach The prodrug approach is another potential strategy for targeting CNS diseases,84-85 where undesirable groups of the lead compound are coupled with a promoiety, resulting in altered


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physicochemical properties and a prodrug more likely to reach CNS. After penetration of the BBB, the promoiety must be cleaved off by enzymes or oxidation/reduction whereby the compound is liberated to interact with the target protein.85 A typical prodrug strategy for carboxylic acids is esterification as this modifies the physicochemical properties of the compound, the chemical linking of the promoiety is wellestablished, and they are easily cleaved by enzymes, such as esterases and peptidases.86 Small alkyl groups (e.g. methyl or ethyl) would increase the molecular weight only slightly and potentially provide ester prodrugs with more favorable physicochemical properties for reaching CNS. However, the main caveat of this strategy is the difficulty in finding esters that are stable to esterases in blood plasma but sensitive to enzyme-cleavage in the brain.84 Nevertheless, successful applications of this strategy for targeting CNS have been reported. For example, the BBB permeability of the non-steroidal anti-inflammatory drug (NSAID) ketoprofen is very low, due to its lipophilicity and the ionization of its carboxylic acid group. However, coupling of a diacetylglyceride to the acid of ketoprofen led to improved delivery to the brain via increased permeability through the BBB and rapid hydrolysis of the prodrug within the brain.87 Also, the carboxylic acid of another NSAID, Dexibuprofen, was modified via different esterifications and the prodrugs showed improved BBB permeability compared to the parent compound.88 The prodrug and esterification approach could theoretically be used to improve the CNS profiles of current Keap1 inhibitors, but is also challenging as the conversion of the CNS prodrugs needs to be selective and rapid enough to compete with elimination from the target tissue.85 Also, the molecular size of the compounds is a critical factor, and could be a problem for the published Keap1 inhibitors, which are generally already large. Another strategy that could be further pursued is exemplified by the previously mentioned reversible Keap1-Nrf2 peptide inhibitor,


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Tat-Cal-DEETGE, where Cal is cleaved off in the cell resulting in the active peptide.24 Similarly, hybrids of small molecules and cell-penetrating peptides, such as Tat, which can lead to some BBB permability,89-90 could be attempted for targeting Keap1 in CNS. 7. SUMMARY Over the past years, the PPI between Keap1 and Nrf2 has been widely studied, and Keap1 has emerged as a promising drug target against inflammation and CNS diseases. Identification of brain-targeting small-molecule Keap1-Nrf2 inhibitors is of great interest as activation of Nrf2 is found to protect neurons against stroke (ischemic and hemorrhagic), traumatic brain injury, and neurodegenerative disorders. Several non-covalent small-molecule Keap1-Nrf2 inhibitors with promising potencies and biological activities have been developed. However, none of these have been shown able to penetrate the BBB and the majority contain carboxylic acids, which usually prevents CNS permeability. Unfortunately, the binding pocket of the Keap1-Kelch domain is polar and relatively large compared to traditional targets making it a challenging task to target Keap1 in the CNS. Different strategies can be attempted to circumvent this major drug discovery challenge, such as FBDD, carboxylic acid bioisosteric replacement, and the prodrug approach. During optimization using any of these strategies it is essential to balance the physicochemical properties (e.g. guided by CNS MPO scoring algorithms) to increase the chances of obtaining CNS permeability in the search of brain-targeting small-molecule Keap1-Nrf2 inhibitors.

AUTHOR INFORMATION Corresponding Author *Phone: +4521288604. Email: [email protected]


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests. Biographies Jakob Staun Pallesen obtained his Bachelor’s degree from School of Pharmaceutical Sciences, University of Copenhagen, Denmark, in 2012 and his Master’s degree from Department of Drug Design and Pharmacology (University of Copenhagen) in 2015. After employment as a scientific assistant, he began his Ph.D. in 2016 in medicinal chemistry under the supervision of associate professor Anders Bach. His work focuses on employing fragment-based drug discovery, including medicinal chemistry, in silico methods, and biochemical- and biophysical testing, to find new Keap1-Nrf2 small-molecule inhibitors. Kim Tai Tran obtained his bachelor’s degree within pharmacy in 2016 at the University of Copenhagen, Denmark. Currently, he is a master student in medicinal chemistry at the Department of Drug Design and Pharmacology (University of Copenhagen) under supervision of associate professor Anders Bach. His work focuses on employing fragment-based drug discovery, including medicinal chemistry and in silico methods, to find new small-molecule inhibitors against Keap1. Anders Bach obtained his bachelor’s degree within biochemistry in 2002 and his master’s degree within human biology in 2005 from University of Copenhagen, Denmark. He received his Ph.D.


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in medicinal chemistry from Department of Drug Design and Pharmacology (University of Copenhagen) in 2009. He continued as a postdoc at University of Copenhagen, until he later in 2012 joined the Italian Institute of Technology, Department of Drug Discovery and Development (Genoa, Italy) also as a postdoc. Currently, he is an associate professor at Department of Drug Design and Pharmacology where he recently started up his own research group focusing on fragment-based drug discovery and protein-protein interactions involved in CNS diseases.

ACKNOWLEDGMENT The Lundbeck Foundation is acknowledged for its financial support (Grant R190-2014-3710 for AB; the Drug Research Academy/Lundbeck Foundation scholarship for KTT).

ABBREVIATIONS Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2; PPI, Protein-Protein Interaction; ROS, reactive oxygen species;

NQO1, NAD(P)H

dehydrogenase (quinone) 1; GST, glutathione S-transferaseioxidant; ARE, antioxidant response element; DMF, dimethyl fumarate; MMF, monomethyl fumarate; Cal, calpain; AKBA, acetyl11-keto-β-boswellic acid; MCAO, middle cerebral artery occlusion; BBB, blood-brain barrier; PAINS, pan-assay interference compounds; CNS MPO, CNS multiparameter optimization; Neh, Nrf2-ECH homology; BTB, Broad complex, Tramtrack, and Bric-a-Brac; IVR, intervening region; DGR, double glycine repeat; Cul3, cullin 3; Rbx1, RING-box protein 1; CRL3, CullinRING ligase 3; FRET, Förster resonance energy transfer; SF, sulforaphane; HTS, highthroughput screening; FP, fluorescence polarization; SPR, surface plasmon resonance; BLI,


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biolayer interferometry; FBDD, fragment-based drug discovery; COPD, chronic obstructive pulmonary disease; TR-FRET, time-resolved FRET; cLogP, partition coefficient; cLogD, distribution coefficient; MW, molecular weight; tPSA, topological polar surface area; HBD, hydrogen bond donors; P-gp, P-glycoprotein; ER, efflux ratio; LE, ligand efficiency; NSAID, non-steroidal anti-inflammatory drug


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