BACKGROUND:Over the past decades, NRF2 has evolved from a redox/detox regulator to a central orchestrator of cellular stability, governing protein integrity, metabolism, immunity, and iron homeostasis, with roles in cancer, metabolic, inflammatory, and neurodegenerative diseases. It exhibits a "Taiji-like" duality: a cytoprotective "Yang phase" under stress versus a tumor-promoting "Yin phase" (featuring metabolic reprogramming and chemoresistance) upon aberrant activation. As a promising cancer target, specific NRF2 inhibitors remain largely in the preclinical stage, necessitating an integrated synthesis of its biological roles and translational potential. AIM OF REVIEW:This review systematically delineates the multifaceted biological functions of NRF2, with a focused emphasis on its context-dependent cancer-promoting effects. It not only integrates fragmented insights into NRF2's dualistic roles in cellular homeostasis and tumor progression but also highlights underexplored nuances within its regulatory network. Furthermore, it discusses potential NRF2 inhibition strategies, profiles investigational NRF2-targeting agents, and examines key therapeutic challenges that hinder the translation of these inhibitors into clinical practice. KEY SCIENTIFIC CONCEPTS OF REVIEW:We contextualize NRF2's dualism as a core barrier to its therapeutic targeting: while its cytoprotective functions are indispensable for physiological homeostasis, its overactivation fuels tumor progression. We further survey the landscape of preclinical NRF2 inhibitors, analyzing their mechanisms of action and limitations in specificity or efficacy. A central focus is placed on how resolving the paradox of NRF2's dual roles-through precision strategies that selectively abrogate its tumor-promoting Yin phase-could unlock its therapeutic potential in cancer. By bridging insights into NRF2's biological regulation with translational oncology and precision medicine frameworks, this review outlines a path to advance NRF2-targeted therapies.

PIM kinases, as members of the serine/threonine kinase family, regulate key cellular processes such as proliferation, apoptosis, and metabolism by phosphorylating multiple substrates, making them important therapeutic targets for cancer treatment. In this study, we reported a series of structurally novel PIM-1 kinase inhibitors based on a scaffold-hopping strategy. After multiple rounds of structural optimization, the highly active compound C2 was obtained, exhibiting an IC50 of 33.02 ± 1.31 nM against PIM-1 kinase. Molecular docking results revealed that compound C2 stably bound to the hydrophobic cavity of the PIM-1 protein and formed hydrogen bond interactions with polar residues in the hinge region, thereby effectively inhibiting kinase activity. In vitro antitumor assessment demonstrated significant proliferation inhibition of the hematological tumor cell line MM.1S (IC50 = 1.87 μM), comparable to the positive control SGI-1776 (IC50 = 1.71 μM). In addition, compound C2 possessed favorable drug-like properties and excellent stability in simulated gastrointestinal fluids and rat plasma. This study provides promising lead compounds for the development of novel PIM-1-targeted anticancer drugs, which can be further optimized.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pleiotropic transcription factor essential for cellular defense. Extensive research has demonstrated its oncogenic role, positioning Nrf2 as a promising target for cancer therapy. However, achieving tumor-specific Nrf2 inhibition remains a challenge. Herein, we report the discovery of 13, a biased inhibitor of Kelch-like ECH-associated protein 1-phosphorylated p62 (Keap1-p-p62) interaction, as a selective and effective Nrf2 inhibitor. In fluorescence polarization assays, 13 showed potent Keap1-p-p62 inhibitory activity (IC50 = 0.11 μM) and high selectivity over the inhibition of Keap1-Nrf2 interaction. Notably, 13 specifically inhibited Nrf2 activity in p62 aberrant hepatocellular carcinoma (HCC) cells by selectively disrupting Keap1-p-p62 interaction and normalizing Nrf2 ubiquitination. Furthermore, cotreatment with 13 sensitized p62 aberrant HCC to ferroptosis induced by sorafenib (TGI = 95.3%). Overall, our study identifies a biased Keap1-p-p62 inhibitor 13 as a targeted Nrf2 inhibition therapy and provides valuable insights into HCC treatment.

Myeloid cell leukemia 1 (Mcl-1) is a pivotal anti-apoptotic protein, whose overexpression drives tumorigenesis, progression and drug resistance in multiple hematological malignancies. Herein, guided by a bioisosteric design, we replaced the sulfonyl group with a drug-like sulfonimide linker and synthesized a novel series of Mcl-1 inhibitors based on our previous lead compound DDO-8201. Among them, compound 52 displayed potent and selective Mcl-1 inhibition (Ki = 0.024 μM) with at least 400-fold selectivity over other Bcl-2 family proteins. It showed an IC50 of 0.45 μM against Mcl-1-sensitive Molm-13 cells, superior to A1210477, and retained good activity against Venetoclax-resistant Molm-13 cells (Molm-13_VenR, IC50 = 1.16 μM). Moreover, 52 exhibited favorable drug-like properties, including good membrane permeability (Pe = 11.11 × 10-6 cm/s), water solubility (78.5 μg/mL), high stability (HLM, t1/2 = 1.83 h) and oral bioavailability (F% = 32%). This study provided a new strategy for Mcl-1 inhibitor development, and 52 represents a promising candidate for overcoming Venetoclax resistance.

INTRODUCTION:Kelch-like ECH-associated protein 1 (Keap1), an E3 ligase negatively regulating the nuclear factor erythroid 2-related factor 2 (Nrf2), has emerged as an auspicious drug target for treating ailments associated with oxidative stress and inflammation. Discovery of Keap1 inhibitors have attracted significant interest. AREAS COVERED:This review covers patents on Keap1 inhibitors from 2019 to 2024, providing a comprehensive analysis of their structural characteristics, optimization strategies, pharmacological properties and clinical progress. EXPERT OPINION:Extensive efforts have been devoted to enhance potency and drug-like properties of Keap1 inhibitors. Strategies such as ROS-cleavable prodrug design, bivalent inhibition and PROTACs are emerging. As the range of drug types and applications expands, Keap1 inhibitors are becoming a sagacious option for disease treating.

The NRF2 transcription factor is constitutively active in various cancers, functioning as an oncogenic driver for tumor progression and chemo/radiotherapy resistance. Despite the well-documented role of NRF2 overactivation in cancer, no targeted therapy is currently available. In this study, using a combination of phenotypic screening, chemoproteomics, and biochemical and cellular assays, we identified WS3 as a potent allosteric inhibitor of 14-3-3 that selectively inhibits NRF2 activity in tumor cells. Mechanistically, WS3 binds allosterically to the 14-3-3 dimer, inducing a conformational change and disrupting the 14-3-3-pGSK3β interaction, thereby releasing pGSK3β for dephosphorylation. This activation of GSK3β subsequently enhances the ubiquitination and degradation of NRF2 by the CUL1-β-TrCP E3 ligase. WS3 effectively elicits oxidative stress and potentiates chemotherapeutics and ferroptosis in NRF2-driven cancers. Our findings uncover a previously unrecognized role of 14-3-3 in the hyperactivation of NRF2 and present a first-in-class sub-micromolar 14-3-3 allosteric inhibitor as an effective therapeutic strategy to suppress NRF2 overactivation, especially in Keap1 defective cancers.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular defense system against oxidative insults. Directly inhibiting the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 protein-protein interaction (PPI) has emerged as a promising approach to activate Nrf2 for the treatment of diseases associated with oxidative stress. Herein, we identified beta-amino acids as privileged structural fragments for designing novel naphthalene sulfonamide-based Keap1-Nrf2 PPI inhibitors. Comprehensive structure-activity relationship (SAR) exploration identified compound 19 as the optimal inhibitor with an IC50 of 0.55 mu M for disrupting the Keap1-Nrf2 interaction and a Kd of 0.50 mu M for binding to Keap1. Further studies demonstrated that 19 effectively activated the Nrf2-regulated cytoprotective system and provided protective effects against dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in both in vitro and in vivo models. These findings highlight the potential of beta-amino acid substituted naphthalene sulfonamide Keap1-Nrf2 inhibitor 19 as a prospective therapeutic agent for UC via Keap1 targeting.

Most BTB-containing E3 ligases homodimerize to recognize a single substrate by engaging multiple degrons, represented by E3 ligase KEAP1 dimer and its substrate NRF2. Inactivating KEAP1 to hinder ubiquitination-dependent NRF2 degradation activates NRF2. While various KEAP1 inhibitors have been reported, all reported inhibitors bind to KEAP1 in a monovalent fashion and activate NRF2 in a lagging manner. Herein, we report a unique bivalent KEAP1 inhibitor, biKEAP1 (3), that engages cellular KEAP1 dimer to directly release sequestered NRF2 protein, leading to an instant NRF2 activation. 3 promotes the nuclear translocation of NRF2, directly suppressing proinflammatory cytokine transcription. Data from in vivo experiments showed that 3, with unprecedented potency, reduced acute inflammatory burden in several acute inflammation models in a timely manner. Our findings demonstrate that the bivalent KEAP1 inhibitor can directly enable sequestered substrate NRF2 to suppress inflammatory transcription response and dampen various acute inflammation injuries.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a pleiotropic transcription factor which regulates the constitutive and inducible transcription of a wide array of genes and confers protection against a variety of pathologies. Directly disrupting Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 protein-protein interaction (PPI) has been explored as a promising strategy to activate NRF2. We reported here the first identification of a series of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 inhibitors. Our efforts led to the potent small molecule KEAP1-NRF2 inhibitor, 20c, which exhibited a Kd of 24 nM to KEAP1 and an IC50 of 75 nM in disrupting KEAP1-NRF2 interaction. Subsequent biological studies provided consistent evidence across mouse macrophage cell-based and in vivo models that 20c induced NRF2 target gene expression and enhanced downstream antioxidant and anti-inflammatory activities. Our study not only demonstrated that small molecule KEAP1-NRF2 PPI inhibitors can be potential preventive and therapeutic agents for diseases and conditions involving oxidative stress and inflammation but also enriched the chemical diversity of the KEAP1-NRF2 inhibitors.

Transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) and its negative regulator, the E3 ligase adaptor Kelch-like ECH-associated protein 1 (Keap1), control the redox and metabolic homeostasis and oxidative stress. Inhibitors of Keap1-Nrf2 interaction are promising in oxidative stress related inflammatory diseases but now hit hurdles. By utilizing thiazolidinone moiety to shield the key carboxyl pharmacophore in Keap1-Nrf2 inhibitor, a hydrogen peroxide (H2O2)-responsive prodrug pro2 was developed. The prodrug modification improved the physicochemical properties and cell membrane permeability of the parent drug. Pro2 was stable and stayed inactive under various physiological conditions, while became active by stimulation of H2O2 or inflammation derived reactive oxygen species. Moreover, pro2 exhibited proper pharmacokinetic profile suitable for oral administration and enhanced anti-inflammatory efficiency in vivo. Thus, this novel prodrug approach may not only provide an important advance in the therapy of chronic inflammatory diseases with high level of H2O2, but also offer a fresh solution to improve the drug-like and selectivity issues of Keap1-Nrf2 inhibitors.

Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable azobenzene-proteolysis targeting chimeras (Azo-PROTACs) by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photoswitchability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off properties.

The Keap1-Nrf2-ARE pathway regulates the constitutive and inducible transcription of various genes that encode detoxification enzymes, antioxidant proteins and anti-inflammatory proteins and has pivotal roles in the defence against cellular oxidative stress. In this study, we investigated the therapeutic potential of CPUY192018, a potent small-molecule inhibitor of the Keap1-Nrf2 protein-protein interaction (PPI), in renal inflammation. In human proximal tubular epithelial HK-2 cells, CPUY192018 treatment significantly increased Nrf2 protein level and Nrf2 nuclear translocation, which enhanced Nrf2-ARE transcription capacity and the downstream protein content in a Nrf2 dependent manner. In lipopolysaccharide (LPS)-challenged human HK-2 cells, CPUY192018 exhibited cytoprotective effects by enhancing the Nrf2-ARE regulated antioxidant system and diminished the LPS-induced inflammatory response by hindering the ROS-mediated activation of the NF-κB pathway. In the LPS-induced mouse model of chronic renal inflammation, by activating Nrf2, CPUY192018 treatment balanced renal oxidative stress and suppressed inflammatory responses. Hence, administration of CPUY192018 reduced kidney damage and ameliorated pathological alterations of the glomerulus. Taken together, our study suggested that small-molecule Keap1-Nrf2 PPI inhibitors can activate the Nrf2-based cytoprotective system and protect the kidney from inflammatory injury, raising a potential application of Keap1-Nrf2 PPI inhibitors in the treatment of inflammatory kidney disorders.

The transcription factor Nrf2 is a key regulator of cytoprotective system, and enhancing Nrf2 activity can protect cells from various insults and threats. Directly disrupting Keap1-Nrf2 protein-protein interactions has been regarded as a promising way to activate Nrf2. We reported here the first identification of amino acids as preferred substituents to design potent Keap1-Nrf2 inhibitors. Comprehensive structure-activity analysis identified Pro as a preferred substituent, obtaining a potent inhibitor 35 with an IC50 of 43 nM in the competitive fluoresce polarization (FP) assay and a Kd value of 53.7 nM for Keap1 protein in the isothermal titration calorimetry (ITC) assay. The Pro analogue 35 exhibited tight and prolonged Keap1 binding in vitro and in cells, and treatment with 35 activated Nrf2-regulated cytoprotective response and antagonized acetaminophen-induced liver injury both in cellular and in vivo models. This work not only provides a useful tool to further explore the therapeutic potential of Keap1-Nrf2 inhibition but also enriches the diversity of chemical structures suitable for the Keap1-Nrf2 interface.

Liquid chromatography-mass spectrometry based profiling of microbial metabolites has been a challenging task due to their diverse physicochemical properties and wide concentration ranges. This study is aimed to develop a systematic platform for the broad-scale profiling of microbial metabolites by integrating aqueous-lipophilic biphasic extractions and chemical derivatizations with a data-dependent automatable metabolite annotation algorithm. This complementary strategy of detection will not only largely expand the metabolite coverage, but also facilitate the drawing out of interested submetabolome using designed chemical derivatizations. Then, the data-dependent metabolite annotation algorithm is able to automatically match the raw MS/MS data with those of compounds in the self-collected databases. The performance of this platform is illustrated through the analysis of two representative bacteria (Escherichia coli and Pseudomonas aeruginosa) and intestinal contents samples from experimental colitis mice. As a result, 292 metabolites corresponding to 875 annotated features distributing over 25 chemical families were putatively annotated in a short time. Of these metabolites, 197 and 218 are respectively from the bacteria and intestinal contents, and 107 are identified in all three biological samples. This systematic platform could be used to accomplete high-coverage detection and high-quality data processing of microbial metabolites. At the same time, chemical derivatization design and the establishment of self-collected databases will facilitate self-driven untargeted analysis.

Directly disrupting Keap1-Nrf2 protein-protein interaction (PPI) has emerged as a novel way to activate Nrf2. Peptide Keap1-Nrf2 PPI inhibitors have been reported with high Keapi binding affinity. However, these peptide inhibitors show weak activity in cells. In this study, the head-to-tail cyclic strategy was applied in the development of peptide inhibitors. The privileged residue sequence with minimal acidic residues was used as the template for the cyclic peptide, and the appropriate conjugation method was designed based on the peptide-Keap1 binding mode. The glycine was introduced as the linker to connect both sides, which can avoid the terminal charge, enhance the peptide stability and constrain the binding conformation simultaneously. The obtained novel cyclic peptide 3 showed high binding affinity with Keapl and possessed high potency in Nrf2 activation at cellular level. We also showed that peptide 3 exhibited effective anti-inflammatory effects in mouse RAW 264.7 cells by activating the Nrf2-regulated defense system and enhancing the antioxidant capacity. This study proved that the head-to-tail cyclic strategy is quite useful in improving the cell potency of peptide Keap1-Nrf2 inhibitors and provided a possible way to develop drug-like peptides as therapeutic Nrf2 activators. (C) 2017 Elsevier Masson SAS. All rights reserved.

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric (containing α and β subunits) transcription factor, is involved in hypoxia response pathway that regulates the expression of many tumorrelated genes. The stabilized HIF-1 heterodimer couples to the general co-activators p300/CBP (CREB binding protein), forming an active transcription factor to initiate hypoxic responses. Inhibiting the transcription factor-coactivator HIF-1α-p300/CBP interaction represents an attractive approach for blocking hypoxia pathway in tumors. Recently, diverse HIF-1α-p300/CBP inhibitors have been designed and their anti-tumor activities have been evaluated. The developments of inhibitors of HIF-1α- p300/CBP are discussed in this review. An outline of structures and biological activities of these inhibitors can be traced, along with the approaches for inhibitors discovery. The challenges in identifying novel and selective potent inhibitors of HIF-1α-p300/CBP are also put forward.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pleiotropic transcription factor, especially for its complex and dual effects in cancer. With the continuous growing research, new regulatory modes and new functions of Nrf2 and tumor-promoting effects of Nrf2 in malignant transformed tumors have become increasingly clear. Accumulating evidence has established that Nrf2 contributes to the whole process of pathogenesis, progression, metastasis, and prognosis of cancer, and Nrf2 could be a promising target in cancer therapy. However, the development of Nrf2 inhibitor is still limited. In this perspective, we will briefly describe the biological function and modulating network of Nrf2, stress its oncogenic role, and point out possible ways to inhibit Nrf2, as well as summarize the reported Nrf2 inhibitors.

Induced protein degradation by PROTACs has emerged as a promising strategy to target nonenzymatic proteins inside the cell. The aim of this study was to identify Keap1, a substrate adaptor protein for ubiquitin E3 ligase involved in oxidative stress regulation, as a novel candidate for PROTACs that can be applied in the degradation of the nonenzymatic protein Tau. A peptide PROTAC by recruiting Keap1-Cul3 ubiquitin E3 ligase was developed and applied in the degradation of intracellular Tau. Peptide 1 showed strong in vitro binding with Keap1 and Tau. With proper cell permeability, peptide 1 was found to colocalize with cellular Keap1 and resulted in the coimmunoprecipitation of Tau and Keap1. The results of flow cytometry and western blotting assays showed that peptide 1 can downregulate the intracellular Tau level in both time- and concentration-dependent manner. The application of Keap1 siRNA silencing and the proteasome inhibitor MG132 confirmed that peptide 1 could promote the Keap1-dependent poly-ubiquitination and proteasome-dependent degradation of Tau. The results suggested that using PROTACs to recruit Keap1 to induce the degradation of Tau may show promising character in the treatment of neurodegenerative disease. Besides, our research demonstrated that Keap1 should be a promising E3 ligase adaptor to be used in the design of novel PROTACs.

Directly disrupting Keap1-Nrf2 protein-protein interaction (PPI) has emerged as a novel way to activate Nrf2. Peptide Keap1-Nrf2 PPI inhibitors have been reported with high Keap1 binding affinity. However, these peptide inhibitors show weak activity in cells. In this study, the head-to-tail cyclic strategy was applied in the development of peptide inhibitors. The privileged residue sequence with minimal acidic residues was used as the template for the cyclic peptide, and the appropriate conjugation method was designed based on the peptide-Keap1 binding mode. The glycine was introduced as the linker to connect both sides, which can avoid the terminal charge, enhance the peptide stability and constrain the binding conformation simultaneously. The obtained novel cyclic peptide 3 showed high binding affinity with Keap1 and possessed high potency in Nrf2 activation at cellular level. We also showed that peptide 3 exhibited effective anti-inflammatory effects in mouse RAW 264.7 cells by activating the Nrf2-regulated defense system and enhancing the antioxidant capacity. This study proved that the head-to-tail cyclic strategy is quite useful in improving the cell potency of peptide Keap1-Nrf2 inhibitors and provided a possible way to develop drug-like peptides as therapeutic Nrf2 activators.