RESUMO
Polypharmaceutical regimens often impair treatment of patients with metabolic syndrome (MetS), a complex disease cluster, including obesity, hypertension, heart disease, and type II diabetes. Simultaneous targeting of soluble epoxide hydrolase (sEH) and peroxisome proliferator-activated receptor γ (PPARγ) synergistically counteracted MetS in various in vivo models, and dual sEH inhibitors/PPARγ agonists hold great potential to reduce the problems associated with polypharmacy in the context of MetS. However, full activation of PPARγ leads to fluid retention associated with edema and weight gain, while partial PPARγ agonists do not have these drawbacks. In this study, we designed a dual partial PPARγ agonist/sEH inhibitor using a structure-guided approach. Exhaustive structure-activity relationship studies lead to the successful optimization of the designed lead. Crystal structures of one representative compound with both targets revealed potential points for optimization. The optimized compounds exhibited favorable metabolic stability, toxicity, selectivity, and desirable activity in adipocytes and macrophages.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , PPAR gama/agonistas , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Polimedicação , Ratos , Relação Estrutura-AtividadeRESUMO
Non-alcoholic steatohepatitis (NASH) presents as an epidemic chronic liver disease that is closely associated with metabolic disorders and involves hepatic steatosis, inflammation, and fibrosis as key factors. Despite the enormous global prevalence of NASH, effective pharmacological interventions are lacking. Based on the hypothesis that the multifactorial condition NASH may benefit from combined multiple modes of action for enhanced therapeutic efficacy, we have previously developed dual FXR activators/sEH inhibitors (FXRa/sEHi) and observed remarkable antifibrotic effects upon their use in rodent NASH models. However, these first-generation FXRa/sEHi were characterized by moderate metabolic stability and short in vivo half-life. Aiming to overcome these pharmacokinetic drawbacks, we have systematically studied the structure-activity and structure-stability relationships of the chemotype and obtained second-generation FXRa/sEHi with improved pharmacokinetic parameters. With high plasma exposure, a half-life greater than 5 h, and similar dual potency on the intended targets, 13 presents as a substantially optimized FXRa/sEHi for late-stage preclinical development.
Assuntos
Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Benzamidas/síntese química , Benzamidas/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Epóxido Hidrolases/metabolismo , Células Hep G2 , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/genética , Relação Estrutura-AtividadeRESUMO
Nonalcoholic fatty liver disease (NAFLD) is an epidemic chronic liver disease and may progress over nonalcoholic steatohepatitis (NASH) to liver cirrhosis and hepatocellular carcinoma. The multiple metabolic, environmental, and genetic factors that are involved in NAFLD/NASH pathogenesis and progression suggest a need for multimechanistic interventions. We have developed and preliminarily characterized a concept of dual farnesoid X receptor (FXR) and soluble epoxide hydrolase (sEH) modulation as a promising polypharmacological strategy to counteract NASH. Here we report the profiling of FXR activation, sEH inhibition, and simultaneous FXR/sEH modulation as an interventional treatment in pre-established NASH in mice with diet-induced obesity (DIO). We found that full FXR activation was required to obtain antisteatosis effects but also worsened ballooning degeneration and fibrosis. In contrast, sEH inhibition and dual FXR/sEH modulation, despite a lack of antisteatosis activity, had anti-inflammatory effects and efficiently counteracted hepatic fibrosis. These results demonstrate great therapeutic potential of sEH inhibition to counteract hepatic fibrosis and validate the designed polypharmacology concept of dual FXR/sEH modulation as a potentially superior avenue for the effective treatment of the multifactorial condition NASH.
RESUMO
The metabolic syndrome (MetS) is a constellation of cardiovascular and metabolic symptoms involving insulin resistance, steatohepatitis, obesity, hypertension, and heart disease, and patients suffering from MetS often require polypharmaceutical treatment. PPARγ agonists are highly effective oral antidiabetics with great potential in MetS, which promote adipocyte browning and insulin sensitization. However, the application of PPARγ agonists in clinics is restricted by potential cardiovascular adverse events. We have previously demonstrated that the racemic dual sEH/PPARγ modulator RB394 (3) simultaneously improves all risk factors of MetS in vivo. In this study, we identify and characterize the eutomer of 3. We provide structural rationale for molecular recognition of the eutomer. Furthermore, we could show that the dual sEH/PPARγ modulator is able to promote adipocyte browning and simultaneously exhibits cardioprotective activity which underlines its exciting potential in treatment of MetS.
Assuntos
Adipócitos/efeitos dos fármacos , Benzamidas/farmacologia , Butiratos/farmacologia , Cardiotônicos/farmacologia , Epóxido Hidrolases/metabolismo , PPAR gama/agonistas , Animais , Benzamidas/síntese química , Butiratos/síntese química , Cardiotônicos/síntese química , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , EstereoisomerismoRESUMO
The nuclear farnesoid X receptor (FXR) and the enzyme soluble epoxide hydrolase (sEH) are validated molecular targets to treat metabolic disorders such as non-alcoholic steatohepatitis (NASH). Their simultaneous modulation inâ vivo has demonstrated a triad of anti-NASH effects and thus may generate synergistic efficacy. Here we report dual FXR activators/sEH inhibitors derived from the anti-asthma drug Zafirlukast. Systematic structural optimization of the scaffold has produced favorable dual potency on FXR and sEH while depleting the original cysteinyl leukotriene receptor antagonism of the lead drug. The resulting polypharmacological activity profile holds promise in the treatment of liver-related metabolic diseases.
Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/agonistas , Compostos de Tosil/química , Sítios de Ligação , Domínio Catalítico , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Avaliação Pré-Clínica de Medicamentos , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Indóis , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Acoplamento Molecular , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Fenilcarbamatos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Sulfonamidas , Compostos de Tosil/metabolismo , Compostos de Tosil/farmacologiaRESUMO
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
Multitarget design offers access to bioactive small molecules with potentially superior efficacy and safety. Particularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment. By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor γ. The triple modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multitarget agents.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Desenho de Fármacos , Polifarmacologia , Compostos de Tosil/farmacologia , Células 3T3 , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Domínio Catalítico , Epóxido Hidrolases/química , Epóxido Hidrolases/metabolismo , Células Hep G2 , Humanos , Indóis , Camundongos , Simulação de Acoplamento Molecular , PPAR gama/química , PPAR gama/metabolismo , Fenilcarbamatos , Sulfonamidas , Compostos de Tosil/metabolismoRESUMO
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
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.
Assuntos
Benzamidas/síntese química , Benzamidas/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Síndrome Metabólica/tratamento farmacológico , PPAR gama/efeitos dos fármacos , Células 3T3 , Administração Oral , Animais , Benzamidas/farmacocinética , Células COS , Chlorocebus aethiops , Diabetes Mellitus Tipo 2/tratamento farmacológico , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacocinética , Humanos , Hipertensão/tratamento farmacológico , Técnicas In Vitro , Camundongos , Microssomos Hepáticos/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
Increased activity of efflux transporters, e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier is a pathological hallmark of many neurological diseases, and the resulting multiple drug resistance represents a major clinical challenge. Noninvasive imaging of transporter activity can help to clarify the underlying mechanisms of drug resistance and facilitate diagnosis, patient stratification, and treatment monitoring. We have developed a metabolically activated radiotracer for functional imaging of P-gp/BCRP activity with positron emission tomography (PET). In preclinical studies, the tracer showed excellent initial brain uptake and clean conversion to the desired metabolite, although at a sluggish rate. Blocking with P-gp/BCRP modulators led to increased levels of brain radioactivity; however, dynamic PET did not show differential clearance rates between treatment and control groups. Our results provide proof-of-concept for development of prodrug tracers for imaging of P-gp/BCRP function in vivo but also highlight some challenges associated with this strategy.