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Review
. 2015 Jul;5(4):285-99.
doi: 10.1016/j.apsb.2015.05.008. Epub 2015 Jul 2.

Discovery of direct inhibitors of Keap1-Nrf2 protein-protein interaction as potential therapeutic and preventive agents

Affiliations
Review

Discovery of direct inhibitors of Keap1-Nrf2 protein-protein interaction as potential therapeutic and preventive agents

Dhulfiqar Ali Abed et al. Acta Pharm Sin B. 2015 Jul.

Abstract

The Keap1-Nrf2-ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1-Nrf2 protein-protein interaction (PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of Keap1-Nrf2 PPI and they are electrophilic species that act by modifying the sulfhydryl groups of Keap1׳s cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of Keap1-Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target Keap1 Kelch domain protein. These non-covalent direct inhibitors of Keap1-Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions.

Keywords: 1O2, singlet oxygen; AD, Alzheimer׳s disease; ARE, antioxidant response element; BTB, broad complex, tramtrack and bric-a-brac; Bach1, BTB and CNC homology 1; CBP, cAMP response element binding (CREB) protein; CDDO-Me, bardoxolone methyl; COPD, chronic obstructive pulmonary disease; CTR, C-terminal region; CVD, cardiovascular disease; DGR, double glycine repeats; Direct inhibitors of protein–protein interaction; FITC, flurescein isothiocyanate; FP, fluorescence polarization; GCL, glutamate-cysteine ligase; GPx, glutathione peroxidase; GST, glutathione S-transferase; H2O2, hydrogen peroxide; HO-1, heme-oxygenase-1; HTS, high-throughput screening; High throughput screening assays; IBS, inflammatory bowel disease; IVR, intervening region; Keap1; Keap1, Kelch-like ECH-associated protein 1; MD, molecular dynamics; NMR, .; NO, nitric oxide; NQO1, NAD(P)H quinone oxidoreductase I; NTR, N-terminal region; Nrf2; Nrf2, nuclear factor erythroid 2–related factor 2; Oxidative stress; PD, Parkinson׳s disease; PPI, protein–protein interaction; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; SPR, surface plasmon resonance; STZ, streptozotocin; Structure–activity relationships; THIQ, tetrahydroisoquinoline; TRX, thioredoxin; X-ray crystallography; [Formula: see text], peroxynitrate; [Formula: see text], superoxide, OH·, hydroxyl radical; vitamin C, ascorbate; vitamin E, tocopherols.

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Figures

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Graphical abstract
Figure 1
Figure 1
The antioxidant defense system employed by our body to defend against and neutralize the damaging effects of oxidative stress. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are constantly produced by normal cellular processes and environmental sources. Their damaging effects are mitigated through direction reduction by dietary or endogenous antioxidants or through the more efficient catalytic detoxification by various antioxidant enzymes under the control of transcription factor Nrf2. Keap1 serves as an important redox sensor involved in the feedback regulation of oxidative stress response.
Figure 2
Figure 2
The organization and domain structure of Nrf2.
Figure 3
Figure 3
The organization and domain structure of Keap1.
Figure 4
Figure 4
The Keap1-Nrf-ARE pathway. In the "hinge" and "latch" mechanism of Nrf2 regulation, the high affinity ETGE motif of Nrf2 initially binds to Kelch domain of Keap1 and the lower affinity affinity DLG motif binds to the second Keap1 to close the conformation. Nrf2 is polyubiquitinated at its Lys rich (7K) region and targeted for subsequent degradation by the 26S proteasome.
Figure 5
Figure 5
Summary of structure–activity relationship data for the DLG motif (residues 24–30; blue) and the ETGE motif (residues 76–83; red).
Figure 6
Figure 6
Structure–activity relationships around LH601A (1). The activity noted was based on the solution competition SPR assay.
Figure 7
Figure 7
Structures of direct inhibitors of Keap1–Nrf2 PPI that have recently been reported.
Figure 8
Figure 8
Structures of the Kelch domain of human Keap1 bound to an Nrf2 peptide. (A) A top-down view showing the six-bladed β-propeller structure in red ribbon and the peptide as a yellow tube. Each blade of the β-propeller is numbered I–VI. Both the N- and C-termini of the domain are located in blade I and are labeled N and C, respectively. The four β-strands found in each blade are designated A–D as shown in white font on blade VI. (B) A surface representation of the Kelch propeller (gray) and peptide (yellow tube). Selected residues are shown in blue (basic), orange (polar) and green (apolar). (C) Charge–charge and H-bonding interactions between the side chain atoms of the Nrf2 peptide and residues in the Kelch domain. Not shown are 5 H-bond interactions between the peptide backbone atoms and residues in the Kelch domain (reproduced with permission from reference108).
Figure 9
Figure 9
The interactions observed in X-ray co-crystal structures of Keap1 Kelch domain with small molecule direct inhibitors of Keap1–Nrf2 PPI: (A) 1 or (B) 2 or (C) 3. Ionic interactions are indicated with red dotted lines and hydrophobic interactions are indicated with blue dotted double sided arrows. There are two ligands occupying the binding site in the co-crystal structure of Keap1 Kelch domain with 2. (A was reproduced with permission from reference113).

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