NOVEL MODULATORS OF NRF2 AND USES THEREOF

Abstract

There is provided modulators of Nrf2 protein which comprises a compound which binds at least one of the BTB domain, IVR domain and Kelch domain of Keap1 protein, activating or inhibiting Nrf2. There is also provided pharmaceutical compositions containing the modulators, as well as uses and method of use of the modulators for the treatment of conditions.

Description

BACKGROUND

[0001] (a) Field

[0002] The subject matter disclosed generally relates to modulators of Nrf2, and more specifically to compounds which binds at least one of the BTB domain, IVR domain and Kelch domain of Keap1 protein, activating or inhibiting Nrf2.

[0003] (b) Related Prior Art

[0004] As chemical carcinogenesis likely plays a role in cancer development, cytoprotective enzyme induction is believed to be an important means of cancer chemoprevention. Transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates a battery of genes encoding carcinogen-detoxifying enzymes and antioxidant proteins by binding to the antioxidant response element (ARE) promoter regulatory sequence. Under basal conditions, in which the redox homeostasis is maintained in cells, Nrf2 is sequestered in the cytoplasm by a protein known as Keap1, which targets Nrf2 for ubiquitination and degradation by the proteasome, and thus controls both the subcellular localization and steady-state levels of Nrf2. In response to oxidative stress or chemopreventive compounds, Keap1-mediated ubiquitination of Nrf2 is decreased significantly and the Nrf2 pathway is turned on. Thus, Keap1 is a molecular switch that senses various stimuli and turns the Nrf2 pathway on and off. Administration of Nrf2-inducing agents has been shown to result in decreased carcinogenesis in animal models and altered carcinogen metabolism in humans. Clinical interventions have shown that Nrf2 inducers increase cytoprotective enzyme expression, resulting in modulation of aflatoxin disposition.

[0005] Interestingly, Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, it may be necessary to inhibit the Nrf2 pathway during chemotherapy, and thereby favor the action of concomitantly administered chemotherapeutic agents.

[0006] Keap1 contains the BTB domain mediating Keap1 homodimer formation, the `intervening region` (IVR) (amino acids 180-314), and the C-terminal Kelch domain that mediates binding to the Neh2 domain of Nrf2. Human Keap1 contains 27 cysteine residues. Three key cysteine residues (C151, C273, and C288) have been identified. C151 is required for several Nrf2 inducers, such as sulforaphane (SFN) and tert-butylhydroquinone, to manifest their effect. Importantly, residues C273 and C288 at the IVR domain are necessary for Keap1 to repress Nrf2. A single cysteine to serine mutation C273S or C288S render Keap1 unable to repress Nrf2. The transgenic expression of mutant Keap1(C273A) and/or Keap1(C288A) protein in Keap1 null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo. This suggests a critical role of the domains of Keap1 in the regulation of the functional interaction of Keap1 with Nrf2.

[0007] Therefore, there is a need for the identification of small molecules to modulate the interaction of Keap1 with Nrf2.

[0008] There is a need for the identification of small molecules to induce Nrf2 activity and stimulate or at least positively influence its activity.

[0009] There is a need for the identification of small molecules to inhibit Nrf2 activity and prevent or at least negatively influence its activity.

SUMMARY

[0010] In a first embodiment there is disclosed a modulator of Nrf2 protein comprising a compound which binds at least one of a BTB domain, an IVR domain and a Kelch domain of Keap1 protein. The modulator restores, enhances, reduces or impairs protein-protein interactions among the Keap1 protein and at least one of the Nrf2 protein and Cul3 protein, for inhibiting or activating the activity of Nrf2.

[0011] The modulator may be a compound of formula (I), (II), (III), (IV) or (V):

##STR00001##

[0012] wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁0, R₁₁, R₁2, R₁₃, R₁4, R₁₅, R₁6, R₁₇, R₁8, R₁₉, R₂0, R₂₁, R₂2, R₂₃, R₂4, R₂₅, R₂6, R₂₇ and R₂₈ may be independently chosen or identical, and may be chosen from hydrogen, halide, hydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, alkyloxide, substituted alkyloxide, halogenated alkyl, halogenated alkenyl, halogenated alkyloxide, halogenated substituted alkyloxide, amine, substituted amine, cycloalkyl, substituted cycloalkyl, heterocyclic aromatic or non-aromatic 5- to 10-membered ring containing 1-4 heteroatoms selected from N, O or S, wherein N and S can be oxidized and N can be quaternized, ═O, CH₃, OCH₃, OC₂H₅, NO₂, CN, F, Cl, Br, SH, CF₃, OCF₃, O(CF₂)₂H, NH₂, N(CH₃)₂, N(C₂H₄OH)₂, CH(OC₂H₅)₂,

##STR00002##

[0013] The indicates an attachment point, and R' is chosen from hydrogen, halide, hydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, alkyloxide, substituted alkyloxide, halogenated alkyl, halogenated alkenyl, halogenated alkyloxide, halogenated substituted alkyloxide, amine, substituted amine, cycloalkyl, substituted cycloalkyl, heterocyclic aromatic or non-aromatic 5- to 10-membered ring containing 1-4 heteroatoms selected from N, O or S, wherein N and S can be oxidized and N can be quaternized, ═O, CH₃, OCH₃, OC₂H₅, NO₂, CN, F, Cl, Br, SH, CF₃, OCF₃, O(CF₂)₂H, NH₂, N(CH₃)₂, N(C₂H₄OH)₂, CH(OC₂H₅)₂,

##STR00003##

[0014] The is a single bond or a double bond;

[0015] pharmaceutically acceptable salt, racemic mixture, enantiomer, diastereoisomer, isomer, and tautomer thereof.

[0016] The compound of formula (I) may be:

##STR00004## ##STR00005##

[0017] The compound of formula (II) may be:

##STR00006## ##STR00007## ##STR00008## ##STR00009##

[0018] The compound of formula (II) may be:

##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##

[0019] The compound of formula (III) may be:

##STR00015##

[0020] The compound of formula (III) may be:

##STR00016##

[0021] The compound of formula (III) may be:

##STR00017##

[0022] The compound of formula (III) may be:

##STR00018## ##STR00019##

[0023] The compound of formula (IV) may be:

##STR00020## ##STR00021## ##STR00022##

[0024] The compound of formula (IV) may be:

##STR00023##

[0025] The compound of formula (V) may be:

##STR00024## ##STR00025## ##STR00026## ##STR00027##

[0026] The modulator may be an inhibitor of Nrf2.

[0027] The modulator may be an activator of Nrf2.

[0028] In another embodiment, there is disclosed a pharmaceutical composition for activating Nrf2 protein which comprises a therapeutically effective amount of a modulator of the present invention, in association with a pharmaceutically acceptable carrier.

[0029] In another embodiment, there is disclosed a pharmaceutical composition for neuroprotection which comprises a therapeutically effective amount of a compound of formula (I), (II), (III), (IV) or (V) of the present invention, in association with a pharmaceutically acceptable carrier.

[0030] In yet another embodiment, there is disclosed a pharmaceutical composition for the inhibition of Nrf2 protein which comprises a therapeutically effective amount of a compound of formula (I), (II), (III), (IV) or (V) of the present invention in association with a pharmaceutically acceptable carrier.

[0031] In yet another embodiment, there is disclosed a pharmaceutical composition for overcoming drug resistance in cancer chemotherapy, and for the inhibition of tumor growth which comprises a therapeutically effective amount of a compound of formula (I), (II), (III), (IV) or (V) of the present invention, in association with a pharmaceutically acceptable carrier.

[0032] In another embodiment, there is disclosed a use of a compound of formula (I), (II), (III), (IV) or (V) of the present invention, for the treatment or prevention of a condition which involves the abnormal activity and/or expression level of Nrf2 protein.

[0033] The condition may be an oxidative stress associated disease.

[0034] The oxidative stress associated disease may be Parkinson's disease, Parkinson's disease with dementia with Lewy body, Huntington's disease, multiple system atrophy (MSA), progressive supranuclear palsy (PSA), corticobasal degeneration (CBD), and frontotemporal lobe degeneration.

[0035] The oxidative stress associated disease may be atherosclerosis, heart failure, myocardial infarction, Alzheimer's disease, Fragile X syndrome, and chronic fatigue.

[0036] The condition may be a cancer.

[0037] The cancer may be liver cancer, lung cancer, breast cancer, prostate cancer, colon cancer, neuroblastoma or leukemia.

[0038] In another embodiment, there is disclosed a use of a compound of formula (I), (II), (III), (IV) or (V) of the present invention, for the fabrication of a medicament for treatment or prevention of a condition which involves the abnormal activity and/or expression level of Nrf2 protein.

[0039] In yet another embodiment, there is disclosed a method for effecting neuroprotection in a patient in need thereof by administering a therapeutically effective amount of a compound of formula (I), (II), (III), (IV) or (V) of the present invention.

[0040] In another embodiment, there is disclosed a method for treating a disease which involves the abnormal activation and or expression level of a Nrf2 protein in a patient in need thereof by administering a therapeutically effective amount of a compound of formula (I), (II), (III), (IV) or (V) of the present invention.

[0041] In another embodiment, there is disclosed a method for treating a disease which involves at least one of 1) the abnormal activation and or expression level of a Nrf2 protein, and 2) the abnormal inhibition of a Nrf2 protein, by administering a therapeutically effective amount of a compound of formula (I), (II), (III), (IV) or (V) of the present invention.

[0042] The disease may be an oxidative stress associated disease chosen from Parkinson's disease, Parkinson's disease with dementia with Lewy body, Huntington's disease, multiple system atrophy (MSA), progressive supranuclear palsy (PSA), corticobasal degeneration (CBD), and frontotemporal lobe degeneration.

[0043] The disease may be an oxidative stress associated disease chosen from atherosclerosis, heart failure, myocardial infarction, Alzheimer's disease, Fragile X syndrome, and chronic fatigue.

[0044] In another embodiment, there is disclosed a method for treating a cancer in a subject in need thereof by administering a therapeutically effective amount of a compound of formula (I), (II), (III), (IV) or (V) of the present invention.

[0045] In yet another embodiment, there is disclosed a method of preventing a cancer in a subject in need thereof by administering a therapeutically effective amount of a compound of formula (I), (II), (III), (IV) or (V) of the present invention.

[0046] The cancer may be liver cancer, lung cancer, breast cancer, prostate cancer, colon cancer, neuroblastoma or leukemia.

[0047] The following terms are defined below.

[0048] The term "oxidative stress" is intended to mean an imbalance between the production of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. All forms of life maintain a reducing environment within their cells. This reducing environment is preserved by enzymes that maintain the reduced state through a constant input of metabolic energy. Disturbances in this normal redox state can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA.

[0049] The term "modulator" is intended to mean a means to regulate, adjust or adapt the activity of a given target (e.g. Nrf2). Modulation may be of varying intensity and may range from weak to strong. The modulator may be inhibitory (preventing the activity of the target) or stimulatory (inducing the activity of the target).

[0050] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 illustrates a structural model of the intervening region (IVR, amino acids 180-314) of human Keap1 according to the present invention.

[0052] FIG. 2 illustrates the ARE-driven luciferase activity in the cell-based system exposed to the DMSO vehicle, ethoxyquin (EQ) at 10 μM, sulforaphane (SFN) at 10 μM and compounds BM4, 5, 6, 9, 10, 18 and 19 at 10 and 50 μM according to the present invention.

[0053] FIG. 3 illustrates (A) Nrf2 protein detected in nuclear proteins by Western blotting and (B) endogenous γ-glutamylcysteine synthetase (GCS), a Nrf2-regulated protein, in whole cell protein extracts, detected by Western blotting. HepG2 cells were treated with EQ, SFN, DMSO, BM10, BM31 and BM40 at designated concentrations for 24 h.

[0054] FIG. 4 illustrates the ARE-driven luciferase assays in 293T cells, demonstrating that Keap1 C297S mutant did not affect the potency of SFN, but abolished the Nrf2 induction activity of BM31.

[0055] FIG. 5 illustrates 293T cell lysates immunoprecipitated with Keap1 antibody and the eluted proteins were immunoblotted with Nrf2 antibody. Lane 1: Nrf2 transfection; Lanes 2, 3, 4: Nrf2 and Keap1 co-transfection. Lanes 2, 3, 4: treated with DMSO, BM31 (50 μM) and SFN (5 μM), respectively.

[0056] FIG. 6 illustrates the protective effect of BM31 for MCF-7 cells against the cytotoxic effect of 160 nM B[a]P. DMSO without B[a]P is included as a control. *P<0.05, **P<0.006 when compared with control 0 (DMSO with B[a]P). Experiments were performed in triplicate.

[0057] FIG. 7 illustrates ribbon representation (A) and molecular surface (B) of the crystal structure of Keap1 Kelch domain (ribbon in white and red) in complex with Nrf2-derived peptide (ribbon in cyan) where the mutation distribution at Keap1 Kelch domain are labeled and shown in red ribbon.

[0058] FIG. 8 illustrates Nrf2 protein detected in nuclear proteins by Western blotting (A, B, C). HepG2 cells were treated with EQ, SFN, DMSO, BM compounds at designated concentrations for 24 h. BM9 at 50 μM has modest activity in inducing Nrf2 nuclear translocation in HepG2 cells.

[0059] FIG. 9 illustrates MCF-7 cell survival curves for BM10, BM31, BM40 and SFN. MCF-7 cells were treated with test compounds at designated concentrations for 72 hours. Viable cells were evaluated by MTT assays. Experiments were performed in triplicate and repeated twice.

[0060] FIG. 10 illustrates the sequence of Full length sequence of human Keap1 (1-624)(SEQ ID NO:1), the BTB domain (amino acids 67-170) of human Keap1 (SEQ ID NO:2), the IVR domain (amino acids 180-314) of human Keap1 (SEQ ID NO:3), and the Kelch domain (amino acids 315-598) of human Keap1 (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] In embodiments there are disclosed modulators of the interaction between Nrf2 protein and Keap1 protein. The modulators may comprises a compound which binds at least one of the BTB domain (amino acids 67-170) (SEQ ID NO:2), an IVR domain (amino acids 180-314) (SEQ ID NO:3) and Kelch domain (amino acids 315-598) of the Keap1 protein (SEQ ID NO:4).

[0062] The present invention combines in silico screening with cell based assays to identify novel agents that modulate the Keap1-Nrf2 signalling pathway. A three-dimensional (3D) structural model of the Keap1 IVR domain was built and utilized to virtually screen the chemical databases for putative Nrf2 modulators, which were subsequently tested by luciferase-based reporter gene assays and/or Western blot analyses.

[0063] The modulators may modulate the interaction of Keap1 with Nrf2 to prevent keap1 from binding to Nrf2, hence stimulating the activity of Nrf2, or they may modulate the interaction between Keap1 and Cul3, and reduce Nrf2 degradation, hence stimulating the activity and/or expression level of Nrf2. On the other hand, they may favor the interaction of Keap1 with Nrf2, hence preventing the release of Nrf2 from keap1. In addition, they may restore the association of Keap1 and Nrf2 that is weakened or lost due to mutations at Keap1, Nrf2 or both of them.

[0064] The modulators of the present invention may be useful for the treatment of conditions in which the activity of Nrf2 is misregulated or detrimental to a biological or treatment process. Nrf2 is an important molecular switch for activating genes important in the cellular adaptation to redox stress. Modulators stimulating the activity of Nrf2 may therefore be useful for the treatment conditions in which this adaptation is reduced, disrupted or absent and reestablishment of a response to oxidative stress is required. Several human conditions have been linked to such disruption. These include Parkinson's disease, Parkinson's disease with dementia with Lewy body, as well as other related diseases, such as multiple system atrophy (MSA), progressive supranuclear palsy (PSA), corticobasal degeneration (CBD), frontotemporal lobe degeneration and huntington's disease. Other diseases in which oxidative stress is involved include but are not limited to atherosclerosis, heart failure, myocardial infarction, Alzheimer's disease, Fragile X syndrome, and chronic fatigue.

[0065] The modulators of the present invention may be seen as playing a protective role (i.e. neuroprotective, cardioprotective, etc) by at least slowing, if not preventing the progress of neurodegenerative or cardiac diseases. In addition, the modulators of the present invention may act as cancer chemoprevention agents.

[0066] The modulators of the present invention may inhibit the activity of Nrf2. The modulator may prevent the release of Nrf2 from Keap1 and/or stimulate its degradation. Multiple Keap1 and Nrf2 mutations have been identified in cancers. These mutations affect the interaction of Keap1 with Nrf2 by interfering with the repressive activity of Keap1 toward Nrf2. Therefore, the modulator of the present invention may be useful for the treatment of conditions in which the repressive activity of Keap1 towards Nrf2 is weakened or impaired due to mutations in Keap1, Nrf2 or both of them. Examples of mutations of Keap1 include, but not limit to, S104A, H200P, D236H, C273A, R272C, Q284L, C288A, T314M, G333S, G350S, G364C, G379D, R413L, R415G, A427V, and G430C. Selected mutations are indicated in FIG. 7. The mutations at the BTB domain and IVR region may affect the Keap1/Cul3 interaction as well as the Keap1/Nrf2 interaction.

[0067] Such conditions include but are not limited to cancers. The cancers include but are not limited to liver cancer, lung cancer, breast cancer, prostate cancer, colon cancer, neuroblastoma or leukemia. In such conditions, the reduction in or the inability of Keap1 of repressing Nrf2 (which may or may not be caused by mutations) results in increased Nrf2 activity. This increased activity over-stimulates the cellular Nrf2-dependent response, and cause tumor cells to become resistant to drugs with which they are treatment. Thus, tumor cells could hijack the Nrf2 pathway to their survival advantage, conferring resistance to chemotherapeutic agents. The Nrf2 modulators of the present invention are useful for overcoming drug resistance in cancer chemotherapies, and they are also useful for inhibiting tumor growth since tumor cells may depend on aberrant activation of Nrf2.

[0068] Novel Nrf2 modulators are identified, BM4, BM5, BM6, BM9, BM10, BM18, BM19, BM31 and BM40 that triggered Nrf2 nuclear translocation. (Scheme 1) It is demonstrated that BM10, BM31, and BM40 triggered Nrf2 nuclear translocation and induced expression of ARE-regulated enzymes, such as γ-glutamylcysteine synthetase (GCS). The present invention includes BM31 that in the low dose range (50-200 nM) provides protections for the MCF-7 cells from cytotoxic damage of carcinogen benzo[a]pyrene (B[a]P).

Alternative Embodiments

Part A

Construction of the 3D Model of Keap1 IVR and Virtual Screening

[0069] Using a novel fold recognition technique as implemented in program Prospect (Oak Ridge National Laboratory, Oak Ridge, Tenn., USA), the crystal structure of FadR (PDB entry: 1e2x), a fatty acid-responsive transcription factor is identified as a structure template for the construction of the 3D model of Keap1 IVR (residues 180-314). The percentage of the sequence identify and strong similarity between Keap1 IVR (residues 180-314) and the template (1e2x, residues 25-225) is 33.65%. Based on this template, 100 structural models of Keap1 IVR are generated using software MODELLER v8 (9) and the model with lowest objective function is selected for further study. As shown in FIG. 1, residues Cys-297 and CYS-288 (red surface) are inside the cavities, whereas Cys-273 (red surface) and Cys-257 (on the other side of the surface) are on the surface but not inside a cavity. Ethoxyquin (in green sticks) was docked into the surface cavity that includes Cys-297.

[0070] Docking of ethoxyquin (EQ) and virtual screening of chemical database are performed using software GOLD v3.0 (The Cambridge Crystallographic Data Centre, Cambridge, UK). The ligand is treated as fully flexible whereas the protein is kept rigid except that each serine, threonine, and tyrosine hydroxyl group are allowed to rotate to optimize hydrogen bonding. The scoring function GoldScore and the GOLD standard default parameter setting were selected.

Cell Culture

[0071] The HepG2, MCF-7, and 293T cell lines are purchased from the American Type Culture Collection (ATCC; Manassas, Va., USA). The HepG2 cells are grown in alpha-minimal essential media supplemented with non-essential amino acids, sodium pyruvate, 90% Earle's balanced salt solution, and 10% fetal bovine serum. MCF-7 cells are maintained in RPMI-1640 medium. The 293T cells are grown in DMEM supplemented with 10% fetal bovine serum.

[0072] Luciferase Assay

[0073] One representative ARE (A2-ARE) from GSTA2 is used in this work, and A2-ARE are cloned into the KpnI and MluI sites of pGL3-promoter vector, which contains a heterologous SV40 promoter and a firefly luciferase reporter gene. Cells are seeded at 1×10⁵ per well using 24-well plate, grown overnight before being transfected transfected with pGL3 and other designated constructs using LipofectAMINE (Invitrogen) for 5 h. The plasmid pRL, containing a renilla reniformis luciferase reporter gene, is cotransfected as internal control to correct transfection efficiency. After transfection, cells are treated with test compounds at designated concentrations for 24 h prior to harvesting. The cells are then washed twice with phosphate-buffered saline and harvested in 100 μL of 1× passive lysis buffer (Promega, Madison, Wis., USA). The luciferase activities are analyzed in 20-uL cell extracts with the Dual luciferase assay® kit (Promega) on a Lumat LB 9507 luminometer (Berthold Technologies, Oak Ridge, Tenn., USA). The related luciferase activities are expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. The fold of induction is expressed as ratio of induction from the treated cells versus the untreated. Values represent measurements from three independent experiments performed in triplicate and are presented as the mean±SEM.

Western Blot Analysis

[0074] Cells are treated with test compounds at designated concentrations and are harvested at 24 h. Sixty microgram of cell-lysate protein and 35 ug of nuclear extract are loaded into each lane, and electrophoresed through a 4-20% gradient SDS-PAGE gel (Bio-Rad, Mississauga, ON, Canada), and transferred onto a nitrocellulose membrane. The membrane is incubated with primary antibody and subsequently incubated with HRP-conjugated secondary antibody. Finally, ECL detection is carried out, and the results were recorded on X-ray film by autophotography.

Cell Viability and Capability of Compounds in Protecting Cells from Carcinogen

[0075] Cells are seeded at 4×10³ per well using 96-well plate, grown overnight before being treated with test compounds for 72 h. Viable cells are measured by MTT assays. To evaluate the ability of test compounds in protecting MCF-7 cells from the damage of B[a]P, MCF-7 cells are seeded at 2×10³ per well using 96-well plate, grown overnight before being treated with test compounds at designed concentration for 48 h. Next, cells are treated with 160 nM B[a]P in the presence of test compound for another 72 h. The viable cells are measured using MTT assays. Experiments are performed in triplicate and repeated twice.

Statistical Analysis

[0076] GRAPHPAD PRISM 5 software (San Diego, Calif., USA) is used to perform statistical analysis by t-test. p<0.05 is considered statistical significant.

Structural Model of Keap1 IVR Domain and Virtual Screening

[0077] Residues C257, C273, C288, and C297, all of which are in IVR region, have been proposed as the direct sensors of inducers of the ARE-regulated enzymes. According to the 3D IVR model, residues C288 and C297 are found to be inside surface cavities whereas C273 and C257 are on the surface, but they are not inside a cavity (FIG. 1). The Keap1 IVR model suggested residues C297 and C288 as two possible direct sensors of Nrf2 inducers. EQ, an effective inducer of ARE-regulated enzymes, is docked into the IVR structural model. Without any preset preference between cavities C288 and C297, EQ is docked into cavity C297 automatically using software GOLD, probably because of the fact that the cavity around C288 is shallow.

[0078] Next, the Keap1 IVR EQ complex is employed to virtually screen 90 000 compounds extracted from the NCI-3D chemical database (NCI, US) for putative Nrf2 inducers. The chemical structures and the predicted binding modes for each of the 50 top-score compounds are visually inspected. A multiple-cycle screening strategy is used. In the first cycle, chemical samples for 23 top-score compounds are obtained. Of the 23 compounds, seven hits are identified using ARE-driven luciferase assays. In the second cycle, the NCI-3D database for the analogues of the initial hits by a ligand-based approach using the tanimoto index (with a cutoff of 0.9) is screened. Chemical samples of 17 analogues are obtained of the hits from the first cycle and then they are subjected to cell-based assays. The two cycles of screenings identify 9 novel Nrf2 inducers (Scheme 1 below).

##STR00028## ##STR00029##

ARE-driven luciferase assays and Western blot analyses of the top-score compounds have identified novel Nrf2 inducers

[0079] The 23 top-score molecules from the first round of virtual screening are initially tested in the cell-based ARE-driven luciferase assay. Specifically, HepG2 cell line are transfected with ARE-driven pGL3 luciferase vector and then exposed to individual test compounds at 10, 50 μM. Classical Nrf2 inducers EQ and SFN were used as the positive controls. Our results showed that 7 out of 23 top-score compounds are significantly active in inducing ARE-driven luciferase activities (FIG. 3). To examine the effect of these active compounds on Nrf2 nuclear translocation, Western blot analysis is used to measure the nuclear level of Nrf2 after cells are exposed to these compounds. Among the 7 hits, compound BM10 is most potent in inducing Nrf2 nuclear translocation (FIG. 8). However, although compounds BM4, BM6, and BM18 potently induce ARE-driven luciferase activities (FIG. 2), it is found they only have modest activity in inducing Nrf2 nuclear translocation (FIG. 8), suggesting the Western blot analysis of Nrf2 nuclear translocation is a more reliable approach to detect Nrf2 inducers. Consequently, the 17 compounds from the second cycle of screening are directly subjected to Western blot analysis, which leads to identification of BM31 and BM40 as potent Nrf2 inducers. Overall, our results show BM10, BM31, and BM40 potently elevate the nuclear Nrf2 in HepG2 cells, and their potencies are comparable to that of SFN (FIG. 3A).

Compounds BM10, 31 and 40 Induce Downstream Carcinogen-Detoxifying Enzymes

[0080] Nrf2 is a master transcription factor regulating multiple ARE-regulated antioxidant and detoxifying enzymes, such as GCS, glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase-1 (NQO1), and UDP glucuronosyltransferase (UGT), etc. The effects of our lead compounds on GCS are examined. Our results demonstrate that GCS was indeed induced by BM10, 31, 40 (FIG. 3B). The potencies of BM31 and BM40 are comparable to that of SFN and EQ. It is of note that despite BM10 potently inducing nuclear translocation of Nrf2, it shows modest potency in inducing the GCS.

Impact of Keap1 Mutation on the Nrf2-Inducing Potency of BM31 and SFN

[0081] Keap1 is a multi-domain protein, in which the IVR domain contains 4 cysteine residues, C257, C273, C288 and C297. The known Nrf2 inducers may interact with these cysteine residues in the IVR domain, resulting in conformational change of Keap1 and ultimately activating Nrf2. To investigate the role of these active cysteine residues in the Nrf2-inducing activity of BM31, 4 Keap1 mutants (C257S, C273S, C288S and C297S) are tested. BM31 is selected for this and further investigation because the dose-response is consistent for its capability in inducing Nrf2 nuclear translocation and induction of enzyme GCS (FIG. 3). Co-transfection experiments to investigate the effects of Keap1 IVR cysteine mutants on ARE-driven luciferase activities are performed. Specifically, 293T cells are co-transfected with ARE-driven pGL3 luciferase report vector, Keap1 vector with and without the Nrf2 expression vector (FIG. 4). After transfection, the cells are exposed to BM31 at 50 μM for 16 h before harvest. The cell lysates are used for measurement of ARE-driven luciferase activities. The data demonstrate that the compound BM31 potently induces the ARE-driven luciferase activities when cells are co-transfected with ARE-pGL3, Nrf2 and the C257S, C273S or C288S mutant Keap1 vectors. The results further demonstrate that the C273S and C288S Keap1 mutations led to the partial loss of basal level inhibition on Nrf2 by Keap1 and could constitutively activate the ARE-driven luciferase activities (FIG. 4), consistent with previous findings. However, it is found that the C273S and C288S mutations do not completely abolish the activity of BM31 or SFN in activating the ARE-driven luciferase activities (FIG. 4). In particular, our data demonstrated that Keap1 C297S mutant limits the potency of BM31 but not SFN in activating the ARE-driven luciferase activities (FIG. 4). Our conclusion is that BM31 targets the binding site around C297 in the IVR domain of Keap1, and mutation of residue C297 partially abolishes the Nrf2-inducing effect of BM31.

BM31 and SFP enhance the Keap1-Nrf2 association

[0082] To study the effect of BM31 on the interaction of Keap1 and Nrf2, Co-IP experiments are performed. 293T cells are successfully transfected and achieved high Nrf2 expression in this cell line. In the co-IP experiments, 293T cells are co-transfected with Nrf2 and Keap1 expression vectors. After transfection, the cells are treated with SFN and BM31 for 24 h before harvesting. The cell lysates are used for IP with Keap1 antibody. The eluted proteins are immunoblotted with Nrf2 antibody. The results reveal that treatment with BM31 or SFN increases the Nrf2/Keap1 complex (FIG. 5), indicating that BM31 may be inducing Nrf2 by modulating interaction between Keap1 and another protein partner in Keap1-Nrf2 pathway. For example, arsenic induces the Nrf2-dependent response through markedly enhancing the interaction between Keap1 and Cul3, subunits of the E3 ubiquitin ligase for Nrf2, which leads to impaired dynamic assembly/disassembly of the E3 ubiquitin ligase and thus decreases its ligase activity, resulting in inhibition of Nrf2 ubiquitination and degradation. Our further work will determine whether BM31 induces Nrf2 via a mechanism similar to that of arsenic.

BM31 Protects MCF-7 Cells from the Cytotoxic Damage of the Carcinogen B[a]P

[0083] To test the capability of BM compounds in protecting against the cytotoxic effect of B[a]P, cellular assays using MCF-7 cells are performed. Incubation of MCF-7 cells with various concentrations of BM31 for 48 hours is followed by incubation with 160 nM B[a]P+the BM31 for 72 hours. MTT assays are performed to evaluate the protection effect of the BM31. DMSO without B[a]P was used as a control. As shown in FIG. 6, in the low dose range (from 50 nM to 200 nM), BM31 provides significant protection for the MCF-7 cells from the cytotoxic damage of the carcinogen B[a]P (FIG. 6). In addition, it is found that BM10, BM31 and BM40 do not have cytotoxic effects on the proliferation of MCF-7 cells in a broad dose range (10 nM to 10 μM), which distinguishes them from SFN (FIG. 9).

Sequence CWU 1

1

41624PRTHomo sapiens 1Met Gln Pro Asp Pro Arg Pro Ser Gly Ala Gly Ala Cys Cys Arg Phe 1 5 10 15 Leu Pro Leu Gln Ser Gln Cys Pro Glu Gly Ala Gly Asp Ala Val Met 20 25 30 Tyr Ala Ser Thr Glu Cys Lys Ala Glu Val Thr Pro Ser Gln His Gly 35 40 45 Asn Arg Thr Phe Ser Tyr Thr Leu Glu Asp His Thr Lys Gln Ala Phe 50 55 60 Gly Ile Met Asn Glu Leu Arg Leu Ser Gln Gln Leu Cys Asp Val Thr 65 70 75 80 Leu Gln Val Lys Tyr Gln Asp Ala Pro Ala Ala Gln Phe Met Ala His 85 90 95 Lys Val Val Leu Ala Ser Ser Ser Pro Val Phe Lys Ala Met Phe Thr 100 105 110 Asn Gly Leu Arg Glu Gln Gly Met Glu Val Val Ser Ile Glu Gly Ile 115 120 125 His Pro Lys Val Met Glu Arg Leu Ile Glu Phe Ala Tyr Thr Ala Ser 130 135 140 Ile Ser Met Gly Glu Lys Cys Val Leu His Val Met Asn Gly Ala Val 145 150 155 160 Met Tyr Gln Ile Asp Ser Val Val Arg Ala Cys Ser Asp Phe Leu Val 165 170 175 Gln Gln Leu Asp Pro Ser Asn Ala Ile Gly Ile Ala Asn Phe Ala Glu 180 185 190 Gln Ile Gly Cys Val Glu Leu His Gln Arg Ala Arg Glu Tyr Ile Tyr 195 200 205 Met His Phe Gly Glu Val Ala Lys Gln Glu Glu Phe Phe Asn Leu Ser 210 215 220 His Cys Gln Leu Val Thr Leu Ile Ser Arg Asp Asp Leu Asn Val Arg 225 230 235 240 Cys Glu Ser Glu Val Phe His Ala Cys Ile Asn Trp Val Lys Tyr Asp 245 250 255 Cys Glu Gln Arg Arg Phe Tyr Val Gln Ala Leu Leu Arg Ala Val Arg 260 265 270 Cys His Ser Leu Thr Pro Asn Phe Leu Gln Met Gln Leu Gln Lys Cys 275 280 285 Glu Ile Leu Gln Ser Asp Ser Arg Cys Lys Asp Tyr Leu Val Lys Ile 290 295 300 Phe Glu Glu Leu Thr Leu His Lys Pro Thr Gln Val Met Pro Cys Arg 305 310 315 320 Ala Pro Lys Val Gly Arg Leu Ile Tyr Thr Ala Gly Gly Tyr Phe Arg 325 330 335 Gln Ser Leu Ser Tyr Leu Glu Ala Tyr Asn Pro Ser Asp Gly Thr Trp 340 345 350 Leu Arg Leu Ala Asp Leu Gln Val Pro Arg Ser Gly Leu Ala Gly Cys 355 360 365 Val Val Gly Gly Leu Leu Tyr Ala Val Gly Gly Arg Asn Asn Ser Pro 370 375 380 Asp Gly Asn Thr Asp Ser Ser Ala Leu Asp Cys Tyr Asn Pro Met Thr 385 390 395 400 Asn Gln Trp Ser Pro Cys Ala Pro Met Ser Val Pro Arg Asn Arg Ile 405 410 415 Gly Val Gly Val Ile Asp Gly His Ile Tyr Ala Val Gly Gly Ser His 420 425 430 Gly Cys Ile His His Asn Ser Val Glu Arg Tyr Glu Pro Glu Arg Asp 435 440 445 Glu Trp His Leu Val Ala Pro Met Leu Thr Arg Arg Ile Gly Val Gly 450 455 460 Val Ala Val Leu Asn Arg Leu Leu Tyr Ala Val Gly Gly Phe Asp Gly 465 470 475 480 Thr Asn Arg Leu Asn Ser Ala Glu Cys Tyr Tyr Pro Glu Arg Asn Glu 485 490 495 Trp Arg Met Ile Thr Ala Met Asn Thr Ile Arg Ser Gly Ala Gly Val 500 505 510 Cys Val Leu His Asn Cys Ile Tyr Ala Ala Gly Gly Tyr Asp Gly Gln 515 520 525 Asp Gln Leu Asn Ser Val Glu Arg Tyr Asp Val Glu Thr Glu Thr Trp 530 535 540 Thr Phe Val Ala Pro Met Lys His Arg Arg Ser Ala Leu Gly Ile Thr 545 550 555 560 Val His Gln Gly Arg Ile Tyr Val Leu Gly Gly Tyr Asp Gly His Thr 565 570 575 Phe Leu Asp Ser Val Glu Cys Tyr Asp Pro Asp Thr Asp Thr Trp Ser 580 585 590 Glu Val Thr Arg Met Thr Ser Gly Arg Ser Gly Val Gly Val Ala Val 595 600 605 Thr Met Glu Pro Cys Arg Lys Gln Ile Asp Gln Gln Asn Cys Thr Cys 610 615 620 2104PRTHomo sapiens 2Met Asn Glu Leu Arg Leu Ser Gln Gln Leu Cys Asp Val Thr Leu Gln 1 5 10 15 Val Lys Tyr Gln Asp Ala Pro Ala Ala Gln Phe Met Ala His Lys Val 20 25 30 Val Leu Ala Ser Ser Ser Pro Val Phe Lys Ala Met Phe Thr Asn Gly 35 40 45 Leu Arg Glu Gln Gly Met Glu Val Val Ser Ile Glu Gly Ile His Pro 50 55 60 Lys Val Met Glu Arg Leu Ile Glu Phe Ala Tyr Thr Ala Ser Ile Ser 65 70 75 80 Met Gly Glu Lys Cys Val Leu His Val Met Asn Gly Ala Val Met Tyr 85 90 95 Gln Ile Asp Ser Val Val Arg Ala 100 3135PRTHomo sapiens 3Asp Pro Ser Asn Ala Ile Gly Ile Ala Asn Phe Ala Glu Gln Ile Gly 1 5 10 15 Cys Val Glu Leu His Gln Arg Ala Arg Glu Tyr Ile Tyr Met His Phe 20 25 30 Gly Glu Val Ala Lys Gln Glu Glu Phe Phe Asn Leu Ser His Cys Gln 35 40 45 Leu Val Thr Leu Ile Ser Arg Asp Asp Leu Asn Val Arg Cys Glu Ser 50 55 60 Glu Val Phe His Ala Cys Ile Asn Trp Val Lys Tyr Asp Cys Glu Gln 65 70 75 80 Arg Arg Phe Tyr Val Gln Ala Leu Leu Arg Ala Val Arg Cys His Ser 85 90 95 Leu Thr Pro Asn Phe Leu Gln Met Gln Leu Gln Lys Cys Glu Ile Leu 100 105 110 Gln Ser Asp Ser Arg Cys Lys Asp Tyr Leu Val Lys Ile Phe Glu Glu 115 120 125 Leu Thr Leu His Lys Pro Thr 130 135 4284PRTHomo sapiens 4Gln Val Met Pro Cys Arg Ala Pro Lys Val Gly Arg Leu Ile Tyr Thr 1 5 10 15 Ala Gly Gly Tyr Phe Arg Gln Ser Leu Ser Tyr Leu Glu Ala Tyr Asn 20 25 30 Pro Ser Asp Gly Thr Trp Leu Arg Leu Ala Asp Leu Gln Val Pro Arg 35 40 45 Ser Gly Leu Ala Gly Cys Val Val Gly Gly Leu Leu Tyr Ala Val Gly 50 55 60 Gly Arg Asn Asn Ser Pro Asp Gly Asn Thr Asp Ser Ser Ala Leu Asp 65 70 75 80 Cys Tyr Asn Pro Met Thr Asn Gln Trp Ser Pro Cys Ala Pro Met Ser 85 90 95 Val Pro Arg Asn Arg Ile Gly Val Gly Val Ile Asp Gly His Ile Tyr 100 105 110 Ala Val Gly Gly Ser His Gly Cys Ile His His Asn Ser Val Glu Arg 115 120 125 Tyr Glu Pro Glu Arg Asp Glu Trp His Leu Val Ala Pro Met Leu Thr 130 135 140 Arg Arg Ile Gly Val Gly Val Ala Val Leu Asn Arg Leu Leu Tyr Ala 145 150 155 160 Val Gly Gly Phe Asp Gly Thr Asn Arg Leu Asn Ser Ala Glu Cys Tyr 165 170 175 Tyr Pro Glu Arg Asn Glu Trp Arg Met Ile Thr Ala Met Asn Thr Ile 180 185 190 Arg Ser Gly Ala Gly Val Cys Val Leu His Asn Cys Ile Tyr Ala Ala 195 200 205 Gly Gly Tyr Asp Gly Gln Asp Gln Leu Asn Ser Val Glu Arg Tyr Asp 210 215 220 Val Glu Thr Glu Thr Trp Thr Phe Val Ala Pro Met Lys His Arg Arg 225 230 235 240 Ser Ala Leu Gly Ile Thr Val His Gln Gly Arg Ile Tyr Val Leu Gly 245 250 255 Gly Tyr Asp Gly His Thr Phe Leu Asp Ser Val Glu Cys Tyr Asp Pro 260 265 270 Asp Thr Asp Thr Trp Ser Glu Val Thr Arg Met Thr 275 280

Claims

1. A modulator of a Nrf2 protein comprising a compound which binds at least one of a BTB domain, an IVR domain and a Kelch domain of a Keap1 protein, wherein said BTB domain has an amino acid sequence as set forth in SEQ ID NO:2, said IVR domain has an amino acid sequence as set forth in SEQ ID NO:3, and said Kelch domain has an amino acid sequence as set forth in SEQ ID NO:4, and wherein said modulator restores, enhances, reduces or impairs protein-protein interactions among said Keap1 protein and at least one of said Nrf2 protein and a Cul3 protein, for inhibiting or activating the activity of Nrf2; and wherein said compound is a compound of formula: ##STR00030## wherein R₁₇, R₁8, R₁₉, and R₂0 are independently chosen or identical, and are chosen from hydrogen, halide, hydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, alkyloxide, substituted alkyloxide, halogenated alkyl, halogenated alkenyl, halogenated alkyloxide, halogenated substituted alkyloxide, amine, substituted amine, cycloalkyl, substituted cycloalkyl, heterocyclic aromatic or non-aromatic 5- to 10-membered ring containing 1-4 heteroatoms selected from N, O or S, wherein N and S can be oxidized and N can be quaternized, ═O, CH₃, OCH₃, OC₂H₅, NO₂, CN, F, Cl, Br, SH, CF₃, OCF₃, O(CF₂)₂H, NH₂, N (CH₃)₂, N (C₂H₄OH)₂, CH(OC₂H₅)₂, ##STR00031## wherein indicates an attachment point, and R' is chosen from hydrogen, halide, hydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, alkyloxide, substituted alkyloxide, halogenated alkyl, halogenated alkenyl, halogenated alkyloxide, halogenated substituted alkyloxide, amine, substituted amine, cycloalkyl, substituted cycloalkyl, heterocyclic aromatic or non-aromatic 5- to 10-membered ring containing 1-4 heteroatoms selected from N, O or S, wherein N and S can be oxidized and N can be quaternized, ═O, CH₃, OCH₃, OC₂H₅, NO₂, CN, F, Cl, Br, SH, CF₃, OCF₃, O(CF₂)₂H, NH₂, N (CH₃)₂, N(C₂H₄OH)₂, CH(OC₂H₅)₂, ##STR00032## wherein is a single bond or a double bond; with the proviso that R₁₇ is different than O, S, NYZ, wherein Y and Z is H, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted heteroaryl, R₁₉ is different than ##STR00033## wherein X is O, N, or S, and Y is H, a C₁-C₁0 alkyl group, alkenyl, vinyl, aryl, or heteroaryl, and pharmaceutically acceptable salt, racemic mixture, enantiomer, diastereoisomer, isomer, and tautomer thereof.

2. (canceled)

3. (canceled)

4. (canceled)

5. The modulator according to claim 1, wherein said compound of formula (III) is: ##STR00034##

6. The modulator according to claim 1, wherein said compound of formula (III) is: ##STR00035##

7. The modulator according to claim 1, wherein said compound of formula (III) is: ##STR00036##

8. The modulator according to claim 1, wherein said compound of formula (III) are: ##STR00037## ##STR00038##

9. (canceled)

10. (canceled)

11. (canceled)

12.-14. (canceled)

15. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (III) according to claim 1, in association with a pharmaceutically acceptable carrier.

16. (canceled)

17. (canceled)

18. (canceled)

19.-26. (canceled)

27. A method for preventing or treating a disease which involves the abnormal activation and or expression level of a Nrf2 protein in a patient in need thereof, and/or the abnormal inhibition of a Nrf2 protein, the method comprising administering a therapeutically effective amount of a compound of formula as defined in claim 1.

28. (canceled)

29. The method according to claim 27, wherein said disease is an oxidative stress associated disease chosen from Parkinson's disease, Parkinson's disease with dementia with Lewy body, Huntington's disease, multiple system atrophy (MSA), progressive supranuclear palsy (PSA), corticobasal degeneration (CBD), frontotemporal lobe degeneration, atherosclerosis, heart failure, myocardial infarction, Alzheimer's disease, Fragile X syndrome, and chronic fatigue.

30. (canceled)

31. The method of claim 27, wherein said disease is a cancer.

32. (canceled)

33. The method as claimed in claim 31, wherein said cancer is liver cancer, lung cancer, breast cancer, prostate cancer, colon cancer, neuroblastoma or leukemia.

34. The method of claim 27, wherein said administering is for effecting neuroprotection.

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