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.

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.

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.

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.

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.

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.

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.

Enhancing cancer immunotherapy via inhibition of soluble epoxide hydrolase.

Cancer therapy, including immunotherapy, is inherently limited by chronic inflammation-induced tumorigenesis and toxicity within the tumor microenvironment. Thus, stimulating the resolution of inflammation may enhance immunotherapy and improve the toxicity of immune checkpoint inhibition (ICI). As epoxy-fatty acids (EpFAs) are degraded by the enzyme soluble epoxide hydrolase (sEH), the inhibition of sEH increases endogenous EpFA levels to promote the resolution of cancer-associated inflammation. Here, we demonstrate that systemic treatment with ICI induces sEH expression in multiple murine cancer models. Dietary omega-3 polyunsaturated fatty acid supplementation and pharmacologic sEH inhibition, both alone and in combination, significantly enhance anti-tumor activity of ICI in these models. Notably, pharmacological abrogation of the sEH pathway alone or in combination with ICI counter-regulates an ICI-induced pro-inflammatory and pro-tumorigenic cytokine storm. Thus, modulating endogenous EpFA levels through dietary supplementation or sEH inhibition may represent a unique strategy to enhance the anti-tumor activity of paradigm cancer therapies.

Role of soluble epoxide hydrolase in pain and depression comorbidity.

The coexistence of chronic pain and depression in clinical practice places a substantial social burden and profoundly impacts in patients. Although a clear correlation exists, the underlying mechanism of comorbidity between chronic pain and depression remains elusive. Research conducted in recent decades has uncovered that soluble epoxide hydrolase, a pivotal enzyme in the metabolism of polyunsaturated fatty acids, plays a crucial role in inflammation. Interestingly, this enzyme is intricately linked to the development of both pain and depression. With this understanding, this review aims to summarize the roles of soluble epoxide hydrolase in pain, depression, and their comorbidity. Simultaneously, we will also explore the underlying mechanisms, providing guidance for future research and drug development.

Deciphering the stereo-specific catalytic mechanisms of cis-epoxysuccinate hydrolases producing L(+)-tartaric acid.

Microbial epoxide hydrolases, cis-epoxysuccinate hydrolases (CESHs), have been utilized for commercial production of enantiomerically pure L(+)- and D(-)-tartaric acids for decades. However, the stereo-catalytic mechanism of CESH producing L(+)-tartaric acid (CESH[L]) remains unclear. Herein, the crystal structures of two CESH[L]s in ligand-free, product-complexed, and catalytic intermediate forms were determined. These structures revealed the unique specific binding mode for the mirror-symmetric substrate, an active catalytic triad consisting of Asp-His-Glu, and an arginine providing a proton to the oxirane oxygen to facilitate the epoxide ring-opening reaction, which has been pursued for decades. These results provide the structural basis for the rational engineering of these industrial biocatalysts.

Inhibition of soluble epoxide hydrolase enhances the dentin-pulp complex regeneration mediated by crosstalk between vascular endothelial cells and dental pulp stem cells.

BACKGROUND: Revascularization and restoration of normal pulp-dentin complex are important for tissue-engineered pulp regeneration. Recently, a unique periodontal tip-like endothelial cells subtype (POTCs) specialized to dentinogenesis was identified. We have confirmed that TPPU, a soluble epoxide hydrolase (sEH) inhibitor targeting epoxyeicosatrienoic acids (EETs) metabolism, promotes bone growth and regeneration by angiogenesis and osteogenesis coupling. We hypothesized that TPPU could also promote revascularization and induce POTCs to contribute to pulp-dentin complex regeneration. Here, we in vitro and in vivo characterized the potential effect of TPPU on the coupling of angiogenesis and odontogenesis and investigated the relevant mechanism, providing new ideas for pulp-dentin regeneration by targeting sEH. METHODS: In vitro effects of TPPU on the proliferation, migration, and angiogenesis of dental pulp stem cells (DPSCs), human umbilical vein endothelial cells (HUVECs) and cocultured DPSCs and HUVECs were detected using cell counting kit 8 (CCK8) assay, wound healing, transwell, tube formation and RT-qPCR. In vivo, Matrigel plug assay was performed to outline the roles of TPPU in revascularization and survival of grafts. Then we characterized the VEGFR2 + POTCs around odontoblast layer in the molar of pups from C57BL/6 female mice gavaged with TPPU. Finally, the root segments with DPSCs mixed with Matrigel were implanted subcutaneously in BALB/c nude mice treated with TPPU and the root grafts were isolated for histological staining. RESULTS: In vitro, TPPU significantly promoted the migration and tube formation capability of cocultured DPSCs and HUVECs. ALP and ARS staining and RT-qPCR showed that TPPU promoted the osteogenic and odontogenic differentiation of cultured cells, treatment with an anti-TGF-ß blocking antibody abrogated this effect. Knockdown of HIF-1α in HUVECs significantly reversed the effect of TPPU on the expression of angiogenesis, osteogenesis and odontogenesis-related genes in cocultured cells. Matrigel plug assay showed that TPPU increased VEGF/VEGFR2-expressed cells in transplanted grafts. TPPU contributed to angiogenic-odontogenic coupling featured by increased VEGFR2 + POTCs and odontoblast maturation during early dentinogenesis in molar of newborn pups from C57BL/6 female mice gavaged with TPPU. TPPU induced more dental pulp-like tissue with more vessels and collagen fibers in transplanted root segment. CONCLUSIONS: TPPU promotes revascularization of dental pulp regeneration by enhancing migration and angiogenesis of HUVECs, and improves odontogenic differentiation of DPSCs by TGF-ß. TPPU boosts the angiogenic-odontogenic coupling by enhancing VEGFR2 + POTCs meditated odontoblast maturation partly via upregulating HIF-1α, which contributes to increasing pulp-dentin complex for tissue-engineered pulp regeneration.

Novel molecular mechanism driving neuroprotection after soluble epoxide hydrolase inhibition: Insights for Alzheimer's disease therapeutics.

BACKGROUND: Neuroinflammation is widely recognized as a significant hallmark of Alzheimer's disease (AD). To combat neuroinflammation, the inhibition of the soluble epoxide hydrolase (sEH) enzyme has been demonstrated crucial. Importantly, sEH inhibition could be related to other neuroprotective pathways described in AD. AIMS: The aim of the study was to unveil new molecular pathways driving neuroprotection through sEH, we used an optimized, potent, and selective sEH inhibitor (sEHi, UB-SCG-51). MATERIALS AND METHODS: UB-SCG-51 was tested in neuroblastoma cell line, SH-SY5Y, in primary mouse and human astrocytes cultures challenged with proinflammatory insults and in microglia cultures treated with amyloid oligomers, as well as in mice AD model (5XFAD). RESULTS: UB-SCG-51 (10 and 30 µM) prevented neurotoxic reactive-astrocyte conversion in primary mouse astrocytes challenged with TNF-α, IL-1α, and C1q (T/I/C) combination for 24 h. Moreover, in microglial cultures, sEHi reduced inflammation and glial activity. In addition, UB-SCG-51 rescued 5XFAD cognitive impairment, reducing the number of Amyloid-ß plaques and Tau hyperphosphorylation accompanied by a reduction in neuroinflammation and apoptotic markers. Notably, a transcriptional profile analysis revealed a new pathway modulated by sEHi treatment. Specifically, the eIF2α/CHOP pathway, which promoted the endoplasmic reticulum response, was increased in the 5XFAD-treated group. These findings were confirmed in human primary astrocytes by combining sEHi and eIF2α inhibitor (eIF2αi) treatment. Besides, combining both treatments resulted in increased in C3 gene expression after T/I/C compared with the group treated with sEHi alone in cultures. DISCUSSION: Therefore, sEHi rescued cognitive impairment and neurodegeneration in AD mice model, based on the reduction of inflammation and eIF2α/CHOP signaling pathway. CONCLUSIONS: In whole, our results support the concept that targeting neuroinflammation through sEH inhibition is a promising therapeutic strategy to fight against Alzheimer's disease with additive and/or synergistic activities targeting neuroinflammation and cell stress.

Hepatic Transcriptome and Its Regulation Following Soluble Epoxide Hydrolase Inhibition in Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (ALD) is a serious public health problem with limited pharmacologic options. The goal of the current study was to investigate the efficacy of pharmacologic inhibition of soluble epoxide hydrolase (sEH), an enzyme involved in lipid metabolism, in experimental ALD, and to examine the underlying mechanisms. C57BL/6J male mice were subjected to acute-on-chronic ethanol (EtOH) feeding with or without the sEH inhibitor 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB). Liver injury was assessed by multiple end points. Liver epoxy fatty acids and dihydroxy fatty acids were measured by targeted metabolomics. Whole-liver RNA sequencing was performed, and free modified RNA bases were measured by mass spectrometry. EtOH-induced liver injury was ameliorated by TUCB treatment as evidenced by reduced plasma alanine aminotransferase levels and was associated with attenuated alcohol-induced endoplasmic reticulum stress, reduced neutrophil infiltration, and increased numbers of hepatic M2 macrophages. TUCB altered liver epoxy and dihydroxy fatty acids and led to a unique hepatic transcriptional profile characterized by decreased expression of genes involved in apoptosis, inflammation, fibrosis, and carcinogenesis. Several modified RNA bases were robustly changed by TUCB, including N6-methyladenosine and 2-methylthio-N6-threonylcarbamoyladenosine. These findings show the beneficial effects of sEH inhibition by TUCB in experimental EtOH-induced liver injury, warranting further mechanistic studies to explore the underlying mechanisms, and highlighting the translational potential of sEH as a drug target for this disease.

Modulating the sEH/EETs Axis Restrains Specialized Proresolving Mediator Impairment and Regulates T Cell Imbalance in Experimental Periodontitis.

Epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids are short-acting lipids involved in resolution of inflammation. Their short half-life, due to its metabolism by soluble epoxide hydrolase (sEH), limits their effects. Specialized proresolving mediators (SPMs) are endogenous regulatory lipids insufficiently synthesized in uncontrolled and chronic inflammation. Using an experimental periodontitis model, we pharmacologically inhibited sEH, examining its impact on T cell activation and systemic SPM production. In humans, we analyzed sEH in the gingival tissue of periodontitis patients. Mice were treated with sEH inhibitor (sEHi) and/or EETs before ligature placement and treated for 14 d. Bone parameters were assessed by microcomputed tomography and methylene blue staining. Blood plasma metabololipidomics were carried out to quantify SPM levels. We also determined T cell activation by reverse transcription-quantitative PCR and flow cytometry in cervical lymph nodes. Human gingival samples were collected to analyze sEH using ELISA and electrophoresis. Data reveal that pharmacological sEHi abrogated bone resorption and preserved bone architecture. Metabololipidomics revealed that sEHi enhances lipoxin A4, lipoxin B4, resolvin E2, and resolvin D6. An increased percentage of regulatory T cells over Th17 was noted in sEHi-treated mice. Lastly, inflamed human gingival tissues presented higher levels and expression of sEH than did healthy gingivae, being positively correlated with periodontitis severity. Our findings indicate that sEHi preserves bone architecture and stimulates SPM production, associated with regulatory actions on T cells favoring resolution of inflammation. Because sEH is enhanced in human gingivae from patients with periodontitis and connected with disease severity, inhibition may prove to be an attractive target for managing osteolytic inflammatory diseases.

Hyperglycemia disrupted the integrity of the blood-brain barrier following diffuse axonal injury through the sEH/NF-κB pathway.

OBJECTIVES: We aimed to investigate the role of soluble epoxide hydrolase for hyperglycemia induced-disruption of blood-brain barrier (BBB) integrity after diffuse axonal injury (DAI). METHODS: Rat DAI hyperglycemia model was established by a lateral head rotation device and intraperitoneal injection of 50% glucose. Glial fibrillary acidic protein, ionized calcium-binding adapter molecule-1, ß-amyloid precursor protein, neurofilament light chain, and neurofilament heavy chain was detected by immunohistochemistry. Cell apoptosis was examined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay. The permeability of blood-brain barrier (BBB) was assessed by expression of tight junction proteins, leakage of Evans blue and brain water content. The soluble epoxide hydrolase (sEH) pathway was inhibited by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) and the nuclear transcription factor kappa B (NF-κB) pathway was inhibited by pyrrolidine dithiocarbamate and activated by phorbol-12-myristate-13-acetate in vivo and/or vitro, respectively. The inflammatory factors were detected by enzyme-linked immunosorbent assay. RESULTS: Hyperglycemia could exacerbate axonal injury, aggravate cell apoptosis and glial activation, worsen the loss of BBB integrity, increase the release of inflammatory factors, and upregulate the expression of sEH and NF-κB. Inhibition of sEH could reverse all these damages and protect BBB integrity by upregulating the expression of tight junction proteins and downregulating the levels of inflammatory factors in vivo and vitro, while the agonist of NF-κB pathway abrogated the protective effects of TPPU on BBB integrity in vitro. CONCLUSIONS: sEH was involved in mediating axonal injury induced by hyperglycemia after DAI by disrupting BBB integrity through inducing inflammation via the NF-κB pathway.

Determinants of Meal-Induced Changes in Circulating FFA Epoxides, Diols, and Diol-to-Epoxide Ratios as Indices of Soluble Epoxide Hydrolase Activity.

Soluble epoxide hydrolase (sEH) is an important enzyme for metabolic and cardiovascular health. sEH converts FFA epoxides (EpFAs), many of which are regulators of various cellular processes, to biologically less active diols. In human studies, diol (sEH product) to EpFA (sEH substrate) ratios in plasma or serum have been used as indices of sEH activity. We previously showed these ratios profoundly decreased in rats during acute feeding, possibly reflecting decreases in tissue sEH activities. The present study was designed to test which tissue(s) these measurements in the blood represent and if factors other than sEH activity, such as renal excretion or dietary intake of EpFAs and diols, significantly alter plasma EpFAs, diols, and/or their ratios. The results show that postprandial changes in EpFAs and diols and their ratios in plasma were very similar to those observed in the liver but not in other tissues, suggesting that the liver is largely responsible for these changes in plasma levels. EpFAs and diols were excreted into the urine, but their levels were not significantly altered by feeding, suggesting that renal excretion of EpFAs and diols may not play a major role in postprandial changes in circulating EpFAs, diols, or their ratios. Diet intake had significant impacts on circulating EpFA and diol levels but not on diol-to-EpFA (D-to-E) ratios, suggesting that these ratios, reflecting sEH activities, may not be significantly affected by the availability of sEH substrates (i.e., EpFAs). In conclusion, changes in FFA D-to-E ratios in plasma may reflect those in the liver, which may in turn represent sEH activities in the liver, and they may not be significantly affected by renal excretion or the dietary intake of EpFAs and diols.

Role of soluble epoxide hydrolase in the abnormal activation of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by enigmatic pathogenesis. Polyunsaturated fatty acids (PUFAs) are implicated in RA's development and progression, yet their exact mechanisms of influence are not fully understood. Soluble epoxide hydrolase (sEH) is an enzyme that metabolizes anti-inflammatory epoxy fatty acids (EpFAs), derivatives of PUFAs. In this study, we report elevated sEH expression in the joints of CIA (collagen-induced arthritis) rats, concomitant with diminished levels of two significant EpFAs. Additionally, increased sEH expression was detected in both the synovium of CIA rats and in the synovium and fibroblast-like synoviocytes (FLS) of RA patients. The sEH inhibitor TPPU attenuated the migration and invasion capabilities of FLS derived from RA patients and to reduce the secretion of inflammatory factors by these cells. Our findings indicate a pivotal role for sEH in RA pathogenesis and suggest that sEH inhibitors offer a promising new therapeutic strategy for managing RA.

Discovery of Amino Alcohols as Highly Potent, Selective, and Orally Efficacious Inhibitors of Leukotriene A4 Hydrolase.

The discovery of chiral amino alcohols derived from our previously disclosed clinical LTA4H inhibitor LYS006 is described. In a biochemical assay, their optical antipodes showed similar potencies, which could be rationalized by the cocrystal structures of these compounds bound to LTA4H. Despite comparable stabilities in liver microsomes, they showed distinct in vivo PK properties. Selective O-phosphorylation of the (R)-enantiomers in blood led to clearance values above the hepatic blood flow, whereas the (S)-enantiomers were unaffected and exhibited satisfactory metabolic stabilities in vivo. Introduction of two pyrazole rings led to compound (S)-2 with a more balanced distribution of polarity across the molecule, exhibiting high selectivity and excellent potency in vitro and in vivo. Furthermore, compound (S)-2 showed favorable profiles in 16-week IND-enabling toxicology studies in dogs and rats. Based on allometric scaling and potency in whole blood, compound (S)-2 has the potential for a low oral efficacious dose administered once daily.

Pro-inflammatory GPR75 and anti-apoptotic phospholipase signaling pathways contribute to the ameliorating effect of soluble epoxide hydrolase inhibition on chronic experimental autoimmune encephalomyelitis in mice.

Soluble epoxide hydrolase (sEH) inhibition has currently emerged as a therapeutic target in the treatment of various neuroinflammatory neurodegenerative diseases, including multiple sclerosis. Previously, we reported that treatment of mice with a sEH-selective inhibitor, 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea; TPPU), ameliorated chronic experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein 35-55 peptide immunization followed by injection of pertussis toxin to mice via regulating pro-inflammatory and anti-inflammatory pathways in the central nervous system. This study tested the hypothesis that the pro-inflammatory G protein-coupled receptor (GPR) 75 and anti-apoptotic phospholipase C (PLC) signaling pathways also contribute to the ameliorating effect of TPPU on chronic EAE. Brains and spinal cords of phosphate-buffered saline-, dimethyl sulfoxide-, or TPPU (3 mg/kg)-treated mice were used for the measurement of sEH, GPR75, Gaq/11, activator protein (AP)-1, PLC ß4, phosphoinositide 3-kinase (PI3K) p85a, Akt1, mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase (ERK) 1/2, cyclic adenosine monophosphate-response element-binding protein (CREB) 1, B-cell lymphoma (Bcl)-2, semaphorin (SEMA) 3A, and myelin proteolipid protein (PLP) expression and/or activity by using the immunoblotting method. Expression of sEH, GPR75, Gaq/11, c-jun, phosphorylated c-Jun, and SEMA3A was lower, while PLCß4, phosphorylated PI3K p85a, phosphorylated Akt1, phosphorylated MEK1/2, phosphorylated ERK1/2, phosphorylated CREB1, Bcl-2, and myelin PLP expression was higher in the tissues of TPPU (3 mg/kg)-treated mice as compared with the EAE and vehicle control groups. Inhibition of sEH by TPPU ameliorates chronic EAE through suppressing pro-inflammatory GPR75/Gaq/11/AP-1 pathway and reducing expression of the remyelination inhibitor, SEMA3A, as well as increasing anti-apoptotic PLC/PI3K/Akt1/MEK1/2/ERK1/2/CREB1/Bcl-2 pathway activity and myelin PLP expression.

The Generation of a Nanobody-Based ELISA for Human Microsomal Epoxide Hydrolase.

A microsomal epoxide hydrolase (mEH) metabolizes in vivo in both xenobiotic and endogenous epoxides associated with signaling function. Findings in patients suggest that mEH might be a biomarker for several diseases, including metastatic cancer and viral hepatitis. To easily quantify mEH, nanobodies specific to the human mEH were isolated from a phage library of llama VHHs. Four unique clones were obtained and used for developing ELISAs. Three formats of double antibody sandwich assays were investigated using different detection strategies. Using PolyHRP, the signal was strongly amplified, yielding a 22-fold lower LOD (12 pg mL-1) than the 'conventional'. To further validate the performance of the immunoassays, human tissue samples were analyzed by nanobody-based ELISAs and compared to the enzyme activities (R2 > 0.95). The results demonstrate that these nanobodies are powerful tools for the quantification of human mEH and could eventually result in a bedside assay.