Upregulated Nuclear Expression of Soluble Epoxide Hydrolase Predicts Poor Outcome in Breast Cancer Patients: Importance of the Digital Pathology Approach.

Breast cancer (BC) is the most common cancer in women, with incidence rates increasing globally in recent years. Therefore, it is important to find new molecules with prognostic and therapeutic value to improve therapeutic response and quality of life. The polyunsaturated fatty acids (PUFAs) metabolic pathway participates in various physiological processes, as well as in the development of malignancies. Although aberrancies in the PUFAs metabolic pathway have been implicated in carcinogenesis, the functional and clinical relevance of this pathway has not been well explored in BC. To evaluate the clinical significance of soluble epoxide hydrolase (EPHX2) expression in Mexican patients with BC using tissue microarrays (TMAs) and digital pathology (DP). Immunohistochemical analyses were performed on 11 TMAs with 267 BC samples to quantify this enzyme. Using DP, EPHX2 protein expression was evaluated solely in tumor areas. The association of EPHX2 with overall survival (OS) was detected through bioinformatic analysis in public databases and confirmed in our cohort via Cox regression analysis. Clear nuclear expression of EPHX2 was identified. Receiver operating characteristics (ROC) curves revealed the optimal cutoff point at 2.847062 × 10-3 pixels, with sensitivity of 69.2% and specificity of 67%. Stratification based on this cutoff value showed elevated EPHX2 expression in multiple clinicopathological features, including older age and nuclear grade, human epidermal growth factor receptor 2 (HER2) and triple negative breast cancer (TNBC) subtypes, and recurrence. Kaplan-Meier curves demonstrated how higher nuclear expression of EPHX2 predicts shorter OS. Consistently, multivariate analysis confirmed EPHX2 as an independent predictor of OS, with a hazard ratio (HR) of 3.483 and a 95% confidence interval of 1.804-6.724 (p < 0.001). Our study demonstrates for the first time that nuclear overexpression of EPHX2 is a predictor of poor prognosis in BC patients. The DP approach was instrumental in identifying this significant association. Our study provides valuable insights into the potential clinical utility of EPHX2 as a prognostic biomarker and therapeutic target in BC.

Triazine herbicide prometryn alters epoxide hydrolase activity and increases cytochrome P450 metabolites in murine livers via lipidomic profiling.

Oxylipins are a group of bioactive fatty acid metabolites generated via enzymatic oxygenation. They are notably involved in inflammation, pain, vascular tone, hemostasis, thrombosis, immunity, and coagulation. Oxylipins have become the focus of therapeutic intervention since they are implicated in many conditions, such as nonalcoholic fatty liver disease, cardiovascular disease, and aging. The liver plays a crucial role in lipid metabolism and distribution throughout the organism. Long-term exposure to pesticides is suspected to contribute to hepatic carcinogenesis via notable disruption of lipid metabolism. Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. The amounts of prometryn documented in the environment, mainly waters, soil and plants used for human and domestic consumption are significantly high. Previous research revealed that prometryn decreased liver development during zebrafish embryogenesis. To understand the mechanisms by which prometryn could induce hepatotoxicity, the effect of prometryn (185 mg/kg every 48 h for seven days) was investigated on hepatic and plasma oxylipin levels in mice. Using an unbiased LC-MS/MS-based lipidomics approach, prometryn was found to alter oxylipins metabolites that are mainly derived from cytochrome P450 (CYP) and lipoxygenase (LOX) in both mice liver and plasma. Lipidomic analysis revealed that the hepatotoxic effects of prometryn are associated with increased epoxide hydrolase (EH) products, increased sEH and mEH enzymatic activities, and induction of oxidative stress. Furthermore, 9-HODE and 13-HODE levels were significantly increased in prometryn treated mice liver, suggesting increased levels of oxidation products. Together, these results support that sEH may be an important component of pesticide-induced liver toxicity.

Evaluation of the Therapeutic Potential of Sulfonyl Urea Derivatives as Soluble Epoxide Hydrolase (sEH) Inhibitors.

The inhibition of soluble epoxide hydrolase (sEH) can reduce the level of dihydroxyeicosatrienoic acids (DHETs) effectively maintaining endogenous epoxyeicosatrienoic acids (EETs) levels, resulting in the amelioration of inflammation and pain. Consequently, the development of sEH inhibitors has been a prominent research area for over two decades. In the present study, we synthesized and evaluated sulfonyl urea derivatives for their potential to inhibit sEH. These compounds underwent extensive in vitro investigation, revealing their potency against human and mouse sEH, with 4f showing the most promising sEH inhibitory potential. When subjected to lipopolysaccharide (LPS)-induced acute lung injury (ALI) in studies in mice, compound 4f manifested promising anti-inflammatory efficacy. We investigated the analgesic efficacy of sEH inhibitor 4f in a murine pain model of tail-flick reflex. These results validate the role of sEH inhibition in inflammatory diseases and pave the way for the rational design and optimization of sEH inhibitors based on a sulfonyl urea template.

Design and Synthesis of sEH/HDAC6 Dual-Targeting Inhibitors for the Treatment of Inflammatory Pain.

Soluble epoxide hydrolase (sEH) and HDAC6 mediate the NF-κB pathway in inflammatory responses, and their inhibitors exhibit powerful anti-inflammatory and analgesic activities in treating both inflammation and pain. Therefore, a series of dual-targeting inhibitors containing urea or squaramide and hydroxamic acid moieties were designed and synthesized, and their role as a new sEH/HDAC6 dual-targeting inhibitor in inflammatory pain was evaluated in a formalin-induced mice model and a xylene-induced mouse ear swelling model. Among them, compounds 28g and 28j showed the best inhibitory and selectivity of sEH and HDAC6. Compound 28g had satisfactory pharmacokinetic characteristics in rats. Following administration at 30 mg/kg, compound 28g exhibited more effective analgesic activity than either an sEH inhibitor (GL-B437) or an HDAC6 inhibitor (Rocilinostat) alone and coadministration of both inhibitors. Thus, these novel sEH/HDAC6 dual-targeting inhibitors exhibited powerful analgesic activity in nociceptive behavior and are worthy of further development.

Cytochrome P450-soluble epoxide hydrolase oxylipins, depression and cognition in type 2 diabetes.

AIMS: This study examined serum cytochrome P450-soluble epoxide hydrolase (CYP450-sEH) oxylipins and depressive symptoms together in relation to cognitive performance in individuals with type 2 diabetes mellitus (T2DM). METHODS: Clinically cognitively normal T2DM individuals were recruited (NCT04455867). Depressive symptom severity was assessed using the Beck Depression Inventory-II (BDI-II; total scores ≤13 indicated minimal depressive symptoms and ≥ 14 indicated significant depressive symptoms). Executive function and verbal memory were assessed. Fasting serum oxylipins were quantified by ultra-high-performance liquid chromatography tandem mass-spectrometry. RESULTS: The study included 85 participants with minimal depressive symptoms and 27 with significant symptoms (mean age: 63.3 ± 9.8 years, 49 % women). In all participants, higher concentrations of linoleic acid derived sEH (12,13-dihydroxyoctadecamonoenoic acid; DiHOME) and CYP450 (12(13)-epoxyoctadecamonoenoic acid; EpOME) metabolites were associated with poorer executive function (F1,101 = 6.094, p = 0.015 and F1,101 = 5.598, p = 0.020, respectively). Concentrations of multiple sEH substrates interacted with depressive symptoms to predict 1) poorer executive function, including 9(10)-EpOME (F1,100 = 12.137, p < 0.001), 5(6)-epoxyeicosatrienoic acid (5(6)-EpETrE; F1,100 = 6.481, p = 0.012) and 11(12)-EpETrE (F1,100 = 4.409, p = 0.038), and 2) verbal memory, including 9(10)-EpOME (F1,100 = 4.286, p = 0.041), 5(6)-EpETrE (F1,100 = 6.845, p = 0.010), 11(12)-EpETrE (F1,100 = 3.981, p = 0.049) and 14(15)-EpETrE (F1,100 = 5.019, p = 0.027). CONCLUSIONS: Associations of CYP450-sEH metabolites and depressive symptoms with cognition highlight the biomarker and therapeutic potential of the CYP450-sEH pathway in T2DM.

Inhibition of soluble epoxide hydrolase by natural isothiocyanates.

GOAL: The long-term goal of our research is to develop safe and effective soluble epoxide hydrolase (sEH) inhibitors. The objective of this study is to evaluate the potency and selectivity of six natural isothiocyanates (ITCs) as sEH inhibitors. METHODS: Molecular docking was used to model likely interactions between the ligands and receptors. The sEH inhibitory activity was tested using a validated fluorescence-based assay and PHOME as a substrate. To evaluate their selectivity as sEH inhibitors, the inhibitory potential of the ITCs was determined on microsomal epoxide hydrolase (mEH) and cytochrome P450 (CYP) enzymes in human liver microsomes. Probe substrates such as styrene oxide (mEH substrate) and established substrates for CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were used in this study. The metabolites of these substrates were analyzed using validated LC-MS/MS and HPLC-UV assays. RESULTS: Molecular Docking revealed significant differences in binding site preference among the ITCs in silico and pointed to important interactions between the ligands and the catalytic residues of the sEH enzyme. In vitro, the ITCs showed varying degrees of sEH inhibition, but sulforaphane (SFN) and phenyl isothiocyanate (PITC) were the most potent inhibitors with IC50 values of 3.65 and 7.5 µM, respectively. mEH was not significantly inhibited by any of the ITCs. Erucin and iberin were the only ITCs that did not inhibit the activity of any of the tested CYP enzymes. CONCLUSION: Our results demonstrate that natural ITCs have the potential to offer safe, selective, and potent sEH inhibition.

PharmaCore: The Automatic Generation of 3D Structure-Based Pharmacophore Models from Protein/Ligand Complexes.

In this work, we present PharmaCore: a new, completely automatic workflow aimed at generating three-dimensional (3D) structure-based pharmacophore models toward any target of interest. The proposed approach relies on using cocrystallized ligands to create the input files for generating the pharmacophore hypotheses, integrating not only the three-dimensional structural information on the ligand but also data concerning the binding mode of these molecules put in the protein cavity. We developed a Python library that, starting from the specific UniProt ID of the protein under investigation as the only element that requires user intervention, subsequently collects and aligns the corresponding structures bearing a known ligand in a fully automated fashion, bringing them all into the same coordinate system. The protocol includes a final phase in which the aligned small molecules are used to produce the pharmacophore hypotheses directly onto the protein structure using a specific software, e.g., Phase (Schrödinger LLC). To validate the entire procedure and highlight the possible applications in the field of drug discovery and repositioning, we first generated pharmacophores for soluble epoxide hydrolase (sEH) and compared with already-published ones. Then, we reproduced the binding profile of a reported selective binder of ATAD2 bromodomain (AM879), testing it against a panel of 1741 pharmacophores related to 16 epigenetic proteins and automatically generated with PharmaCore, finally disclosing putative unprecedented off-targets. The computational predictions were successfully validated with AlphaScreen assays, highlighting the applicability of the proposed workflow in drug discovery and repositioning. Finally, the process was also validated on tankyrase 2 and SARS-CoV-2 MPro, confirming the robustness of PharmaCore.

Epoxygenase Cyp2c44 Regulates Hepatic Lipid Metabolism and Insulin Signaling by Controlling FATP2 Localization and Activation of the DAG/PKCδ Axis.

Cytochrome P450 epoxygenase Cyp2c44, a murine epoxyeicosatrienoic acid (EET)-producing enzyme, promotes insulin sensitivity, and Cyp2c44-/- mice show hepatic insulin resistance. Because insulin resistance leads to hepatic lipid accumulation and hyperlipidemia, we hypothesized that Cyp2c44 regulates hepatic lipid metabolism. Standard chow diet (SCD)-fed male Cyp2c44-/- mice had significantly decreased EET levels and increased hepatic and plasma lipid levels compared with wild-type mice. We showed increased hepatic plasma membrane localization of the FA transporter 2 (FATP2) and total unsaturated fatty acids and diacylglycerol (DAG) levels. Cyp2c44-/- mice had impaired glucose tolerance and increased hepatic plasma membrane-associated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Surprisingly, SCD and high-fat diet (HFD)-fed Cyp2c44-/- mice had similar glucose tolerance and hepatic plasma membrane PKCδ levels, suggesting that SCD-fed Cyp2c44-/- mice have reached their maximal glucose intolerance. Inhibition of PKCδ resulted in decreased IRS-1 serine phosphorylation and improved insulin-mediated signaling in Cyp2c44-/- hepatocytes. Finally, Cyp2c44-/- HFD-fed mice treated with the analog EET-A showed decreased hepatic plasma membrane FATP2 and PCKδ levels with improved glucose tolerance and insulin signaling. In conclusion, loss of Cyp2c44 with concomitant decreased EET levels leads to increased hepatic FATP2 plasma membrane localization, DAG accumulation, and PKCδ-mediated attenuation of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling.

Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity.

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 µM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

Tissue-, Region-, and Gene-Specific Induction of Microsomal Epoxide Hydrolase Expression and Activity in the Mouse Intestine by Arsenic in Drinking Water.

This study aimed to characterize the effects of arsenic exposure on the expression of microsomal epoxide hydrolase (mEH or EPHX1) and soluble epoxide hydrolase (sEH or EPHX2) in the liver and small intestine. C57BL/6 mice were exposed to sodium arsenite in drinking water at various doses for up to 28 days. Intestinal, but not hepatic, mEH mRNA and protein expression was induced by arsenic at 25 ppm, in both males and females, whereas hepatic mEH expression was induced by arsenic at 50 or 100 ppm. The induction of mEH was gene specific, as the arsenic exposure did not induce sEH expression in either tissue. Within the small intestine, mEH expression was induced only in the proximal, but not the distal segments. The induction of intestinal mEH was accompanied by increases in microsomal enzymatic activities toward a model mEH substrate, cis-stilbene oxide, and an epoxide-containing drug, oprozomib, in vitro, and by increases in the levels of PR-176, the main hydrolysis metabolite of oprozomib, in the proximal small intestine of oprozomib-treated mice. These findings suggest that intestinal mEH, playing a major role in converting xenobiotic epoxides to less reactive diols, but not sEH, preferring endogenous epoxides as substrates, is relevant to the adverse effects of arsenic exposure, and that further studies of the interactions between drinking water arsenic exposure and the disposition or possible adverse effects of epoxide-containing drugs and other xenobiotic compounds in the intestine are warranted. SIGNIFICANCE STATEMENT: Consumption of arsenic-contaminated water has been associated with increased risks of various adverse health effects, such as diabetes, in humans. The small intestinal epithelial cells are the main site of absorption of ingested arsenic, but they are not well characterized for arsenic exposure-related changes. This study identified gene expression changes in the small intestine that may be mechanistically linked to the adverse effects of arsenic exposure and possible interactions between arsenic ingestion and the pharmacokinetics of epoxide-containing drugs in vivo.

Effects of sEH inhibition on the eicosanoid and cytokine storms in SARS-CoV-2-infected mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves an initial viral infection phase followed by a host-response phase that includes an eicosanoid and cytokine storm, lung inflammation and respiratory failure. While vaccination and early anti-viral therapies are effective in preventing or limiting the pathogenic host response, this latter phase is poorly understood with no highly effective treatment options. Inhibitors of soluble epoxide hydrolase (sEH) increase levels of anti-inflammatory molecules called epoxyeicosatrienoic acids (EETs). This study aimed to investigate the impact of sEH inhibition on the host response to SARS-CoV-2 infection in a mouse model with human angiotensin-converting enzyme 2 (ACE2) expression. Mice were infected with SARS-CoV-2 and treated with either vehicle or the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU). At day 5 post-infection, SARS-CoV-2 induced weight loss, clinical signs, a cytokine storm, an eicosanoid storm, and severe lung inflammation with ~50% mortality on days 6-8 post-infection. SARS-CoV-2 infection induced lung expression of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX) pathway genes, while suppressing expression of most cytochrome P450 genes. Treatment with the sEH inhibitor TPPU delayed weight loss but did not alter clinical signs, lung cytokine expression or overall survival of infected mice. Interestingly, TPPU treatment significantly reversed the eicosanoid storm and attenuated viral-induced elevation of 39 fatty acids and oxylipins from COX, LOX and P450 pathways, which suggests the effects at the level of PLA2 activation. The suppression of the eicosanoid storm by TPPU without corresponding changes in lung cytokines, lung inflammation or mortality reveals a surprising dissociation between systemic oxylipin and cytokine signaling pathways during SARS-CoV-2 infection and suggests that the cytokine storm is primarily responsible for morbidity and mortality in this animal model.

Soluble epoxide hydrolase inhibition reverses cognitive dysfunction in a mouse model of metabolic syndrome by modulating inflammation.

Midlife metabolic syndrome (MetS) is associated with cognitive impairment in late life. The mechanism of delayed MetS-related cognitive dysfunction (MetSCD) is not clear, but it has been linked to systemic inflammation and chronic cerebral microangiopathy. Currently there is no treatment for late life MetSCD other than early risk factor modification. We investigated the effect of soluble epoxide hydrolase (sEH) inhibitor 4-[[trans-4-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]cyclohexyl]oxy]-benzoic acid (t-AUCB) on cognitive performance, cerebral blood flow (CBF), and central and peripheral inflammation in the high-fat diet (HFD) model of MetS in mice. At 6 weeks of age, male mice were randomly assigned to receive either HFD or standard chow (STD) for 6 months. Mice received either t-AUCB or vehicle for 4 weeks. Cognitive performance was evaluated, followed by CBF measurement using magnetic resonance imaging (MRI). At the end of the study, blood was collected for measurement of eicosanoids and inflammatory cytokines. The brains were then analyzed by immunohistochemistry for glial activation markers. The HFD caused a significant impairment in novel object recognition. Treatment with t-AUCB increased plasma levels of 14,15-EET, prevented this cognitive impairment and modified hippocampal glial activation and plasma cytokine levels, without affecting CBF in mice on HFD. In conclusion, sEH inhibition for four weeks prevents cognitive deficits in mice on chronic HFD by modulating inflammatory processes without affecting CBF.

The Inhibition Activity of Natural Methoxyflavonoid from Inula britannica on Soluble Epoxide Hydrolase and NO Production in RAW264.7 Cells.

Soluble epoxide hydrolase (sEH) is an enzyme targeted for the treatment of inflammation and cardiovascular diseases. Activated inflammatory cells produce nitric oxide (NO), which induces oxidative stress and exacerbates inflammation. We identify an inhibitor able to suppress sEH and thus NO production. Five flavonoids 1-5 isolated from Inula britannica flowers were evaluated for their abilities to inhibit sEH with IC50 values of 12.1 ± 0.1 to 62.8 ± 1.8 µM and for their effects on enzyme kinetics. A simulation study using computational chemistry was conducted as well. Furthermore, five inhibitors (1-5) were confirmed to suppress NO levels at 10 µM. The results showed that flavonoids 1-5 exhibited inhibitory activity in all tests, with compound 3 exhibiting the most significant efficacy. Thus, in the development of anti-inflammatory inhibitors, compound 3 is a promising natural candidate.

Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (R)-Tebuconazole in High Optical Purity Mediated by an Epoxide Hydrolase from Rhodotorula paludigensis.

Tebuconazole is a chiral triazole fungicide used globally in agriculture as a racemic mixture, but its enantiomers exhibit significant enantioselective dissimilarities in bioactivity and environmental behaviors. The steric hindrance caused by the tert-butyl group makes it a great challenge to synthesize tebuconazole enantiomers. Here, we designed a simple chemoenzymatic approach for the asymmetric synthesis of (R)-tebuconazole, which includes the biocatalytic resolution of racemic epoxy-precursor (2-tert-butyl-2-[2-(4-chlorophenyl)ethyl] oxirane, rac-1a) by Escherichia coli/Rpeh whole cells expressed epoxide hydrolase from Rhodotorula paludigensis (RpEH), followed by a one-step chemocatalytic synthesis of (R)-tebuconazole. It was observed that (S)-1a was preferentially hydrolyzed by E. coli/Rpeh, whereas (R)-1a was retained with a specific activity of 103.8 U/g wet cells and a moderate enantiomeric ratio (E value) of 13.4, which was remarkably improved to 43.8 after optimizing the reaction conditions. Additionally, a gram-scale resolution of 200 mM rac-1a was performed using 150 mg/mL E. coli/Rpeh wet cells, resulting in the retention of (R)-1a in a 97.0% ees, a 42.5% yields, and a 40.5 g/L/d space-time yield. Subsequently, the synthesis of highly optical purity (R)-tebuconazole (>99% ee) was easily achieved through the chemocatalytic ring-opening of the epoxy-precursor (R)-1a with 1,2,4-triazole. To elucidate insight into the enantioselectivity, molecular docking simulations revealed that the unique L-shaped substrate-binding pocket of RpEH plays a crucial role in the enantioselective recognition of bulky 2,2-disubstituted oxirane 1a.

Comprehensive analysis of the immune implication of EPHX4 gene in laryngeal squamous cell carcinoma.

OBJECTIVES: The role of Epoxide Hydrolase-4 (EPHX4), a member of epoxide hydrolase family, has not been investigated in cancer. The purpose of this article is to explore the application value of EPHX4 in laryngeal cancer and its relationship with immune infiltration. METHODS: We observed that EPHX4 expression and its survival assays in laryngeal cancer specimens based on The Cancer Genome Atlas (TCGA) cohorts. We also analyzed the correlation between immune cell infiltration levels and EPHX4 gene copy number in laryngeal cancer. Finally, we conducted in vitro assay to evaluate the functions of EPHX4 in laryngeal cancer cell line. RESULTS: EPHX4 is highly expressed in laryngeal cancer specimens and has a poor prognosis. EPHX4 related immune cell analysis showed that it participated in NK Natural killer cell mediated cytotoxicity. Finally, Cell experiments indicate that EPHX4 could promote laryngeal cancer cell line proliferation, colony formation and invasion. CONCLUSIONS: Our research results suggest that EPHX4 may be a potential immunotherapy target for laryngeal cancer. The nominated immune signature is a helpful and promising prognostic indicator in laryngeal cancer. LEVELS OF EVIDENCE: Level 3.

Cardioprotective response and senescence in aged sEH null female mice exposed to LPS.

Deterioration of physiological systems, like the cardiovascular system, occurs progressively with age impacting an individual's health and increasing susceptibility to injury and disease. Cellular senescence has an underlying role in age-related alterations and can be triggered by natural aging or prematurely by stressors such as the bacterial toxin lipopolysaccharide (LPS). The metabolism of polyunsaturated fatty acids by CYP450 enzymes produces numerous bioactive lipid mediators that can be further metabolized by soluble epoxide hydrolase (sEH) into diol metabolites, often with reduced biological effects. In our study, we observed age-related cardiac differences in female mice, where young mice demonstrated resistance to LPS injury, and genetic deletion or pharmacological inhibition of sEH using trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid attenuated LPS-induced cardiac dysfunction in aged female mice. Bulk RNA-sequencing analyses revealed transcriptomics differences in aged female hearts. The confirmatory analysis demonstrated changes to inflammatory and senescence gene markers such as Il-6, Mcp1, Il-1ß, Nlrp3, p21, p16, SA-ß-gal, and Gdf15 were attenuated in the hearts of aged female mice where sEH was deleted or inhibited. Collectively, these findings highlight the role of sEH in modulating the aging process of the heart, whereby targeting sEH is cardioprotective.NEW & NOTEWORTHY Soluble epoxide hydrolase (sEH) is an essential enzyme for converting epoxy fatty acids to their less bioactive diols. Our study suggests deletion or inhibition of sEH impacts the aging process in the hearts of female mice resulting in cardioprotection. Data indicate targeting sEH limits inflammation, preserves mitochondria, and alters cellular senescence in the aged female heart.

Structure-Guided Elaboration of a Fragment-Like Hit into an Orally Efficacious Leukotriene A4 Hydrolase Inhibitor.

Leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have provided strong evidence that LTA4H is an attractive drug target for the treatment of chronic inflammatory diseases. Here, we describe the transformation of compound 2, a fragment-like hit, into the potent inhibitor of LTA4H 3. Our strategy involved two key steps. First, we aimed to increase the polarity of fragment 2 to improve its drug-likeness, particularly its solubility, while preserving both its promising potency and low molecular weight. Second, we utilized structural information and incorporated a basic amino function, which allowed for the formation of an essential hydrogen bond with Q136 of LTA4H and consequently enhanced the potency. Compound 3 exhibited exceptional selectivity and showed oral efficacy in a KRN passive serum-induced arthritis model in mice. The anticipated human dose to achieve 90% target engagement at the trough concentration was determined to be 40 mg administered once daily.

Identification and characterization of the in-vivo metabolites of the novel soluble epoxide hydrolase inhibitor EC5026 using liquid chromatography quadrupole time of flight mass spectrometry.

EC5026 is a novel soluble epoxide hydrolase inhibitor being developed clinically to treat neuropathic pain and inflammation. In the current study, we employed the LC-ESI-Q-TOF-MS/MS technique to identify four in-vivo phase-I metabolites of EC5026 in rat model, out of which three were found to be novel. The identified metabolites include aliphatic hydroxylation, di-hydroxylation, terminal desaturation, and carboxylation. No phase-II metabolites were found. The pharmacokinetic profile of identified metabolites was established after a single oral dose of EC5026 to Wistar rats. The Tmax of the drug and metabolites were found to be in the range of 1-2 hours and 4-12 hours, respectively. The major metabolites M1 and M2 were found to have more than 2-fold (263.87% AUC) and equivalent exposure (96.33% AUC) compared to the parent drug, respectively. Further, the docking study revealed that the mono-hydroxylated and terminally desaturated metabolites possess better binding affinity than the parent drug. Therefore, these metabolites may hold sEH inhibition potential and can be followed through future research.

Regulation of soluble epoxide hydrolase in renal-associated diseases: insights from potential mechanisms to clinical researches.

In recent years, numerous experimental studies have underscored the pivotal role of soluble epoxide hydrolase (sEH) in renal diseases, demonstrating the reno-protective effects of sEH inhibitors. The nexus between sEH and renal-associated diseases has garnered escalating attention. This review endeavors to elucidate the potential molecular mechanisms of sEH in renal diseases and emphasize the critical role of sEH inhibitors as a prospective treatment modality. Initially, we expound upon the correlation between sEH and Epoxyeicosatrienoic acids (EETs) and also addressing the impact of sEH on other epoxy fatty acids, delineate prevalent EPHX2 single nucleotide polymorphisms (SNPs) associated with renal diseases, and delve into sEH-mediated potential mechanisms, encompassing oxidative stress, inflammation, ER stress, and autophagy. Subsequently, we delineate clinical research pertaining to sEH inhibition or co-inhibition of sEH with other inhibitors for the regulation of renal-associated diseases, covering conditions such as acute kidney injury, chronic kidney diseases, diabetic nephropathy, and hypertension-induced renal injury. Our objective is to validate the potential role of sEH inhibitors in the treatment of renal injuries. We contend that a comprehensive comprehension of the salient attributes of sEH, coupled with insights from clinical experiments, provides invaluable guidance for clinicians and presents promising therapeutic avenues for patients suffering from renal diseases.

Association between CYP4A11 and EPHX2 genetic polymorphisms and chronic kidney disease progression in hypertensive patients.

BACKGROUND: There are evidence indicating that some metabolites of arachidonic acid produced by cytochromes P450 (CYP) and epoxide hydroxylase (EPHX2), such as hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs) or dihydroxyeicosatrienoic acids (DHETEs), play an important role in blood pressure regulation and they could contribute to the development of hypertension (HT) and kidney damage. Therefore, the main aim of the study was to evaluate whether the genetic polymorphisms of CYP2C8, CYP2C9, CYP2J2, CYP4F2, CYP4F11 and EPHX2, responsible for the formation of HETEs, EETs and DHETEs, are related to the progression of impaired renal function in a group of patients with hypertension. METHODS: 151HT patients from a hospital nephrology service were included in the study. Additionally, a group of 87 normotensive subjects were involved in the study as control group. For HT patients, a general biochemistry analysis, estimated glomerular filtration rate and genotyping for different CYPs and EPHX2 variant alleles was performed. RESULTS: CYP4A11 rs3890011, rs9332982 and EPHX2 rs41507953 polymorphisms, according to the dominant model, presented a high risk of impaired kidney function, with odds ratios (OR) of 2.07 (1.00-4.32; P=0.049) 3.02 (1.11-8.23; P=0.030) and 3.59 (1.37-9.41; P=0.009), respectively, and the EPHX2 rs1042032 polymorphism a greater risk according to the recessive model (OR=6.23; 95% CI=1.50-25.95; P=0.007). However, no significant differences in allele frequencies between HT patients and in normotensive subjects for any of the SNP analysed. In addition, the patients with diagnosis of dyslipidemia (n=90) presented higher frequencies of EPHX2 K55R (rs41507953) and *35A>G (rs1042032) variants than patients without dyslipidemia, 4% vs. 14% (P=0.005) and 16 vs. 27% (P=0.02), respectively. CONCLUSIONS: In this study has been found higher odds of impaired renal function progression associated with rs3890011 and rs9332982 (CYP4A11) and rs41507953 and rs1042032 (EPHX2) polymorphisms, which may serve as biomarkers for improve clinical interventions aimed at avoiding or delaying, in chronic kidney disease patients, progress to end-stage kidney disease needing dialysis or kidney transplant.