Inhibitor Report for: t-AUCB

Objectives: Brown adipose tissue has recently emerged as a novel target for obesity treatment and prevention. In contrast to the lipid storing function of white adipocytes, brown adipocytes are responsible for dissipating energy as heat, a process involving uncoupling protein 1 (UCP1). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme that converts epoxy fatty acids (EpFAs) into less active diols. By stabilizing endogenous EpFAs, potent small molecule sEH inhibitors have been shown to be beneficial for many chronic diseases. Several recent papers have reported that sEH inhibitors are able to reduce diet-induced obesity, possibly by upregulating UCP1 expression. In the current study, we sought to study the mechanisms by which sEH inhibitor acts on brown preadipocytes. Methods: The effects of a potent sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), on murine brown adipocyte differentiation were evaluated by lipid accumulation and expression of brown adipocyte marker genes. PPAR alpha and PPAR gamma activation by t-AUCB was measured by their respective transactivation assays. The roles of PPARs were further studied by pharmacological antagonism and knockdown experiments by small RNA interference. Results: We report that sEH expression was increased during murine brown adipocyte differentiation. t-AUCB dose-dependently promoted brown adipocyte differentiation. Moreover, we demonstrate that t-AUCB activated PPAR alpha, but not PPAR gamma. t-AUCB-induced upregulation of thermogenic gene Ucp1 and Pgc1 alpha and the general differentiation marker Fabp4 were significantly attenuated by the antagonist of PPAR alpha, GW6471. In contrast, they were only partially attenuated by the antagonist of PPAR gamma, GW9662, and specific knockdown of PPAR gamma. Conclusions: Our findings suggest that sEH may regulate brown adipogenesis and sEH pharmacological inhibition by t-AUCB promotes brown adipogenesis, possibly through activation of PPAR alpha. Funding Sources: The work is supported by NIH 1R15DK114790-01A1 (to LZ), R00DK100736 (to AB) and R01ES002710 (to BDH).

Although sEH inhibitors are well studied in inflammatory and cardiovascular diseases, their effects on gliomas are unclear. In this study, we investigated the effects of t-AUCB, a more potent and selective sEH inhibitor, on U251 and U87 human glioblastoma cell lines and the HepG2 human hepatocellular carcinoma cell line. Our results showed that t-AUCB efficiently inhibited sEH activities in all three cell lines (the inhibition rate was more than 80% in each) and suppressed U251 and U87 cell growth in a dose-dependent manner, but exhibited no cell growth inhibition on HepG2. We detected high levels of phosphorylated NF-kappaB-p65 (Ser536) in t-AUCB-treated U251 and U87 cells, and then found that the NF-kappaB inhibitor PDTC can completely abolish t-AUCB-induced growth inhibition. This indicated that t-AUCB suppresses U251 and U87 cell growth by activating NF-kappaB-p65. Moreover, we found that t-AUCB induces cell-cycle G0/G1 phase arrest by regulating Cyclin D1 mRNA and protein levels and CDC2 (Thr161) phosphorylation level. We propose to further test this promising reagent for its anti-glioma activity in clinical relevant orthotopic brain glioma models.

We previously reported that sEH inhibitor t-AUCB suppresses the growth of human glioblastoma U251 and U87 cell lines and induces cell-cycle G0/G1 phase arrest. In present study, we found even 96 h-treatment of 200 muM t-AUCB can not induce apoptosis in U251 and U87 cells. We also revealed that 200 muM t-AUCB significantly elevates the activation of p38 MAPK, MAPKAPK2 and Hsp27. The p38 MAPK inhibitor SB203580 and the inhibitor of Hsp27 phosphorylation, KRIBB3, were used to investigate the mechanism of the apoptosis-resistance. The results showed that, after blocking the activation of Hsp27 by SB203580 or KRIBB3, 200 muM t-AUCB significantly induces apoptosis and increases caspase-3 activities in U251 and U87 cells. Our data demonstrated that t-AUCB induces cell apoptosis after blocking itself-induced activation of Hsp27, and that the activation of Hsp27 may confer chemoresistance in GBM cells. The combination of t-AUCB and the inhibitor of Hsp27 phosphorylation may be a potential strategy for treatment of glioblastoma.

Objectives: Brown adipose tissue has recently emerged as a novel target for obesity treatment and prevention. In contrast to the lipid storing function of white adipocytes, brown adipocytes are responsible for dissipating energy as heat, a process involving uncoupling protein 1 (UCP1). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme that converts epoxy fatty acids (EpFAs) into less active diols. By stabilizing endogenous EpFAs, potent small molecule sEH inhibitors have been shown to be beneficial for many chronic diseases. Several recent papers have reported that sEH inhibitors are able to reduce diet-induced obesity, possibly by upregulating UCP1 expression. In the current study, we sought to study the mechanisms by which sEH inhibitor acts on brown preadipocytes. Methods: The effects of a potent sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), on murine brown adipocyte differentiation were evaluated by lipid accumulation and expression of brown adipocyte marker genes. PPAR alpha and PPAR gamma activation by t-AUCB was measured by their respective transactivation assays. The roles of PPARs were further studied by pharmacological antagonism and knockdown experiments by small RNA interference. Results: We report that sEH expression was increased during murine brown adipocyte differentiation. t-AUCB dose-dependently promoted brown adipocyte differentiation. Moreover, we demonstrate that t-AUCB activated PPAR alpha, but not PPAR gamma. t-AUCB-induced upregulation of thermogenic gene Ucp1 and Pgc1 alpha and the general differentiation marker Fabp4 were significantly attenuated by the antagonist of PPAR alpha, GW6471. In contrast, they were only partially attenuated by the antagonist of PPAR gamma, GW9662, and specific knockdown of PPAR gamma. Conclusions: Our findings suggest that sEH may regulate brown adipogenesis and sEH pharmacological inhibition by t-AUCB promotes brown adipogenesis, possibly through activation of PPAR alpha. Funding Sources: The work is supported by NIH 1R15DK114790-01A1 (to LZ), R00DK100736 (to AB) and R01ES002710 (to BDH).

Epoxide hydrolases (EHs) are enzymes involved in the metabolism of endogenous and exogenous epoxides, and the development of EH inhibitors has important applications in the medicine. In humans, EH inhibitors are being tested in the treatment of cardiovascular diseases and show potent anti-inflammatory effects. EH inhibitors are also considerate promising molecules against infectious diseases. EHs are functionally very well studied, but only a few members have its three-dimensional structures characterized. Recently, a new EH from the filamentous fungi Trichoderma reseei (TrEH) was reported, and a series of urea or amide-based inhibitors were identified. In this study, we describe the crystallographic structures of TrEH in complex with five different urea or amide-based inhibitors with resolutions ranging from 2.6 to 1.7A. The analysis of these structures reveals the molecular basis of the inhibition of these compounds. We could also observe that these inhibitors occupy the whole extension of the active site groove and only a few conformational changes are involved. Understanding the structural basis EH interactions with different inhibitors might substantially contribute for the study of fungal metabolism and in the development of novel and more efficient antifungal drugs against pathogenic Trichoderma species.

Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the metabolism of endogenous and exogenous epoxides, and thus have application in pharmacology and biotechnology. In this work, we describe the substrates and inhibitors selectivity of an epoxide hydrolase recently cloned from the filamentous fungus Trichoderma reesei QM9414 (TrEH). We also studied the TrEH urea-based inhibitors effects in the fungal growth. TrEH showed high activity on radioative and fluorescent surrogate and natural substrates, especially epoxides from docosahexaenoic acid. Using a fluorescent surrogate substrate, potent inhibitors of TrEH were identified. Interestingly, one of the best compounds inhibit up to 60% of T. reesei growth, indicating an endogenous role for TrEH. These data make TrEH very attractive for future studies about fungal metabolism of fatty acids and possible development of novel drugs for human diseases.

BACKGROUND: It has been demonstrated that soluble epoxide hydrolase inhibitors (sEHIs) are protective against ischemia-induced lethal arrhythmias, but the mechanisms involved are unknown. Previously, we showed that sEHIs might reduce the incidence of ischemic arrhythmias by suppressing microRNA-1 (miR-1) in the myocardium. As miR-1 and miR-133 have the same proarrhythmic effects in the heart, we assumed that the beneficial effects of sEHIs might also relate to the regulation of miR-133. METHODS: A mouse model of myocardial infarction (MI) was established by ligating the coronary artery. The sEHI t-AUCB (trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid) was administered daily for 7 days before MI. Myocardial infarct size and cardiac function was assessed at 24 h post-MI. The miRNA expression profiles of sham and MI mice treated with or without t-AUCB were determined by microarray and verified by real-time PCR. The incidence of arrhythmias was assessed by in vivo electrophysiologic studies. The mRNA levels of miR-133, its target genes (KCNQ1 [potassium voltage-gated channel subfamily Q member 1] and KCNH2 [potassium voltage-gated channel subfamily H member 2]), and serum response factor (SRF) were measured by real-time PCR; KCNQ1, KCNH2, and SRF protein levels were assessed by western blotting. RESULTS: We demonstrated that the treatment with sEHIs could reduce infarct size, improve cardia function, and prevent the development of cardiac arrhythmias in MI mice. The expression levels of 14 miRNAs differed between the sham and MI groups. t-AUCB treatment altered the expression of eight miRNAs: two were upregulated and six were downregulated. Of these, the muscle-specific miR-133 was downregulated in the ischemic myocardium. In line with this, up-regulation of miR-133 and down-regulation of KCNQ1 and KCNH2 mRNA/protein were observed in ischemic myocaridum, whereas administration of sEHIs produced an opposite effect. In addition, miR-133 overexpression inhibited expression of the target mRNA, whereas t-AUCB reversed the effects. Furthermore, SRF might participate in the negative regulation of miR-133 by t-AUCB. CONCLUSIONS: In MI mice, sEHI t-AUCB can repress miR-133, consequently stimulating KCNQ1 and KCNH2 mRNA and protein expression, suggesting a possible mechanism for its potential therapeutic application in ischemic arrhythmias.

Fragment-based drug discovery relies upon structural information for efficient compound progression, yet it is often challenging to generate structures with bound fragments. A summary of recent literature reveals that a wide repertoire of experimental procedures is employed to generate ligand-bound crystal structures successfully. We share in-house experience from setting up and executing fragment crystallography in a project that resulted in 55 complex structures. The ligands span five orders of magnitude in affinity and the resulting structures are made available to be of use, for example, for development of computational methods. Analysis of the results revealed that ligand properties such as potency, ligand efficiency (LE) and, to some degree, clogP influence the success of complex structure generation.

Soluble epoxide hydrolase (sEH) is a component of the arachidonic acid cascade and is a candidate target for therapies for hypertension or inflammation. Although many sEH inhibitors are available, their scaffolds are not structurally diverse, and knowledge of their specific interactions with sEH is limited. To obtain detailed structural information about protein-ligand interactions, we conducted fragment screening of sEH, analyzed the fragments using high-throughput X-ray crystallography, and determined 126 fragment-bound structures at high resolution. Aminothiazole and benzimidazole derivatives were identified as novel scaffolds that bind to the catalytic triad of sEH with good ligand efficiency. We further identified fragment hits that bound to subpockets of sEH called the short and long branches. The water molecule conserved in the structure plays an important role in binding to the long branch, whereas Asp496 and the main chain of Phe497 form hydrogen bonds with fragment hits in the short branch. Fragment hits and their crystal structures provide structural insights into ligand binding to sEH that will facilitate the discovery of novel and potent inhibitors of sEH.

BACKGROUND: Epoxyeicosatrienoic acids (EETs) are natural angiogenic mediators regulated by soluble epoxide hydrolase (sEH). Inhibitors of sEH can stabilize EETs levels and were reported to reduce atherosclerosis and inhibit myocardial infarction in animal models. In this work, we investigated whether increasing EETs with the sEH inhibitor t-AUCB would increase angiogenesis related function in endothelial progenitor cells (EPCs) from patients with acute myocardial infarction (AMI). METHODS AND
RESULTS: EPCs were isolated from 50 AMI patients and 50 healthy subjects (control). EPCs were treated with different concentrations of t-AUCB for 24h with or without peroxisome proliferator activated receptor gamma (PPARgamma) inhibitor GW9662. Migration of EPCs was assayed in trans-well chambers. Angiogenesis assays were performed using a Matrigel-Matrix in vitro model. The expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1alpha (HIF-1alpha) mRNA and protein in EPCs was measured by real-time PCR or Western blot, respectively. Also, the concentration of EETs in the culture supernatant was detected by ELISA. The activity of EPCs in the AMI patient group was reduced compared to healthy controls. Whereas increasing EET levels with t-AUCB promoted a dose dependent angiogenesis and migration in EPCs from AMI patients. Additionally, the t-AUCB dose dependently increased the expression of the angiogenic factors VEGF and HIF-alpha. Lastly, we provide evidence that these effects were PPARgamma dependent. CONCLUSION: The results demonstrate that the sEH inhibitor positively modulated the functions of EPCs in patients with AMI through the EETs-PPARgamma pathway. The present study suggests the potential utility of sEHi in the therapy of ischemic heart disease.

Although sEH inhibitors are well studied in inflammatory and cardiovascular diseases, their effects on gliomas are unclear. In this study, we investigated the effects of t-AUCB, a more potent and selective sEH inhibitor, on U251 and U87 human glioblastoma cell lines and the HepG2 human hepatocellular carcinoma cell line. Our results showed that t-AUCB efficiently inhibited sEH activities in all three cell lines (the inhibition rate was more than 80% in each) and suppressed U251 and U87 cell growth in a dose-dependent manner, but exhibited no cell growth inhibition on HepG2. We detected high levels of phosphorylated NF-kappaB-p65 (Ser536) in t-AUCB-treated U251 and U87 cells, and then found that the NF-kappaB inhibitor PDTC can completely abolish t-AUCB-induced growth inhibition. This indicated that t-AUCB suppresses U251 and U87 cell growth by activating NF-kappaB-p65. Moreover, we found that t-AUCB induces cell-cycle G0/G1 phase arrest by regulating Cyclin D1 mRNA and protein levels and CDC2 (Thr161) phosphorylation level. We propose to further test this promising reagent for its anti-glioma activity in clinical relevant orthotopic brain glioma models.

We previously reported that sEH inhibitor t-AUCB suppresses the growth of human glioblastoma U251 and U87 cell lines and induces cell-cycle G0/G1 phase arrest. In present study, we found even 96 h-treatment of 200 muM t-AUCB can not induce apoptosis in U251 and U87 cells. We also revealed that 200 muM t-AUCB significantly elevates the activation of p38 MAPK, MAPKAPK2 and Hsp27. The p38 MAPK inhibitor SB203580 and the inhibitor of Hsp27 phosphorylation, KRIBB3, were used to investigate the mechanism of the apoptosis-resistance. The results showed that, after blocking the activation of Hsp27 by SB203580 or KRIBB3, 200 muM t-AUCB significantly induces apoptosis and increases caspase-3 activities in U251 and U87 cells. Our data demonstrated that t-AUCB induces cell apoptosis after blocking itself-induced activation of Hsp27, and that the activation of Hsp27 may confer chemoresistance in GBM cells. The combination of t-AUCB and the inhibitor of Hsp27 phosphorylation may be a potential strategy for treatment of glioblastoma.

OBJECTIVE: To observe the effects of soluble epoxide hydrolase inhibitor t-AUCB on foam cell formation and cholesterol efflux in macrophage.
METHODS: Mouse macrophages RAW264.7 were cultured and stimulated with ox-LDL (80 micromol/L) in the absence (group A) or presence of t-AUCB (1, 10, 50, 100 micromol/L, group B) or t-AUCB (100 micromol/L) pretreated with PPARgamma antagonist GW9662 (5 micromol/L, group C). The foam cell was identified by oil red O staining. The cholesterol efflux rates of (3)H-cholesterol in cells were measured by liquid scintillation counter. mRNA and protein expressions of ABCA1 were detected by real-time PCR or Western blot, respectively.
RESULTS: Oil red O staining showed that t-AUCB (100 micromol/L) significantly inhibited foam cell formation which could be significantly reversed by GW9662 (all P < 0.05). t-AUCB dose-dependently increased cholesterol efflux rates in mouse macrophage [(5.91 +/- 0.18)% in group A, (7.03 +/- 0.33)%, (8.05 +/- 0.32)%, (9.04 +/- 0.14)%, (10.06 +/- 0.85)% in 1, 10, 50, 100 micromol/L t-AUCB groups, all P < 0.05 vs. group A], which could be reversed by pretreatment with GW9662 [(6.33 +/- 0.15)% in 100 micromol/L t-AUCB + GW9662 group].t-AUCB also upregulated ABCA1 mRNA and protein expressions in a dose-dependent manner which could be significantly attenuated by pretreatment with GW9662. CONCLUSION: t-AUCB could inhibit foam cell formation by improving cholesterol efflux through activating PPARgamma-ABCA1 pathway in macrophage.