Keap1-Nrf2 Protein-protein Interaction Research Articles

Novel Inhibitor of Keap1-Nrf2 Protein-Protein Interaction Attenuates Osteoclastogenesis In Vitro and Prevents OVX-Induced Bone Loss In Vivo.

Keap1 interacts with Nrf2 by assisting in its ubiquitination and subsequent proteolysis. By preventing ROS accumulation during RANKL-induced osteoclastogenesis, Nrf2 activation can prevent the differentiation of osteoclasts. Additionally, inhibiting the Keap1-Nrf2 PPI can be an effective strategy for triggering Nrf2 to regulate oxidative stress. Structure-based virtual screening was performed to discover a potentially novel Keap1-Nrf2 PPI inhibitor wherein KCB-F06 was identified. The inhibitory effects of KCB-F06 on osteoclastogenesis were investigated in vitro through TRAP staining and bone resorption assays. An ovariectomy-induced osteoporosis mouse model was applied to evaluate KCB-F06's therapeutic effects in vivo. Lastly, the underlying mechanisms were explored using real-time PCR, Western blotting, and co-IP assays. KCB-F06 was discovered as a novel Keap1-Nrf2 PPI inhibitor. As a result, the expression of antioxidants (HO-1 and NQO1) was suppressed, hence reducing ROS accumulation during osteoclastogenesis. Subsequently, this caused the inactivation of RANKL-induced IKB/NF-kB signaling. This eventually led to the downregulation of osteoclast-specific proteins including NFATc1, which is an essential transcription factor for osteoclastogenesis. These results demonstrated that Nrf2 activation in osteoclasts is a valuable tool for osteoclastic bone loss management. In addition, KCB-F06 presents as an alternative candidate for treating osteoclast-related bone diseases and as a novel small molecule that can serve as a model for further Keap1-NRF2 PPI inhibitor development.

Electrophiles and Keap1-Nrf2 protein-protein interaction inhibitors for prevention and treatment of chronic disease

Electrophiles and Keap1-Nrf2 protein-protein interaction inhibitors for prevention and treatment of chronic disease

Cyclic Peptide Keap1-Nrf2 Protein-Protein Interaction Inhibitors: Design, Synthesis, and In Vivo Treatment of Acute Lung Injury.

Directly blocking the Keap1-Nrf2 pathway is a promising strategy for the mitigation of acute lung injury (ALI). Peptide Keap1-Nrf2 inhibitors have been reported to have a high Keap1 binding affinity. However, these inhibitors showed weak activity in cells and/or animals. In this study, we designed a series of linear peptides from an Nrf2-based 9-mer Ac-LDEETGEFL-NH2. To improve the cellular activity, we further designed cyclic peptides based on the crystal complex of Keap1 with a linear peptide. Among them, cyclic 9-mer ZC9 targeting Keap1 showed a better affinity (KD2 = 51 nM). Specifically, it exhibited an acceptable water solubility (>38 mg/mL), better cell permeability, cell activity, and metabolic stability (serum t1/2 > 24 h). In the in vitro LPS-induced oxidative damages and ALI model, ZC9 showed significant dose-response reversal activity without apparent toxicity. In conclusion, our results suggested ZC9 as a lead cyclic peptide targeting the Keap1-Nrf2 pathway for ALI clinical treatment.

Optimization Efforts for Identification of Novel Highly Potent Keap1-Nrf2 Protein-Protein Interaction Inhibitors.

In research focused on protein-protein interaction (PPI) inhibitors, the optimization process to achieve both high inhibitory activity and favorable physicochemical properties remains challenging. Our previous study reported the discovery of novel and bioavailable Keap1-Nrf2 PPI inhibitor 8 which exhibited moderate in vivo activity in rats. In this work, we present our subsequent efforts to optimize this compound. Two distinct approaches were employed, targeting high energy water molecules and Ser602 as "hot spots" from the anchor with good aqueous solubility, metabolic stability, and membrane permeability. Through ligand efficiency (LE)-guided exploration, we identified two novel inhibitors 22 and 33 with good pharmacokinetics (PK) profiles and more potent in vivo activities, which appear to be promising chemical probes among the existing inhibitors.

Structural modification of C2-substituents on 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N′-diacetic acid derivatives as potent inhibitors of the Keap1-Nrf2 protein-protein interaction

The Keap1-Nrf2-ARE signaling pathway is an attractive therapeutic target for the prevention and treatment of oxidative stress-associated diseases by activating the cellular expression of cytoprotective enzymes and proteins. Small molecule inhibitors can directly disrupt the Keap1-Nrf2 protein-protein interaction (PPI), resulting in elevated levels of Nrf2 protein and subsequent stimulation of related antioxidant responses. Previously, we found that 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N′-diacetic acid derivatives with an ether type C2-substituent on the benzene or naphthalene core exhibited potent inhibitory activities with IC50's in the submicromolar or nanomolar range. We here describe a more detailed structure-activity relationship study around the C2 substituents containing various polar linkers shedding new insight on their binding interactions with the Keap1 Kelch domain. The key observation from our findings is that the substituents at the C2-position of the benzene or naphthalene scaffold impact their inhibitory potencies in biochemical assays as well as activities in cell culture. The biochemical FP and TR-FRET assays revealed that the naphthalene derivatives 17b and 18 with an additional carboxylate at the C2 were the most active inhibitors against Keap1-Nrf2 PPI. In the cell-based assay, the two compounds were shown to be potent Nrf2 activators of the transcription of the Nrf2-dependent genes, such as HMOX2, GSTM3, and NQO1.

Resveratrol improves meat quality traits by activating the lncRNAs-KEAP1-NRF2 axis in pigs

This research aims at uncovering the effects and investigating the molecular mechanisms of dietary resveratrol (RES) supplementation on antioxidant capacity and meat quality of pigs. In this study, 20 μM RES could activate the KEAP1-NRF2 antioxidant defense pathway in response to oxidative stress in porcine skeletal muscle satellite cells was firstly found. Then, twenty-four healthy crossbred castrated boars were allocated to 4 treatments that were fed with a basal diet (control) and a basal diet supplemented with 200 mg, 400 mg or 600 mg RES per Kilogram (kg) of feed for 41 days, respectively. 400 and 600 mg/kg RES-supplemented diet can effectively improve the meat quality traits and activities of antioxidizing enzymes via the KEAP1–NRF2 signaling pathway of pigs. The molecular dynamic simulation further revealed that RES could directly binding to KEAP1 to reduce the tightness of KEAP1–NRF2 protein-protein interaction. More importantly, dietary supplementation of RES also improves antioxidant capacity through a series of KEAP1–NRF2 pathway-related lncRNAs were found by RNA sequencing (RNA-seq). Altogether, this study demonstrated that RES improves meat quality traits by effectively increasing antioxidant levels via the lncRNA-KEAP1–NRF2 axis in vivo and/or in vitro. These results provide new insights into the molecular mechanisms by which RES, as a nutritional agent, regulates antioxidant capacity and improves meat quality in pigs.

In silico approach of bioactive molecule chitosan 501.1 kDa from snail shell as antioxidant and inhibitor of the keap1-nrf2 protein-protein interaction

Background: ROS are created when high levels of oxidative stress occur due to hypercholesterolemia. Nuclear Factor Erythroid 2-related factor (NRF2) controls the expression of antioxidant genes. Kelch-like ECH-associated protein 1 (KEAP1) therapy degrades NRF2. Chitosan 501.1 kDa from snail shells contains bioactive chemicals that can induce NRF2 activity. Objective: To evaluate the potential antioxidant activity of the bioactive compound in Mw 501.1 kDa chitosan by targeting KEAP1 and NRF2 proteins in-silico. Method: The 3D structures of the bioactive compounds chitosan and control 51M were derived from the PubChem database, and the proteins were derived from the RCSB PDB. The biological activity of chitosan bioactive compounds was predicted using the PASS Online server. Molecular docking was performed using Hex 8.0.0 Cuda with Shape+Electro+DARS and visualised with Discovery Studio. The biological activity of chitosan compounds was predicted as lipotropic and antioxidant. Result: The discovery of the bioactive compound chitosan 501.1 kDa interacted strongly with KEAP1. The bioactive compound chitosan also inhibited KEAP1 through residues GLN75 and LEU84 at the 51M-KEAP1 interaction. Conclusion: The bioactive compound chitosan 501.1 kDa could inhibit the interaction of KEAP1-NRF2 proteins so that NRF2 could transcribe antioxidant genes. Therefore, may serve as a suitable alternative.

Medicinal Chemistry Insights into the Development of Small-Molecule Kelch-Like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1-Nrf2) Protein-Protein Interaction Inhibitors.

Oxidative stress has been implicated in a wide range of pathological conditions. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) exerts a central role in regulating the cellular defense system against oxidative and electrophilic insults. Nonelectrophilic inhibition of the protein-protein interaction (PPI) between Kelch-like ECH-associated protein 1 (Keap1) and Nrf2 has become a promising approach to activate Nrf2. Recently, multiple drug discovery strategies have facilitated the development of small-molecule Keap1-Nrf2 PPI inhibitors with potent activity and favorable drug-like properties. In this Perspective, we summarize the latest progress of small-molecule Keap1-Nrf2 PPI inhibitors from medicinal chemistry insights and discuss future prospects and challenges in this field.

A potent phosphodiester Keap1-Nrf2 protein-protein interaction inhibitor as the efficient treatment of Alzheimer's disease

The Keap1-Nrf2 pathway has been established as a therapeutic target for Alzheimer's disease (AD). Directly inhibiting the protein-protein interaction (PPI) between Keap1 and Nrf2 has been reported as an effective strategy for treating AD. Our group has validated this in an AD mouse model for the first time using the inhibitor 1,4-diaminonaphthalene NXPZ-2 with high concentrations. In the present study, we reported a new phosphodiester containing diaminonaphthalene compound, POZL, designed to target the PPI interface using a structure-based design strategy to combat oxidative stress in AD pathogenesis. Our crystallographic verification confirms that POZL shows potent Keap1-Nrf2 inhibition. Remarkably, POZL showed its high in vivo anti-AD efficacy at a much lower dosage compared to NXPZ-2 in the transgenic APP/PS1 AD mouse model. POZL treatment in the transgenic mice could effectively ameliorate learning and memory dysfunction by promoting the Nrf2 nuclear translocation. As a result, the oxidative stress and AD biomarker expression such as BACE1 and hyperphosphorylation of Tau were significantly reduced, and the synaptic function was recovered. HE and Nissl staining confirmed that POZL improved brain tissue pathological changes by enhancing neuron quantity and function. Furthermore, it was confirmed that POZL could effectively reverse Aβ-caused synaptic damage by activating Nrf2 in primary cultured cortical neurons. Collectively, our findings demonstrated that the phosphodiester diaminonaphthalene Keap1-Nrf2 PPI inhibitor could be regarded as a promising preclinical candidate of AD.

Methyl and Fluorine Effects in Novel Orally Bioavailable Keap1-Nrf2 PPI Inhibitor.

Oxidative stress is one of the causes of progression of chronic kidney disease (CKD). Activation of the antioxidant protein regulator Nrf2 by inhibition of the Keap1-Nrf2 protein-protein interaction (PPI) is of interest as a potential treatment for CKD. We report the identification of the novel and weak PPI inhibitor 7 with good physical properties by a high throughput screening (HTS) campaign, followed by structural and computational analysis. The installation of only methyl and fluorine groups successfully provided the lead compound 25, which showed more than 400-fold stronger activity. Furthermore, these dramatic substituent effects can be explained by the analysis of using isothermal titration calorimetry (ITC). Thus, the resulting 25, which exhibited high oral absorption and durability, would be a CKD therapeutic agent because of the dose-dependent manner for up-regulation of the antioxidant protein heme oxigenase-1 (HO-1) in rat kidneys.

A small-molecule inhibitor of Keap1-Nrf2 interaction attenuates sepsis by selectively augmenting the antibacterial defence of macrophages at infection sites.

Macrophages at infection sites are considered as the promising therapeutic targets to prevent sepsis development. The Nrf2/Keap1 system acts as a critical modulator of the antibacterial activity of macrophages. Recently, Keap1-Nrf2 protein-protein interaction (PPI) inhibitors have emerged as safer and stronger Nrf2 activators; however, their therapeutic potential in sepsis remains unclear. Herein, we report a unique heptamethine dye, IR-61, as a Keap1-Nrf2 PPI inhibitor that preferentially accumulates in macrophages at infection sites. A mouse model of acute lung bacterial infection was used to investigate the biodistribution of IR-61. SPR study and CESTA were used to detect the Keap1 binding behaviour of IR-61 invitro and in cells. Established models of sepsis in mice were used to determine the therapeutic effect of IR-61. The relationship between Nrf2 levels and sepsis outcomes was preliminarily investigated using monocytes from human patients. Our data showed that IR-61 preferentially accumulated in macrophages at infection sites, enhanced bacterial clearance, and improved outcomes in mice with sepsis. Mechanistic studies indicated that IR-61 potentiated the antibacterial function of macrophages by activating Nrf2 via direct inhibition of the Keap1-Nrf2 interaction. Moreover, we observed that IR-61 enhanced the phagocytic ability of human macrophages, and the expression levels of Nrf2 in monocytes might be associated with the outcomes of sepsis patients. Our study demonstrates that the specific activation of Nrf2 in macrophages at infection sites is valuable for sepsis management. IR-61 may prove to be a Keap1-Nrf2 PPI inhibitor for the precise treatment of sepsis. This work was supported by the National Natural Science Foundation of China (Major program 82192884), the Intramural Research Project (Grants: 2018-JCJQ-ZQ-001 and 20QNPY018), and the Chongqing National Science Foundation (CSTB2022NSCQ-MSX1222).

Optimization of the C2 substituents on the 1,4-bis(arylsulfonamido)naphthalene-N,N′-diacetic acid scaffold for better inhibition of Keap1-Nrf2 protein-protein interaction

Direct inhibition of the protein-protein interaction (PPI) between Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) reduces the ubiquitination and subsequent degradation of Nrf2, leading to Nrf2 accumulation in the cytosol and the nuclear translocation of Nrf2. Once inside the nucleus, Nrf2 binds to and activates the expression of antioxidant response element (ARE) genes involved in redox homeostasis and detoxification. Herein, we report a series of 1,4-bis(arylsulfonamido)naphthalene-N,N′-diacetic acid analogs with varying C2 substituents to explore the structure-activity relationships at this position of the central naphthalene core. The Keap1-binding activities were first screened with a fluorescence polarization (FP) assay followed by further evaluation of the more potent compounds using a more sensitive time-resolved fluorescence energy transfer (TR-FRET) assay. It was found that compound 24a with C2-phthalimidopropyl group was the most potent in this series showing an IC50 of 2.5 nM in the TR-FRET assay with a Ki value in the subnanomolar range. Our docking study indicated that the C2-phthalimidopropyl group in compound 24a provided an extra hydrogen bonding interaction with the key residue Arg415 that may be responsible for the observed boost in binding affinity. In addition, compounds 12b, 15, and 24a were shown to activate the Nrf2 signaling pathway in NCM460D cells resulting in elevated mRNA levels of GSTM3, HMOX1 and NQO1 by 2.4–11.7 fold at 100 μM as compared to the vehicle control.

Inhibitors of Keap1-Nrf2 protein-protein interaction reduce estrogen responsive gene expression and oxidative stress in estrogen receptor-positive breast cancer

Estrogen contributes to the development of breast cancer through estrogen receptor (ER) signaling and by generating genotoxic metabolites that cause oxidative DNA damage. To protect against oxidative stress, cells activate nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream cytoprotective genes that initiate antioxidant responses and detoxify xenobiotics. Nrf2 activation occurs by inhibiting the protein-protein interaction (PPI) between Nrf2 and its inhibitor Keap1, which otherwise targets Nrf2 for ubiquitination and destruction. In this study, we examined a series of novel direct inhibitors of Keap1-Nrf2 PPI in their role in promoting the availability of Nrf2 for antioxidant activity and attenuating estrogen-mediated responses in breast cancer. ER-positive human breast cancer cells MCF-7 were treated with 17β-estradiol (E2) in the presence or absence of selected Keap1-Nrf2 PPI inhibitors. Keap1-Nrf2 PPI inhibitors suppressed the mRNA and protein levels of estrogen responsive genes induced by E2 exposure, such as PGR. Keap1-Nrf2 PPI inhibitors caused significant activation of Nrf2 target genes. E2 decreased the mRNA and protein level of the Nrf2 target gene NQO1, and the Keap1-Nrf2 PPI inhibitors reversed this effect. The reversal of E2 action by these compounds was not due to binding to ER as ER antagonists. Further, a selected compound attenuated oxidative stress induced by E2, determined by the level of a biomarker 8-oxo-deoxyguanosine. These findings suggest that the Keap1-Nrf2 PPI inhibitors have potent antioxidant activity by activating Nrf2 pathways and inhibit E2-induced gene and protein expression. These compounds may serve as potential chemopreventive agents in estrogen-stimulated breast cancer.

Dehydroeburicoic Acid, a Dual Inhibitor against Oxidative Stress in Alcoholic Liver Disease

Alcoholic liver disease (ALD) is a complicated disease which can lead to hepatocellular carcinoma; however, there is a lack of satisfactory therapeutics. Dehydroeburicoic acid (DEA) (1), a triterpenoid isolated from Antrodia cinnamomea, has been reported to act against ALD, but its mechanisms of action are still not clear. In this study, we report for the first time the use of DEA (1) as a dual inhibitor of the Keap1-Nrf2 protein-protein interaction (PPI) and GSK3β in an in vitro ALD cell model. DEA (1) engages Keap1 to disrupt the Keap1-Nrf2 PPI and inhibits GSK3β to restore Nrf2 activity in a Keap1-independent fashion. DEA (1) promotes Nrf2 nuclear translocation to activate downstream antioxidant genes. Importantly, DEA (1) restores the mitochondrial dysfunction induced by ethanol and generates antioxidant activity in the ALD cell model with minimal toxicity. We anticipate that DEA (1) could be a potential scaffold for the further development of clinical agents for treating ALD.

Development of Noncovalent Small-Molecule Keap1-Nrf2 Inhibitors by Fragment-Based Drug Discovery.

Targeting the protein-protein interaction (PPI) between the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its repressor, Kelch-like ECH-associated protein 1 (Keap1), constitutes a promising strategy for treating diseases involving oxidative stress and inflammation. Here, a fragment-based drug discovery (FBDD) campaign resulted in novel, high-affinity (Ki = 280 nM), and cell-active noncovalent small-molecule Keap1-Nrf2 PPI inhibitors. We screened 2500 fragments using orthogonal assays─fluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR)─and validated the hits by saturation transfer difference (STD) NMR, leading to 28 high-priority hits. Thirteen co-structures showed fragments binding mainly in the P4 and P5 subpockets of Keap1's Kelch domain, and three fluorenone-based fragments featuring a novel binding mode were optimized by structure-based drug discovery. We thereby disclose several fragment hits, including their binding modes, and show how FBDD can be performed to find new small-molecule Keap1-Nrf2 PPI inhibitors.

Design, synthesis and evaluation of novel small molecules acting as Keap1-Nrf2 protein-protein interaction inhibitors

Direct interference with Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 protein-protein interaction (PPI) has recently been introduced as an attractive approach to control life-threatening diseases like myocarditis. The present study aimed to investigate the potential application in myocarditis of a series of novel non-naphthalene derivatives as potential Keap1-Nrf2 PPI inhibitors. Our results indicated that the optimal compound K22 displayed the highest metabolic stability and showed notable Keap1-Nrf2 PPI inhibitory activities in vitro. K22 effectively triggered Nrf2 activation and increased the protein and mRNA expression of Nrf2-regulated genes in H9c2 cells. Moreover, pre-treatment with K22 was shown to mitigate LPS-induced damage to H9c2 cells, causing a marked decrease in the levels of inflammatory factors as well as reactive oxygen species (ROS). Furthermore, K22 was also shown to be non-mutagenic in the Ames test. Overall, our findings suggest that K22 may be a promising drug lead as a Keap1-Nrf2 PPI inhibitor for myocarditis treatment.

KEAP1-NRF2 protein-protein interaction inhibitors: Design, pharmacological properties and therapeutic potential.

The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is considered the master regulator of the phase II antioxidant response. It controls a plethora of cytoprotective genes related to oxidative stress, inflammation, and protein homeostasis, among other processes. Activation of these pathways has been described in numerous pathologies including cancer, cardiovascular, respiratory, renal, digestive, metabolic, autoimmune, and neurodegenerative diseases. Considering the increasing interest of discovering novel NRF2 activators due to its clinical application, initial efforts were devoted to the development of electrophilic drugs able to induce NRF2 nuclear accumulation by targeting its natural repressor protein Kelch-like ECH-associated protein 1 (KEAP1) through covalent modifications on cysteine residues. However, off-target effects of these drugs prompted the development of an innovative strategy, the search of KEAP1-NRF2 protein-protein interaction (PPI) inhibitors. These innovative activators are proposed to target NRF2 in a more selective way, leading to potentially improved drugs with the application for a variety of diseases that are currently under investigation. In this review, we summarize known KEAP1-NRF2 PPI inhibitors to date and the bases of their design highlighting the most important features of their respective interactions. We also discuss the preclinical pharmacological properties described for the most promising compounds.

Pharmacological characterization of a novel peptide inhibitor of the Keap1-Nrf2 protein-protein interaction

LAS200813 is a novel bicyclic lipopeptide that activates Nrf2 by binding to Keap1, thereby antagonising the Keap1-Nrf2 protein–protein interaction. In this work we report the pharmacological characterization of LAS200813 in Nrf2-dependent translational preclinical models. LAS200813 binds to Keap1 with high affinity (IC50: 0.73 nM) and is able to induce the translocation of Nrf2 to the nucleus. Furthermore, LAS200813 increases the expression of Nrf2 target genes in human bronchial epithelial cells (EC50 of 96 and 70 nM for srxn1 and nqo1, respectively). Similarly, the intratracheal administration of LAS200813 to rats increases the expression of Nrf2-dependent genes in lung tissue, an effect that lasts for a few hours. Moreover, in cells exposed to cigarette smoke, LAS200813 shows an antioxidant effect by increasing the production of glutathione and prevents cellular apoptosis. In conclusion, the results described herein demonstrate that LAS200813 is a potent non-electrophilic Nrf2-activating peptide designed to be administered by inhaled route which may be a potential therapeutic strategy for respiratory diseases driven by oxidative stress.

Crystallography-Guided Optimizations of the Keap1-Nrf2 Inhibitors on the Solvent Exposed Region: From Symmetric to Asymmetric Naphthalenesulfonamides.

Directly inhibiting the Keap1-Nrf2 protein-protein interaction has been investigated as a promising strategy to activate Nrf2 for anti-inflammation. We previously reported a naphthalensulfonamide Keap1-Nrf2 inhibitor NXPZ-2, but have not determined the exact binding mode with Keap1. This symmetric naphthalenesulfonamide compound has relatively low solubility. Herein, we first determined a crystal complex (resolution: 2.3 Å) of human Keap1 Kelch domain with NXPZ-2. Further optimizations on the solvent exposed region obtained asymmetric naphthalenesulfonamides and three crystal structures of Keap1 in complex with designed compounds. Among them, the asymmetric piperazinyl-naphthalenesulfonamide 6k with better aqueous solubility showed the best KD2 value of 0.21 μM to block the interaction. The productions of ROS and NO and the expression of TNF-α were inhibited by 6k in the in vitro model. This compound could relieve inflammations by significantly increasing the Nrf2 nuclear translocation in the LPS-induced ALI model with promising pharmacokinetic properties.

Phenyl Bis-Sulfonamide Keap1-Nrf2 Protein-Protein Interaction Inhibitors with an Alternative Binding Mode.

Inhibitors of Kelch-like ECH-associated protein 1 (Keap1) increase the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by stalling its ubiquitination and degradation. This enhances the expression of genes encoding proteins involved in drug detoxification, redox homeostasis, and mitochondrial function. Nrf2 activation offers a potential therapeutic approach for conditions including Alzheimer’s and Parkinson’s diseases, vascular inflammation, and chronic obstructive airway disease. Non-electrophilic Keap1-Nrf2 protein–protein interaction (PPI) inhibitors may have improved toxicity profiles and different pharmacological properties to cysteine-reactive electrophilic inhibitors. Here, we describe and characterize a series of phenyl bis-sulfonamide PPI inhibitors that bind to Keap1 at submicromolar concentrations. Structural studies reveal that the compounds bind to Keap1 in a distinct “peptidomimetic” conformation that resembles the Keap1-Nrf2 ETGE peptide complex. This is different to other small molecule Keap1-Nrf2 PPI inhibitors, including bicyclic aryl bis-sulfonamides, offering a starting point for new design approaches to Keap1 inhibitors.