RESUMO
The protein arginine N-methyltransferase 6 (PRMT6) is overexpressed in a variety of different cancer types and plays a role in human immunodeficiency virus (HIV) infections. Furthermore, the PRMT6 activity might also influence the pathogenesis of neurodegenerative, inflammatory, and cardiovascular diseases, whereby it becomes an interesting target for drug development. Previously reported activity assays for PRMT6 activity are either expensive, time-consuming or use radioactive substrates. To overcome these challenges, we developed a coupled fluorescence-based activity assay using recombinant PRMT6 expressed in E. coli. In the first step of the assay, the fluorogenic substrate Nα-Benzoyl-L-arginine-7-amido-4-methylcoumarin (Bz-Arg-AMC) is methylated by PRMT6, while in a second step the remaining un-methylated substrate is cleaved by trypsin, producing the fluorescent 7-amino-4-methylcoumarin.
Assuntos
Proteínas Nucleares/análise , Peptídeos/química , Proteína-Arginina N-Metiltransferases/análise , Fluorescência , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Núcleo Celular/ultraestrutura , Citosol/ultraestrutura , Isoformas de Proteínas/metabolismo , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/isolamento & purificação , Núcleo Celular/metabolismo , Citosol/metabolismo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Leucotrienos/biossíntese , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificaçãoRESUMO
The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme, containing a peptidase and a hydrolase activity both activities having opposing functions regulating inflammatory response. The hydrolase activity is responsible for the conversion of leukotriene A4 to pro-inflammatory leukotriene B4, and hence, selective inhibitors of the hydrolase activity are of high pharmacological interest. Here we present the thermodynamic characterization of structurally distinct inhibitors of the LTA4H that occupy different regions of the binding site using different biophysical methods. An in silico method for the determination of stabilized water molecules in the binding site of the apo structure of LTA4H is used to interpret the measured thermodynamic data and provided insights for design of novel LTA4H inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Termodinâmica , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Epóxido Hidrolases/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The arachidonic acid cascade is a key player in inflammation, and numerous well-established drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes. Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55 potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.
Assuntos
Anti-Inflamatórios/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Epóxido Hidrolases/antagonistas & inibidores , Hidrocarbonetos Fluorados/farmacologia , Inibidores de Lipoxigenase/síntese química , Inibidores de Lipoxigenase/farmacologia , Ureia/análogos & derivados , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Inflamação/tratamento farmacológico , Leucócitos/metabolismo , Leucotrienos/biossíntese , Lipopolissacarídeos , Inibidores de Lipoxigenase/química , Ureia/síntese química , Ureia/química , Ureia/farmacologiaRESUMO
The pharmacophore concept is commonly employed in virtual screening for hit identification. A pharmacophore is generally defined as the three-dimensional arrangement of the structural and physicochemical features of a compound responsible for its affinity to a pharmacological target. Given a number of active ligands binding to a particular target in the same manner, it can reasonably be assumed that they have some shared features, a common pharmacophore. We present a growing neural gas (GNG)-based approach for the extraction of the relevant features which we called PENG (pharmacophore elucidation by neural gas). Results of retrospective validation indicate an acceptable quality of the generated models. Additionally a prospective virtual screening for leukotriene A4 hydrolase (LTA4H) inhibitors was performed. LTA4H is a bifunctional zinc metalloprotease which displays both epoxide hydrolase and aminopeptidase activity. We could show that the PENG approach is able to predict the binding mode of the ligand by X-ray crystallography. Furthermore, we identified a novel chemotype of LTA4H inhibitors.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Redes Neurais de Computação , Algoritmos , Aminopeptidases/química , Benchmarking , Cristalografia por Raios X , Epóxido Hidrolases/química , Humanos , Ligantes , Modelos Moleculares , Estudos Prospectivos , Ligação Proteica , Reprodutibilidade dos Testes , TermodinâmicaRESUMO
BACKGROUND: To date, no effective immunosuppressive standard for the prevention or treatment of alloantibody production in acute and chronic rejection of renal transplants has been established. Alloantibody formation has been recognized in the well-established rat model of Fischer to Lewis renal transplantation. We used this renal allotransplantation model to test the effectiveness of sirolimus (SRL), bortezomib (BZ) or their combination in an already established humoral rejection situation. METHODS: After 3 weeks, transplanted rats were treated either with placebo (P), SRL, BZ or combination of SRL and BZ. Rats were monitored for donor-specific alloantibodies as well as humoral responses in the spleen and bone marrow, renal function and histological changes in the graft. RESULTS: In all three treatment arms, glomerular, tubulointerstitial and vascular changes of chronic antibody-mediated rejection were ameliorated compared to the P group. Production of alloantibodies against components of glomerular basement membrane was reduced in all three treatment arms. The humoral response was strongly reduced, as shown by decreased numbers of IgG-secreting cells, plasma cells and partially B cells in all treatment groups with a trend of SRL/BZ combination being most effective. Infiltration of the graft with inflammatory cells like cytotoxic T cells, T helper cells, B cells and macrophages was efficiently blocked by BZ and the SRL/BZ combination and, except for B cells, by SRL. CONCLUSIONS: BZ and SRL represent promising drugs with anti-humoral activity in the situation of an already established chronic humoral or mixed alloimmune response after renal transplantation.
Assuntos
Ácidos Borônicos/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Isoanticorpos/imunologia , Transplante de Rim/efeitos adversos , Pirazinas/uso terapêutico , Sirolimo/uso terapêutico , Animais , Bortezomib , Doença Crônica , Depleção Linfocítica , Masculino , Plasmócitos/fisiologia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T/imunologia , Transplante HomólogoRESUMO
In the present article we describe the creation of a small carboranylcarboxamide compound library followed by a screening campaign at the soluble epoxide hydrolase (sEH). We identified meta-carboranyl alkylamides, -anilides, and -benzylamides as potent sEH inhibitors. Furthermore, we optimized the scaffolds and we derived structure-activity relationships. The most potent benzylamide 33 (MS1) was similar to a previously reported adamantane derivative and gave an IC50 value of 0.07⯵M for meta- and 0.08⯵M for para-carborane at isolated sEH. The ortho-derivative suffered deboronation. The results underline the potential of carboranes as non-natural 3-D pharmacophores to extend the chemical space in drug discovery.
Assuntos
Boranos/farmacologia , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Boranos/síntese química , Boranos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Epóxido Hidrolases/metabolismo , Humanos , Estrutura Molecular , Solubilidade , Relação Estrutura-AtividadeRESUMO
Multitarget anti-inflammatory drugs interfering with the arachidonic acid cascade exhibit superior efficacy. In this study, a prototype dual inhibitor of soluble epoxide hydrolase (sEH) and LTA4 hydrolase (LTA4H) with submicromolar activity toward both targets has been designed and synthesized. Preliminary structure-activity relationship studies were performed to identify optimal substitution patterns. X-ray structure analysis of a promising dual inhibitor in complex with sEH, as well as molecular docking with LTA4H provided a rationale for further optimization. Hereby, scaffold extension was successfully applied to yield potent dual sEH/LTA4H inhibitors. The spectrum of pro- and anti-inflammatory lipid mediators was evaluated in M1 and M2 macrophages, stimulated with LPS, and incubated with the most promising compound 14. The effect of 14 on the inflammatory lipid mediator profile characterizes dual sEH/LTA4H inhibitors as an interesting option for future anti-inflammatory agent investigations.
RESUMO
Inhibition of multiple enzymes of the arachidonic acid cascade leads to synergistic anti-inflammatory effects. Merging of 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH) pharmacophores led to the discovery of a dual 5-LOX/sEH inhibitor, which was subsequently optimized in terms of potency toward both targets and metabolic stability. The optimized lead structure displayed cellular activity in human polymorphonuclear leukocytes, oral bioavailability, and target engagement in vivo and demonstrated profound anti-inflammatory and anti-fibrotic efficiency in a kidney injury model caused by unilateral ureteral obstruction in mice. These results pave the way for investigating the therapeutic potential of dual 5-LOX/sEH inhibitors in other inflammation- and fibrosis-related disease models.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Araquidonato 5-Lipoxigenase/metabolismo , Desenho de Fármacos , Epóxido Hidrolases/antagonistas & inibidores , Inibidores de Lipoxigenase/síntese química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Araquidonato 5-Lipoxigenase/genética , Células Cultivadas , Epóxido Hidrolases/genética , Humanos , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
Selective optimization of side activities is a valuable source of novel lead structures in drug discovery. In this study, a computer-aided approach was used to deorphanize the pleiotropic cholesterol-lowering effects of the beta-blocker talinolol, which result from the inhibition of the enzyme soluble epoxide hydrolase (sEH). X-ray structure analysis of the sEH in complex with talinolol enables a straightforward optimization of inhibitory potency. The resulting lead structure exhibited in vivo activity in a rat model of diabetic neuropatic pain.
RESUMO
Cysteinyl leukotriene receptor 1 antagonists (CysLT1RA) are frequently used as add-on medication for the treatment of asthma. Recently, these compounds have shown protective effects in cardiovascular diseases. This prompted us to investigate their influence on soluble epoxide hydrolase (sEH) and peroxisome proliferator activated receptor (PPAR) activities, two targets known to play an important role in CVD and the metabolic syndrome. Montelukast, pranlukast and zafirlukast inhibited human sEH with IC50 values of 1.9, 14.1, and 0.8 µM, respectively. In contrast, only montelukast and zafirlukast activated PPARγ in the reporter gene assay with EC50 values of 1.17 µM (21.9% max. activation) and 2.49 µM (148% max. activation), respectively. PPARα and δ were not affected by any of the compounds. The activation of PPARγ was further investigated in 3T3-L1 adipocytes. Analysis of lipid accumulation, mRNA and protein expression of target genes as well as PPARγ phosphorylation revealed that montelukast was not able to induce adipocyte differentiation. In contrast, zafirlukast triggered moderate lipid accumulation compared to rosiglitazone and upregulated PPARγ target genes. In addition, we found that montelukast and zafirlukast display antagonistic activities concerning recruitment of the PPARγ cofactor CBP upon ligand binding suggesting that both compounds act as PPARγ modulators. In addition, zafirlukast impaired the TNFα triggered phosphorylation of PPARγ2 on serine 273. Thus, zafirlukast is a novel dual sEH/PPARγ modulator representing an excellent starting point for the further development of this compound class.
RESUMO
The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities that are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity, located in the N-terminal phosphatase domain of sEH (sEH-P). Herein we report the discovery and optimization of the first inhibitor of human and rat sEH-P that is applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.
Assuntos
Inibidores Enzimáticos/química , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/química , Animais , Sítios de Ligação , Domínio Catalítico , Desenho de Fármacos , Humanos , Ligantes , Masculino , Oxazóis/química , Monoéster Fosfórico Hidrolases/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , TemperaturaRESUMO
BACKGROUND: Matrix expansion and mesangial proliferation are hallmarks of mesangial proliferative glomerulonephritis. Specific inhibition of PDE-5, an enzyme catalyzing the intracellular degradation of cyclic GMP, can be achieved by the inhibitor vardenafil. In this study, we investigated the effects of PDE-5 inhibition in the anti-Thy1 model in the rat in vivo. METHODS: After disease induction, rats received 10 mg/kg bw vardenafil twice a day via gavage. On Days 2 and 6, renal biopsies, as well as glomerular isolates, urine and blood samples were taken to compare vardenafil- and placebo-treated groups during the course of disease. RESULTS: Small amounts of PDE-5 were detected in healthy kidneys, but induced in a typical mesangial pattern during disease (by IHC and WB). Specific PDE-5 inhibition resulted in increased glomerular levels of cGMP. Treated animals demonstrated inhibition of MC proliferation and matrix accumulation while renal function and influx of inflammatory cells were not affected. Due to PDE-5 inhibition, the endogenous TGF-beta-activating protein TSP-1 and the TGF-beta-signalling protein p-smad-2/3 were decreased suggesting this as an antifibrotic mechanism of action of vardenafil in this model. CONCLUSION: Considering the availability and safety profile of vardenafil, the beneficial antiproliferative and antifibrotic effect in experimental glomerulonephritis may potentially be applicable to the treatment of mesangial proliferative glomerulonephritis in man.
Assuntos
Matriz Extracelular/metabolismo , Glomerulonefrite/metabolismo , Inibidores da Fosfodiesterase 5 , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Anticorpos Monoclonais , Biópsia , Proliferação de Células/efeitos dos fármacos , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Modelos Animais de Doenças , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/patologia , Imidazóis/farmacologia , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Sulfonas/farmacologia , Antígenos Thy-1/imunologia , Triazinas/farmacologia , Dicloridrato de VardenafilaRESUMO
BACKGROUND: The antiproliferative immunosuppressant everolimus adversely affects the acute reversible anti-Thy1 nephritis model. We hypothesized that everolimus treatment started after the acute proliferative phase could even be beneficial in the chronic anti-Thy1 nephritis model in the rat. METHODS: Chronic anti-Thy1 nephritis was induced by injection of the monoclonal antibody 1-22-3 in 20 male Sprague-Dawley rats 7 days after uninephrectomy. Two weeks after disease induction, rats were randomly treated with either everolimus or vehicle for 14 weeks. Changes in progression of renal disease were investigated by immunohistochemistry and real-time PCR 16 weeks after disease induction. RESULTS: During chronic anti-Thy1 nephritis, the formation of focal segmental glomerulosclerosis lesions, the degree of interstitial fibrosis as well as the increase in proteinuria over 14 weeks was ameliorated by everolimus treatment. Increased glomerular hypertrophy observed in the vehicle-treated rats was completely prevented in the everolimus-treated nephritic rats. Increased glomerular fibronectin mRNA and protein as well as the renal influx of monocytes/macrophages was significantly reduced in the everolimus group. Everolimus reduced the pro-angiogenic factor vascular endothelial growth factor (VEGF) and VEGF mRNA in glomeruli, while the transforming growth factor-beta signaling pathway was not affected. CONCLUSION: 'Late' start of everolimus treatment demonstrates beneficial effects on the time course of chronic anti-Thy1 nephritis.
Assuntos
Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/metabolismo , Isoanticorpos/administração & dosagem , Proteínas Quinases/metabolismo , Sirolimo/análogos & derivados , Animais , Doença Crônica , Everolimo , Glomerulonefrite/imunologia , Glomerulonefrite/prevenção & controle , Masculino , Proteínas Quinases/fisiologia , Ratos , Ratos Sprague-Dawley , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR , Fatores de TempoRESUMO
Pathogens, expressing metallo-ß-lactamases (MBLs), become resistant against most ß-lactam antibiotics. Besides the dragging search for new antibiotics, development of MBL inhibitors would be an alternative weapon against resistant bacterial pathogens. Inhibition of resistance enzymes could restore the antibacterial activity of ß-lactams. Various approaches to MBL inhibitors are described; among others, the promising motif of a zinc coordinating thiol moiety is very popular. Nevertheless, since the first report of a thiol-based MBL inhibitor (thiomandelic acid) in 2001, no steps in development of thiol based MBL inhibitors were reported that go beyond clinical isolate testing. In this study, we report on the synthesis and biochemical characterization of thiol-based MBL inhibitors and highlight the challenges behind the development of thiol-based compounds, which exhibit good in vitro activity toward a broad spectrum of MBLs, selectivity against human off-targets, and reasonable activity against clinical isolates.
Assuntos
Descoberta de Drogas/métodos , Compostos de Sulfidrila/isolamento & purificação , Compostos de Sulfidrila/farmacologia , Inibidores de beta-Lactamases/isolamento & purificação , Inibidores de beta-Lactamases/farmacologia , Compostos de Sulfidrila/síntese química , Resistência beta-Lactâmica/efeitos dos fármacos , Inibidores de beta-Lactamases/síntese químicaRESUMO
BACKGROUND: Under certain circumstances the nonnephrotoxic, antiproliferative, immunosuppressive mammalian target of rapamycin (mTOR) inhibitors can cause renal deterioration and proteinuria after conversion from calcineurin inhibitors, especially in long-term renal transplant patients with low glomerular filtration rates. The mTOR inhibitors also show an impaired glomerular healing reaction during acute renal injury in experimental mesangial proliferative glomerulonephritis. In this study, everolimus treatment was investigated in a low nephron number model, the remnant kidney model in rats. METHODS: The remnant kidney model was induced by uninephrectomy and infarction of 2/3 of the remaining kidney in 31 male Sprague-Dawley rats. Three days after disease induction, rats were randomly treated either with everolimus or vehicle. Changes in progression of renal disease were investigated by immunohistochemistry on days 22 and 38 after disease induction. RESULTS: In the remnant kidney model, everolimus treatment worsened chronic disease progression as assessed by increased proteinuria, glomerulosclerosis, interstitial fibrosis, glomerular inflammation as well as decreased creatinine-clearance. This result was due to a markedly increased fraction of glomeruli with a defective glomerular architecture in the everolimus group. Everolimus apparently inhibited the chronic glomerular repair reaction via inhibition of the proliferative but not apoptotic activity of the glomerular endothelial and mesangial cells, which was associated with reduced glomerular vascular endothelial growth factor mRNA and protein. In contrast, the fraction of glomeruli with an intact glomerular architecture within the everolimus group showed clearly less glomerular enlargement compared to vehicle-treated nephrectomy rats. CONCLUSION: This study demonstrates potential mechanisms of mTOR inhibitor induced renal deterioration and proteinuria in the low nephron number remnant kidney model.
Assuntos
Nefropatias/induzido quimicamente , Proteínas Quinases/metabolismo , Proteinúria/induzido quimicamente , Sirolimo/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Everolimo , Mesângio Glomerular/efeitos dos fármacos , Nefropatias/patologia , Masculino , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Trombose/induzido quimicamente , Trombose/genética , Trombose/metabolismo , Trombose/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização/efeitos dos fármacosRESUMO
Nonalcoholic steatohepatitis arising from Western diet and lifestyle is characterized by accumulation of fat in liver causing inflammation and fibrosis. It evolves as serious health burden with alarming incidence, but there is no satisfying pharmacological therapy to date. Considering the disease's multifactorial nature, modulation of multiple targets might provide superior therapeutic efficacy. In particular, farnesoid X receptor (FXR) activation that revealed antisteatotic and antifibrotic effects in clinical trials combined with inhibition of soluble epoxide hydrolase (sEH) as anti-inflammatory strategy promises synergies. To exploit this dual concept, we developed agents exerting partial FXR agonism and sEH inhibitory activity. Merging known pharmacophores and systematic exploration of the structure-activity relationship on both targets produced dual modulators with low nanomolar potency. Extensive in vitro characterization confirmed high dual efficacy in cellular context combined with low toxicity, and pilot in vivo data revealed favorable pharmacokinetics as well as engagement on both targets in vivo.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Anti-Inflamatórios/química , Descoberta de Drogas , Inibidores Enzimáticos/química , Epóxido Hidrolases/metabolismo , Células HeLa , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-AtividadeRESUMO
Arachidonic acid (AA) is metabolized to diverse bioactive lipid mediators. Whereas the 5-lipoxygenase-activating protein (FLAP) facilitates AA conversion by 5-lipoxygenase (5-LOX) to pro-inflammatory leukotrienes (LTs), the soluble epoxide hydrolase (sEH) degrades anti-inflammatory epoxyeicosatrienoic acids (EETs). Accordingly, dual FLAP/sEH inhibition might be advantageous drugs for intervention of inflammation. We present the in vivo pharmacological profile and efficiency of N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N'-(3,4-dichlorophenyl)urea (diflapolin) that dually targets FLAP and sEH. Diflapolin inhibited 5-LOX product formation in intact human monocytes and neutrophils with IC50 = 30 and 170 nM, respectively, and suppressed the activity of isolated sEH (IC50 = 20 nM). Characteristic for FLAP inhibitors, diflapolin (I) failed to inhibit isolated 5-LOX, (II) blocked 5-LOX product formation in HEK cells only when 5-LOX/FLAP was co-expressed, (III) lost potency in intact cells when exogenous AA was supplied, and (IV) prevented 5-LOX/FLAP complex assembly in leukocytes. Diflapolin showed target specificity, as other enzymes related to AA metabolism (i.e., COX1/2, 12/15-LOX, LTA4H, LTC4S, mPGES1, and cPLA2) were not inhibited. In the zymosan-induced mouse peritonitis model, diflapolin impaired vascular permeability, inhibited cysteinyl-LTs and LTB4 formation, and suppressed neutrophil infiltration. Diflapolin is a highly active dual FLAP/sEH inhibitor in vitro and in vivo with target specificity to treat inflammation-related diseases.
Assuntos
Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Inibidores da Proteína Ativadora de 5-Lipoxigenase/química , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Epóxido Hidrolases/metabolismo , Humanos , Masculino , Camundongos , Estrutura Molecular , Transporte ProteicoRESUMO
Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that possesses an epoxide hydrolase and lipid phosphatase activity (sEH-P) at two distinct catalytic domains. While the physiological role of the epoxide hydrolase domain is well understood, the consequences of the phosphatase activity remain unclear. Herein we describe the bacterial expression of the recombinant N-terminal domain of sEH-P and the development of a high-throughput screening protocol using a sensitive and commercially available substrate fluorescein diphosphate. The usability of the assay system was demonstrated and novel inhibitors of sEH-P were identified.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Epóxido Hidrolases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Animais , Domínio Catalítico/genética , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Camundongos , Monoéster Fosfórico Hidrolases/genética , SolubilidadeRESUMO
Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus, and obesity. MetS patients are strongly exposed to polypharmacy; however, the number of pharmacological compounds required for MetS treatment can be reduced by the application of multitarget compounds. This study describes the design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can improve diabetic conditions and hypertension at once. N-Benzylbenzamide derivatives were determined to fit a merged sEH/PPARγ pharmacophore, and structure-activity relationship studies were performed on both targets, resulting in a submicromolar (sEH IC50 = 0.3 µM/PPARγ EC50 = 0.3 µM) modulator 14c. In vitro and in vivo evaluations revealed good ADME properties qualifying 14c as a pharmacological tool compound for long-term animal models of MetS.