Targeted inhibition of NRF2 reduces the invasive and metastatic ability of HIP1 depleted lung cancer cells.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) presents as a highly metastatic disease with Kras and P53 as prevalent oncogenic driver mutations. Endocytosis, through its role in receptor recycling and enrichment, is important for cancer cell proliferation and metastasis. Huntingtin Interacting Protein 1 (HIP1) is a clathrin mediated endocytic adapter protein found overexpressed in different cancers. However, conflicting roles both as a tumour promoter and suppressor are reported. HIP1 expression is found repressed at advanced stages and some HIP1-ALK fusions are reported in NSCLC patients. However, the molecular mechanisms and implications of HIP1 depletion are not completely understood. METHODS: HIP1 depletion was performed using siRNA transient transfection and validated using immunoblotting for each experiment. Gene expression dataset from TCGA, GTEX and GEO databases was analysed to explore HIP1 expression in Lung cancer patients. Kaplan-Meier Plotter database was used to analyse the survival correlation between HIP1 mRNA expression in lung cancer patients. HIP1 depleted A549 cells were analysed for deregulated global proteome using label-free LC-MS and this data is available via ProteomeXchange with identifier PXD054307. Various functional assays such as matrigel based invasion, trans-well migration, soft agar colony and angiogenesis tube formation were performed after HIP1 depletion. NRF2 inhibitor was used after HIP1 knockdown to assess its effect on invasion and soft agar colony formation. RESULTS: In silico analysis of HIP1 transcript expression reveals that it is reduced in high-grade and metastatic lung cancer patients correlating with poor survival. Global proteome profiling reveals that HIP1 depleted A549 cells are enriched in pathways associated with metabolism, proliferation and survival. Molecular and functional analysis indicate higher invasive ability of HIP1 depleted cells. The secretome from HIP1 depleted cells also increases the angiogenic potential of HUVEC cells. NRF2 inhibition significantly reverses invasion of HIP1 depleted NSCLC cells with different driver mutations. CONCLUSION: Our study shows that HIP1 depletion leads to activation of various molecular pathways responsible for cell proliferation and survival. Additionally, enhancement of invasion and anchorage-independent growth in HIP1 depleted subsets of NSCLC cells is via upregulation of NRF2 and can be reversed by its inhibitor.

NRF2 inhibitors: Recent progress, future design and therapeutic potential.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a crucial transcription factor involved in oxidative stress response, which controls the expression of various cytoprotective genes. Recent research has indicated that constitutively activated NRF2 can enhance patients' resistance to chemotherapy drugs, resulting in unfavorable prognosis. Therefore, the development of NRF2 inhibitors has emerged as a promising approach for overcoming drug resistance in cancer treatment. However, there are limited reports and reviews focusing on NRF2 inhibitors. This review aims to provide a comprehensive analysis of the structure and regulation of the NRF2 signaling pathway, followed by a comprehensive review of reported NRF2 inhibitors. Moreover, the current design strategies and future prospects of NRF2 inhibitors will be discussed, aiming to establish a foundation for the development of more effective NRF2 inhibitors.

In vitro cytotoxicity screening of some 3-substituted-4-oxo-imidazolidin-2-(1H)-thione derivatives as anticancer drug.

Aim: This study aimed to investigate the in vitro antitumor activity of new series of 2-thiohydanotin derivatives (7 and 9) against two cancer cell lines.Materials & methods: A new series of 2-thioxoimidazolidine derivatives (3-9) were synthesized and investigated for its structure through spectral analysis and also tested against (HepG-2) and (HCT-116) cell line.Results: Among the synthesized compounds, compound 7 halted liver cancer cells at the G0/G1 phase and triggered apoptosis of liver cancer. Contrarily, compound 9 caused colon cancer cells to be arrested at the S phase and trigger apoptosis. Also, they had a good inhibitory effect on (Nrf2).Conclusion: Both compounds had attractive lead molecules for the creation of colon and liver cancer medications.

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In vitro anti-breast cancer study of hybrid cinnamic acid derivatives bearing 2-thiohydantoin moiety.

Aim: To synthesize new hybrid cinnamic acids (10a, 10b and 11) and ester derivatives (7, 8 and 9) and investigate their anti-breast cancer activities.Materials & methods: Compounds 7-11 were evaluated (in vitro) for their cytotoxic activities against the MCF-7 cell line. A flow cytometry examination was performed. Protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), topoisomerase II and caspase-9 were measured by qRT-PCR. Molecular docking studies were conducted.Results: Several components were discovered to be active, mainly component 11, which induced arrest in the cell cycle at phase S, greatly decreased the expression of Nrf2 and topoisomerase II; and upregulated the expression of caspase-9.Conclusion: The newly thiohydantoin-cinnamic acid hybrids can contribute to creating promising candidates for cancer drugs.

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Arsenic Trioxide Suppresses Angiogenesis in Non-small Cell Lung Cancer via the Nrf2-IL-33 Signaling Pathway.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) ranks as a leading cause of cancer-related mortality, necessitating the urgent search for cost-effective and efficient anti-NSCLC drugs. Our preliminary research has demonstrated that arsenic trioxide (ATO) significantly inhibits NSCLC angiogenesis, exerting anti-tumor effects. In conjunction with existing literature reports, the Nrf2-IL-33 pathway is emerging as a novel mechanism in NSCLC angiogenesis. OBJECTIVE: This study aimed to elucidate whether ATO can inhibit NSCLC angiogenesis through the Nrf2-IL-33 pathway. METHODS: Immunohistochemistry was employed to assess the expression of Nrf2, IL-33, and CD31 in tumor tissues from patients with NSCLC. DETA-NONOate was used as a nitric oxide (NO) donor to mimic high levels of NO in the tumor microenvironment. Western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay were utilized to evaluate the expression of Nrf2 and IL-33 in the NCI-H1299 cell line. Subcutaneous xenograft models were established in nude mice by implanting NCI-H1299 cells to assess the anti-tumor efficacy of ATO. RESULTS: High expression levels of Nrf2 and IL-33 were observed in tumor samples from patients with NSCLC, and Nrf2 expression positively correlated with microvascular density in NSCLC. In vitro, NO (released from 1mM DETA-NONOate) promoted activation of the Nrf2-IL-33 signaling pathway in NCI-H1299 cells, which was reversed by ATO. Additionally, both Nrf2 deficiency and ATO treatment significantly attenuated NOinduced IL-33 expression. In vivo, both ATO and the Nrf2 inhibitor ML385 demonstrated significant inhibitory effects on angiogenesis tumor growth. CONCLUSION: Nrf2-IL-33 signaling is usually activated in NSCLC and positively correlates with tumor angiogenesis. ATO effectively disrupts the activation of the Nrf2-IL-33 pathway in NSCLC and thus inhibits angiogenesis, suggesting its potential as an anti-angiogenic agent for use in the treatment of NSCLC.

A double whammy for KEAP1.

In this issue of Cell Chemical Biology, Lu et al.1 report the discovery of a bivalent KEAP1 inhibitor (biKEAP1), which more rapidly activates NRF2 compared to previously reported monovalent KEAP1 inhibitors. biKEAP1suppresses acute inflammation in animal models.

Spinosin ameliorates osteoarthritis through enhancing the Nrf2/HO-1 signaling pathway.

Osteoarthritis (OA) is a common degenerative joint disease in the elderly, while oxidative stress-induced chondrocyte degeneration plays a key role in the pathologic progression of OA. One possible reason is that the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), which acts as the intracellular defense factor against oxidative stress, is significantly inhibited in chondrocytes. Spinosin (SPI) is a potent Nrf2 agonist, but its effect on OA is still unknown. In this study, we found that SPI can alleviate tert-Butyl hydroperoxide (TBHP)-induced extracellular matrix degradation of chondrocytes. Additionally, SPI can effectively activate Nrf2, heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase 1 (NQO1) in chondrocytes under the TBHP environment. When Nrf2 was silenced by siRNA, the cartilage protective effect of SPI was also weakened. Finally, SPI showed good alleviative effects on OA in mice. Thus, SPI can ameliorate oxidative stress-induced chondrocyte dysfunction and exhibit a chondroprotective effect through activating the Nrf2/HO-1 pathway, which may provide a novel and promising option for the treatment of OA.

Clobetasol propionate, a Nrf-2 inhibitor, sensitizes human lung cancer cells to radiation-induced killing via mitochondrial ROS-dependent ferroptosis.

Combining radiotherapy with Nrf-2 inhibitor holds promise as a potential therapeutic strategy for radioresistant lung cancer. Here, the radiosensitizing efficacy of a synthetic glucocorticoid clobetasol propionate (CP) in A549 human lung cancer cells was evaluated. CP exhibited potent radiosensitization in lung cancer cells via inhibition of Nrf-2 pathway, leading to elevation of oxidative stress. Transcriptomic studies revealed significant modulation of pathways related to ferroptosis, fatty acid and glutathione metabolism. Consistent with these findings, CP treatment followed by radiation exposure showed characteristic features of ferroptosis in terms of mitochondrial swelling, rupture and loss of cristae. Ferroptosis is a form of regulated cell death triggered by iron-dependent ROS accumulation and lipid peroxidation. In combination with radiation, CP showed enhanced iron release, mitochondrial ROS, and lipid peroxidation, indicating ferroptosis induction. Further, iron chelation, inhibition of lipid peroxidation or scavenging mitochondrial ROS prevented CP-mediated radiosensitization. Nrf-2 negatively regulates ferroptosis through upregulation of antioxidant defense and iron homeostasis. Interestingly, Nrf-2 overexpressing A549 cells were refractory to CP-mediated ferroptosis induction and radiosensitization. Thus, this study identified anti-psoriatic drug clobetasol propionate can be repurposed as a promising radiosensitizer for Keap-1 mutant lung cancers.

APE1 inhibition enhances ferroptotic cell death and contributes to hepatocellular carcinoma therapy.

Ferroptosis, a regulated form of cell death triggered by iron-dependent lipid peroxidation, has emerged as a promising therapeutic strategy for cancer treatment, particularly in hepatocellular carcinoma (HCC). However, the mechanisms underlying the regulation of ferroptosis in HCC remain to be unclear. In this study, we have identified a novel regulatory pathway of ferroptosis involving the inhibition of Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme with dual functions in DNA repair and redox regulation. Our findings demonstrate that inhibition of APE1 leads to the accumulation of lipid peroxidation and enhances ferroptosis in HCC. At the molecular level, the inhibition of APE1 enhances ferroptosis which relies on the redox activity of APE1 through the regulation of the NRF2/SLC7A11/GPX4 axis. We have identified that both genetic and chemical inhibition of APE1 increases AKT oxidation, resulting in an impairment of AKT phosphorylation and activation, which leads to the dephosphorylation and activation of GSK3ß, facilitating the subsequent ubiquitin-proteasome-dependent degradation of NRF2. Consequently, the downregulation of NRF2 suppresses SLC7A11 and GPX4 expression, triggering ferroptosis in HCC cells and providing a potential therapeutic approach for ferroptosis-based therapy in HCC. Overall, our study uncovers a novel role and mechanism of APE1 in the regulation of ferroptosis and highlights the potential of targeting APE1 as a promising therapeutic strategy for HCC and other cancers.

Synergistic suppression of ovarian cancer by combining NRF2 and GPX4 inhibitors: in vitro and in vivo evidence.

Ovarian cancer is a significant challenge in women's health due to the lack of effective screening and diagnostic methods, often leading to late detection and the highest mortality rate among all gynecologic tumors worldwide. Recent research has shown that ovarian cancer has an "iron addiction" phenotype which makes it vulnerable to ferroptosis inducers. We tested the combination of NRF2-targeted inhibitors with GPX4-targeted inhibitors in ovarian cancer through in vitro and in vivo experiment. The data showed that combination treatment effectively suppressed adherent cell growth, inhibited suspended cell spheroid formation, and restrained the ability of spheroid formation in 3D-culture. Mechanistically, the combination induced accumulation of ROS, 4-HNE, as well as activation of caspase-3 which indicates that this combination simultaneously increases cell ferroptosis and apoptosis. Notably, inhibition of GPX4 or NRF2 can suppress ovarian cancer spreading and growth in the peritoneal cavity of mice, while the combination of NRF2 inhibitor ML385 with GPX4 inhibitors showed a significant synergistic effect compared to individual drug treatment in a syngeneic mouse ovarian cancer model. Overall, these findings suggest that combining NRF2 inhibitors with GPX4 inhibitors results in a synergy suppression of ovarian cancer in vitro and in vivo, and maybe a promising therapeutic strategy for the treatment of ovarian cancer.

Bivalent inhibitors of the BTB E3 ligase KEAP1 enable instant NRF2 activation to suppress acute inflammatory response.

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.

Synergistic Effects of Tranylcypromine and NRF2 Inhibitor: A Repurposing Strategy for Effective Cancer Therapy.

Drug repurposing has emerged as an attractive strategy for accelerating drug discovery for cancer treatment. In this study, we investigated combining Tranylcypromine (TCP) with a number of well-characterized drugs. Among these combinations, NRF2 inhibitor (ML385) exhibited synergistic effects in combination with TCP. Specifically, our results showed that the combination of TCP and ML385 resulted in a significant reduction in tumor proliferation while neither drug affected cancer cell growth meaningfully on its own. While further studies are needed to understand fully the extent of the synergistic efficacy, the underlying respective mechanisms and the potential side effects of this approach, our study has yielded a promising start for the development of an effective combination cancer therapy.

Cadmium exposure promotes thyroid pyroptosis and endocrine dysfunction by inhibiting Nrf2/Keap1 signaling.

Cadmium (Cd) is a ubiquitous toxic metal and environmental pollutant. Increasing studies have shown that Cd exposure increases the incidence of various endocrine system diseases, including thyrotoxicity reflected by thyroid structural damage and endocrine toxicity. However, the observed outcomes are complex and conflicting, leading to the mechanism of Cd-induced thyrotoxicity remaining obscure. In this study, 4-week-old male C57BL/6 mice were given 2 or 7 mg/kg Cadmium Chloride (CdCl2) intragastrically for 4 and 8 weeks, and the Cd-mediated thyrotoxicity was evaluated by determining alterations in thyroid structure and endocrine function, and alterations of oxidant stress, apoptosis, and pyroptosis. Our data showed that Cd exposure could reduce body weight and induce thyrotoxicity by impairing thyroid follicular morphology and endocrine function, accompanied by elevated oxidative stress and apoptosis, macrophage infiltration, and inflammatory cytokine secretion. Importantly, Cd significantly promoted thyroid follicular cell pyroptosis by increasing Nlrp3, Asc, Caspase-1, Gsdmd, IL-1ß, and IL-18 expression. Mechanistical analysis suggested that Cd treatment could inhibit antioxidant pathway by downregulating antioxidant response protein, Nrf2, and upregulating its negative feedback regulator, Keap1. Collectively, our in vivo findings suggest that Cd exposure could facilitate thyroid follicular cell pyroptosis by inhibiting Nrf2/Keap1 signaling, thereby disrupting thyroid tissue structure and endocrine function, which offers novel insights into the Cd-mediated detrimental consequences on thyroid homeostasis.

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.

Potential Mechanisms by which Glucocorticoids Induce Breast Carcinogenesis through Nrf2 Inhibition.

Breast cancer is the most common malignancy among women worldwide. Several studies indicate that, in addition to established risk factors for breast cancer, other factors such as cortisol release related to psychological stress and drug treatment with high levels of glucocorticoids may also contribute significantly to the initiation of breast cancer. There are several possible mechanisms by which glucocorticoids might promote neoplastic transformation of breast tissue. Among these, the least known and studied is the inhibition of the nuclear erythroid factor 2-related (Nrf2)-antioxidant/electrophile response element (ARE/EpRE) pathway by high levels of glucocorticoids. Specifically, Nrf2 is a potent transcriptional activator that plays a central role in the basal and inducible expression of many cytoprotective genes that effectively protect mammalian cells from various forms of stress and reduce the propensity of tissues and organisms to develop disease or malignancy including breast cancer. Consequently, a loss of Nrf2 in response to high levels of gluco-corticoids may lead to a decrease in cellular defense against oxidative stress, which plays an important role in the initiation of human mammary carcinogenesis. In the present review, we provide a comprehensive overview of the current state of knowledge of the cellular mechanisms by which both glucocorticoid pharmacotherapy and endogenous GCs (cortisol in humans and corticosterone in rodents) may contribute to breast cancer development through inhibition of the Nrf2-ARE/EpRE pathway and the protective role of melatonin against glucocorticoid-induced apoptosis in the immune system.

Novel Keap1-Nrf2 Protein-Protein Interaction Inhibitor UBE-1099 Ameliorates Progressive Phenotype in Alport Syndrome Mouse Model.

Background: Bardoxolone methyl activates nuclear factor erythroid 2-related factor 2 (Nrf2) via covalent binding and irreversible inhibition of Kelch-like ECH-associated protein 1 (Keap1), the negative regulator of Nrf2. Ongoing clinical trials of bardoxolone methyl show promising effects for patients with CKD. However, the direct inhibition of Keap1-Nrf2 protein-protein interaction (PPI) as an approach to activate Nrf2 is less explored. Methods: We developed a noncovalent Nrf2 activator UBE-1099, which highly selectively inhibits Keap1-Nrf2 PPI, and evaluated its efficacy on the progressive phenotype in an Alport syndrome mouse model (Col4a5-G5X). Results: Similar to bardoxolone methyl, UBE-1099 transiently increased proteinuria and reduced plasma creatinine in Alport mice. Importantly, UBE-1099 improved the glomerulosclerosis, renal inflammation, and fibrosis, and prolonged the life span of Alport mice. UBE-1099 ameliorated the dysfunction of Nrf2 signaling in the renal tissue of Alport mice. Moreover, transcriptome analysis in the glomerulus showed that UBE-1099 induced the expression of genes associated with the cell cycle and cytoskeleton, which may explain its unique mechanism of improvement such as glomerular morphologic change. Conclusions: UBE-1099 significantly ameliorates the progressive phenotype in Alport mice. Our results revealed the efficacy of Keap1-Nrf2 PPI inhibitor for glomerulosclerosis and present a potential therapeutic drug for CKD.

Ameliorative effect of betulinic acid against zearalenone exposure triggers testicular dysfunction and oxidative stress in mice via p38/ERK MAPK inhibition and Nrf2-mediated antioxidant defense activation.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin, which mainly contaminates grains and has estrogen-like effects on the reproductive system. Betulinic acid (BA), a natural lupane-type pentacyclic triterpene, has anti-oxidative and anti-inflammatory properties. This study aimed to investigate whether BA alleviates ZEA-induced testicular damage and explore the possible mechanism. Here, BA ameliorated testicular damage by mitigating the disordered arrangement of seminiferous tubules, the exfoliation of lumen cells, and the increase of cell apoptosis caused by ZEA. Meanwhile, BA alleviated ZEA-triggered testicular damage by restoring hormone levels and sperm motility, and reconstructing the blood-testis-barrier. Moreover, BA alleviated ZEA-exposed testicular oxidative stress by activating Nrf2 pathway. Furthermore, BA moderated ZEA-evoked testicular inflammation by inhibiting p38/ERK MAPK pathway. Overall, our results revealed that BA has a therapeutic protective effect on ZEA-induced testicular injury and oxidative stress via p38/ERK MAPK inhibition and Nrf2-mediated antioxidant defense activation, which provides a viable alternative to alleviate ZEA-induced male reproductive toxicology.

In vivo and in vitro protective effects of shengmai injection against doxorubicin-induced cardiotoxicity.

CONTEXT: Shengmai injection (SMI) has been used to treat heart failure. OBJECTIVE: This study determines the molecular mechanisms of SMI against cardiotoxicity caused by doxorubicin (DOX). MATERIALS AND METHODS: In vivo, DOX (15 mg/kg) was intraperitoneally injected in model, Dex (dexrazoxane), SMI-L (2.7 mL/kg), SMI-M (5.4 mL/kg), and SMI-H (10.8 mL/kg) for 7 consecutive days. Hematoxylin-eosin (HE) and Masson staining were used to evaluate histological changes, and cardiomyocyte apoptosis was identified using TdT-mediated dUTP nick-end labelling (TUNEL). Enzymatic indexes were determined. mRNA and protein expressions were analysed through RT-qPCR and Western blotting. In vitro, H9c2 cells were divided into control group, model group (2 mL 1 µM DOX), SMI group, ML385 group, and SMI + ML385 group, the intervention lasted for 24 h. mRNA and protein expressions were analysed. RESULTS: SMI markedly improved cardiac pathology, decreased cardiomyocyte apoptosis, increased creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), decreased superoxide dismutase (SOD). Compared with the model group, the protein expression of nuclear factor erythroid2-related factor 2 (Nrf2) (SMI-L: 2.42-fold, SMI-M: 2.67-fold, SMI-H: 3.07-fold) and haem oxygenase-1(HO-1) (SMI-L: 1.64-fold, SMI-M: 2.01-fold, SMI-H: 2.19-fold) was increased and the protein expression of kelch-like ECH-associated protein 1 (Keap1) (SMI-L: 0.90-fold, SMI-M: 0.77-fold, SMI-H: 0.66-fold) was decreased in SMI groups and Dex group in vivo. Additionally, SMI dramatically inhibited apoptosis, decreased CK, LDH and MDA levels, and enhanced SOD activity. Our results demonstrated that SMI reduced DOX-induced cardiotoxicity via activation of the Nrf2/Keap1 signalling pathway. CONCLUSIONS: This study revealed a new mechanism by which SMI alleviates DOX-induced 45 cardiomyopathy by modulating the Nrf2/Keap1 signal pathway.

Importance of Binding Site Hydration and Flexibility Revealed When Optimizing a Macrocyclic Inhibitor of the Keap1-Nrf2 Protein-Protein Interaction.

Upregulation of the transcription factor Nrf2 by inhibition of the interaction with its negative regulator Keap1 constitutes an opportunity for the treatment of disease caused by oxidative stress. We report a structurally unique series of nanomolar Keap1 inhibitors obtained from a natural product-derived macrocyclic lead. Initial exploration of the structure-activity relationship of the lead, followed by structure-guided optimization, resulted in a 100-fold improvement in inhibitory potency. The macrocyclic core of the nanomolar inhibitors positions three pharmacophore units for productive interactions with key residues of Keap1, including R415, R483, and Y572. Ligand optimization resulted in the displacement of a coordinated water molecule from the Keap1 binding site and a significantly altered thermodynamic profile. In addition, minor reorganizations of R415 and R483 were accompanied by major differences in affinity between ligands. This study therefore indicates the importance of accounting both for the hydration and flexibility of the Keap1 binding site when designing high-affinity ligands.

Blocking iASPP/Nrf2/M-CSF axis improves anti-cancer effect of chemotherapy-induced senescence by attenuating M2 polarization.

The complex interaction between cancer cells and the immune microenvironment is a central regulator of tumor growth and the treatment response. Chemotherapy-induced senescence is accompanied by the senescence-associated secretion phenotype (SASP). However, the mechanisms underlying the regulation of the SASP remain the most poorly understood element of senescence. Here, we show that nuclear erythroid factor 2-like factor 2 (Nrf2), a master antioxidative transcription factor, accumulates upon doxorubicin-induced senescence. This is due to the increased cytoplasmic Inhibitor of Apoptosis Stimulating Protein of P53, iASPP, which binds with Keap1, interrupting Keap1/Nrf2 interaction and promoting Nrf2 stabilization and activation. Activated Nrf2 transactivates a novel target gene of SASP factor, macrophage colony-stimulating factor (M-CSF), which subsequently acts on macrophages and induces polarization from M1 to M2 via a paracrine mechanism. Genetic inhibition of iASPP-Nrf2 suppresses the growth of apoptosis-resistant xenografts, with further analysis revealing that M-CSF/M-CSFR-regulated macrophage polarization is critical for the functional outcomes delineated above. Overall, our data uncover a novel function of iASPP-Nrf2 in skewing the immune microenvironment under treatment-induced senescence. Targeting the iASPP-Nrf2 axis could be a powerful strategy for the implementation of new chemotherapy-based therapeutic opportunities.