The cysteine residues of Keap1 such as C151, C273, and C288 are critical for its repressor activity on Nrf2. However, to date, no molecules have been identified to covalently modify all three cysteine residues for Nrf2 activation. Hence, in this study, our goal is to discover new Keap1 covalent inhibitors that can undergo a Michael addition with all three cysteine residues. The Keap1\'s intervening region was modeled using Modeller v10.4. Covalent docking and binding free energy were calculated using CovDock. Molecular dynamics (MD) was performed using Desmond. Various in-vitro assays were carried out to confirm the neuroprotective effects of the hit molecule in 6-OHDA-treated SH-SY5Y cells. Further, the best hit was evaluated in vivo for its ability to improve rotenone-induced postural instability and cognitive impairment in male rats. Finally, network pharmacology was used to summarize the complete molecular mechanism of the hit molecule. Chalcone and plumbagin were found to form the necessary covalent bonds with all three cysteine residues. However, MD analysis indicated that the binding of plumbagin is more stable than chalcone. Plumbagin displayed neuroprotective effects in 6-OHDA-treated SH-SY5Y cells at concentrations 0.01 and 0.1 μM. Plumbagin at 0.1 µM had positive effects on reactive oxygen species formation and glutathione levels. Plumbagin also improved postural instability and cognitive impairment in rotenone-treated male rats. Our network analysis indicated that plumbagin could also improve dopamine signaling. Additionally, plumbagin could exhibit anti-oxidant and anti-inflammatory activity through the activation of Nrf2. Cumulatively, our study suggests that plumbagin is a novel Keap1 covalent inhibitor for Nrf2-mediated neuroprotection in PD.

Sex differences may play a role in the etiopathogenesis and severity of metabolic dysfunction-associated steatotic liver disease (MASLD), a disorder characterized by excessive fat accumulation associated with increased inflammation and oxidative stress. We previously observed the development of steatosis specifically in female rats fed a high-fat diet enriched with liquid fructose (HFHFr) for 12 weeks. The aim of this study was to better characterize the observed sex differences by focusing on the antioxidant and cytoprotective pathways related to the KEAP1/NRF2 axis. The KEAP1/NRF2 signaling pathway, autophagy process (LC3B and LAMP2), and endoplasmic reticulum stress response (XBP1) were analyzed in liver homogenates in male and female rats that were fed a 12-week HFHFr diet. In females, the HFHFr diet resulted in the initial activation of the KEAP1/NRF2 pathway, which was not followed by the modulation of downstream molecular targets; this was possibly due to the increase in KEAP1 levels preventing the nuclear translocation of NRF2 despite its cytosolic increase. Interestingly, while in both sexes the HFHFr diet resulted in an increase in the levels of LC3BII/LC3BI, a marker of autophagosome formation, only males showed a significant upregulation of LAMP2 and XBP1s; this did not occur in females, suggesting impaired autophagic flux in this sex. Overall, our results suggest that males are characterized by a greater ability to cope with an HFHFr metabolic stimulus mainly through an autophagic-mediated proteostatic process while in females, this is impaired. This might depend at least in part upon the fine modulation of the cytoprotective and antioxidant KEAP1/NRF2 pathway resulting in sex differences in the occurrence and severity of MASLD. These results should be considered to design effective therapeutics for MASLD.

Neurodegenerative diseases constitute a global health issue and a major economic burden. They significantly impair both cognitive and motor functions, and their prevalence is expected to rise due to ageing societies and continuous population growth. Conventional therapies provide symptomatic relief, nevertheless, disease-modifying treatments that reduce or halt neuron death and malfunction are still largely unavailable. Amongst the common hallmarks of neurodegenerative diseases are protein aggregation, oxidative stress, neuroinflammation and mitochondrial dysfunction. Transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) constitutes a central regulator of cellular defense mechanisms, including the regulation of antioxidant, anti-inflammatory and mitochondrial pathways, making it a highly attractive therapeutic target for disease modification in neurodegenerative disorders. Here, we describe the role of NRF2 in the common hallmarks of neurodegeneration, review the current pharmacological interventions and their challenges in activating the NRF2 pathway, and present alternative therapeutic approaches for disease modification.

OBJECTIVE: The goal of the current study was to clarify the potential molecular mechanism underlying the protective effects of silymarin (SIL) administration against diazinon-induced subacute nephrotoxicity, with a special emphasis on the role of the Kelch-like-associated protein-1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway in minimizing the oxidative stress induced by diazinon (DZN).
METHODS: Five equal groups of thirty adult male Wistar rats were created at random. Group 1 (G1) was maintained under typical control conditions and administered saline intragastrically (I/G) once daily for 4 weeks; G2 was administered olive oil I/G for 4 weeks; G3 was I/G administered silymarin daily for 4 weeks; G4 was I/G administered diazinon daily for 4 weeks. G5 was I/G administered silymarin daily 1 h before the I/G administration of the diazinon for 4 weeks. Blood samples were collected at the end of the experiment for the determination of complete blood cell count, and kidney function tests. Kidney specimens were collected for the evaluation of the oxidative markers, mRNA gene expression, protein markers, and histopathological examination.
RESULTS: SIL reduced the renal dysfunction caused by DZN by restoring urea and creatinine levels, as well as oxidative indicators. Although the expression of Keap-1 was also elevated, overexpression of Nrf2 also enhanced the expression of HO-1, a crucial target enzyme of Nrf2.
CONCLUSIONS: SIL is hypothesized to potentially aid in the prevention and management of nephrotoxicity caused by DZN.

Ponicidin is a diterpenoid with demonstrated antitumor activity in clinical trials. However, the specific function and mechanism of action against hepatocellular carcinoma (HCC) remain unknown. In this study, it is found that ponicidin significantly inhibited the proliferation and migration of HCC cells. It is shown that ponicidin targets Keap1 and promotes the formation of the Keap1-PGAM5 complex, leading to the ubiquitination of PGAM5, using biotin-labeled ponicidin for target fishing and the HuProtTM Human Proteome Microarray V4.0. Ponicidin is found to activate the cysteine-dependent mitochondrial pathway via PGAM5, resulting in mitochondrial damage and ROS production, thereby promoting mitochondrial apoptosis in HepG2 cells. The first in vitro cocrystal structure of the PGAM5 IE 12-mer peptide and the Keap1 Kelch domain is obtained. Using molecular dynamics simulations to confirm the binding of ponicidin to the Keap1-PGAM5 complex. Based on the depth-based dynamic simulation, it is found that ponicidin can induce the tightening of the Keap1-PGAM5 interaction pocket, thereby stabilizing the formation of the protein complex. Finally, it is observed that ponicidin effectively inhibited tumor growth and promoted tumor cell apoptosis in a BALB/c nude mouse xenograft tumor model. The results provide insight into the anti-HCC properties of ponicidin based on a mechanism involving the Keap1-PGAM5 complex.

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.

UNASSIGNED: The incidence of endometrial cancer is increasing, and oxidative stress has been suggested to play a vital role in its carcinogenesis. Statins have an impact on the cellular redox-state. The aim of this study was to determine the effects of statin use on redox-state regulating enzymes in endometrial cancer in women with type 2 diabetes.
UNASSIGNED: This retrospective study consisted of 119 women with type 2 diabetes who were diagnosed with endometrial cancer at Oulu University Hospital in Finland between 2007 and 2014. There were 58 statin users and 61 non-users based on medication use at the time of endometrial cancer diagnosis. The expression of redox-state regulating proteins nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in the tumor samples was assessed immunohistochemically, and manganese superoxide dismutase (MnSOD) levels were measured both immunohistochemically and from serum samples.
UNASSIGNED: High MnSOD expression predicted better progression-free survival (PFS) in statin non-users in a univariate analysis (p=0.02). There was no statistical difference in overall survival (OS) or PFS between strong and weak expression of Nrf2 and Keap1. After adjusting for stage and statin use, the results were similar.
UNASSIGNED: Statin non-users with strong MnSOD expression had better PFS compared to statin users which proves that statins have impact on redox-state regulating enzymes. However, these findings are preliminary and require further research.

Sesamolin, a lignan isolated from sesame oils, has been found to possess neuroprotective, anticancer, and free radical scavenging properties. We hypothesized that sesamolin could stimulate the activity of nuclear factor erythroid-derived 2-like 2 (Nrf2) and inhibit adipocyte differentiation of preadipocytes. The objective of this study was to investigate effects of sesamolin on adipocyte differentiation and its underlying molecular mechanisms. In this study, we determined the effects of treatment with 25 to 100 µM sesamolin on adipogenesis in cell culture systems. Sesamolin inhibited lipid accumulation and suppressed the expression of adipocyte markers during adipocyte differentiation of C3H10T1/2, 3T3-L1, and primary preadipocytes. Mechanism studies revealed that sesamolin increased Nrf2 protein expression without inducing its mRNA, leading to an increase in the expression of Nrf2 target genes such as heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 (Nqo1) in C3H10T1/2 adipocytes and mouse embryonic fibroblasts. These effects were significantly attenuated in Nrf2 knockout (KO) mouse embryonic fibroblasts, indicating that effects of sesamolin were dependent on Nrf2. In H1299 human lung cancer cells with KO of Kelch like-ECH-associated protein 1 (Keap1), a negative regulator of Nrf2, sesamolin failed to further increase Nrf2 protein expression. However, upon reexpressing Keap1 in Keap1 KO cells, the ability of sesamolin to elevate Nrf2 protein expression was restored, highlighting the crucial role of Keap1 in sesamolin-induced Nrf2 activation. Taken together, these findings show that sesamolin can inhibit adipocyte differentiation through Keap1-mediated Nrf2 activation.

Breast cancer remains the leading cause of cancer deaths for women. Long-term estrogen exposure is considered carcinogenic due to semiquinone production and to compromised detoxification. Metabolic regulator polymorphisms, such as KEAP1 (rs1048290) and NRF2 (rs35652124, rs6721961, rs6706649), can be valuable in understanding the individual cytoprotection profile. Thus, we aim to genotype these polymorphisms in blood, tumours and surrounding tissue, to identify somatic mutations and correlate it to prognoses. A total of 23 controls and 69 women with histological confirmed breast cancer were recruited, and DNA from blood/surrounding/tumour tissue was genotyped. Genotyping and clinicopathological data were correlated. We verified that rs35652124 presents different genotype distribution between the blood/surrounding tissue (p-value = 0.023) and tumour/surrounding tissues (p-value = 0.041). Apart from rs35652124 and considering the histological grade, the other four polymorphisms have different distributions among different tissues. There is a tendency towards the loss of heterozygosity in the surrounding tissue when compared to blood and tumour tissues, and higher genotype variability in histologic grade 2. These somatic mutations and different distribution patterns may indicate a heterogeneous and active microenvironment, influencing breast cancer outcome. Additionally, it would be pertinent to evaluate the predictive value of the histologic grade 2 considering somatic mutation profiles and distributions.

In response to invading parasites, one of the principal arms of innate immunity is oxidative stress, caused by reactive oxygen species (ROS). However, oxidative stresses play dual functions in the disease, whereby free radicals promote pathogen removal, but they can also trigger inflammation, resulting in tissue injuries. A growing body of evidence has strongly supported the notion that nuclear factor erythroid 2-related factor 2 (NRF) signaling is one of the main antioxidant pathways to combat this oxidative burst against parasites. Given the important role of NRF2 in oxidative stress, in this review, we investigate the activation mechanism of the NRF2 antioxidant pathway in different parasitic diseases, such as malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, entamoebiasis, and trichinosis.