RESUMO
The nuclear transcription factor NF-kappaB is crucial to the expression of numerous cytokines, enzymes, and cell adhesion molecules, all of which can drive inflammatory and autoimmune disorders such as rheumatoid arthritis. The IKK complex plays the most important role in the signal cascade leading to NF-kappaB activation. Recently, inhibition of the interaction between NEMO (NF-kappaB essential modulator) and the catalytic subunits of IKK, especially IKKbeta, has received particular attention as a possible new therapeutic approach to treatment of inflammatory disorders, and several reports have shown the efficacy of cell permeable NEMO binding domain (NBD)-containing peptides in blocking the IKK/NF-kappaB pathway. In this article, we describe in detail the development and validation of two novel binding assays, a homogeneous time-resolved fluorescence (HTRF)-based assay and an enzyme-linked immunosorbent assay (ELISA)-based assay, suitable for the discovery of small molecules that inhibit IKKbeta-NEMO interaction. Using the HTRF-based assay, we screened approximately 15,000 compounds from our chemical library and eliminated false positive hits by the ELISA-based assay and IKK complex kinase assay. As a result, seven positive hit compounds that inhibit IKK complex activity through inhibition of IKKbeta-NEMO interaction were identified. These hit compounds may have a good potential in the treatment of inflammatory and autoimmune disorders such as rheumatoid arthritis.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Ensaios de Triagem em Larga Escala/métodos , Quinase I-kappa B/metabolismo , Inibidores de Proteínas Quinases/química , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Humanos , Quinase I-kappa B/antagonistas & inibidores , Imunoprecipitação , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de TempoRESUMO
Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor γ (PPARγ) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPARγ agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes for discovery of adiponectin secretion modulators. They screened a library of approximately 100 000 small-molecule compounds using this model, performed several follow-up screens, and identified six hit compounds that increase adiponectin secretion without having PPARγ agonistic activity. These compounds may be useful drug candidates for diabetes, obesity, atherosclerosis, and other metabolic syndromes. This HTS assay might be applicable to screening for other adipokine modulators that can be useful for the treatment of other conditions.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adiponectina/metabolismo , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas/farmacologia , Células 3T3-L1 , Adiponectina/genética , Animais , Relação Dose-Resposta a Droga , Humanos , Camundongos , Receptores Ativados por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Elementos Reguladores de Transcrição/genética , Quinase Induzida por NF-kappaBRESUMO
In our search for a novel class of inducible nitric oxide synthase (iNOS) inhibitors, 1,3-oxazolidin-2-imine was found to weakly inhibit iNOS. Further modifications of this compound resulted in a remarkable increase in both the in vivo and in vitro inhibitory activity and selectivity for iNOS.
Assuntos
Inibidores Enzimáticos/farmacocinética , Óxido Nítrico Sintase/antagonistas & inibidores , Oxazolidinonas/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 2 Anéis/administração & dosagem , Compostos Heterocíclicos com 2 Anéis/farmacocinética , Compostos Heterocíclicos com 2 Anéis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase/metabolismo , Oxazolidinonas/administração & dosagem , Oxazolidinonas/farmacologiaRESUMO
Nitric oxide synthase (NOS) has been divided into two major sub-enzymes, i.e. inducible NOS (iNOS) and constitutive NOS (cNOS). Although nitric oxide (NO) plays an important role as host defense mediator, excessive production of NO by iNOS has been involved in the pathology of many inflammatory diseases. Recently, we reported that the 2-imino-1,3-oxazolidine (1a) weakly inhibits iNOS and that introduction of an alkyl moiety on the oxazolidine ring of 1a enhances the inhibitory activity and selectivity for iNOS. In our search for better iNOS inhibitors, we focused our efforts on the 2-aminothiazole scaffold 3 as it possesses a ring similar to that of 1a. In this study, we evaluated the inhibitory activity of a series of 2-aminothiazole derivatives against both iNOS and neuronal NOS (nNOS). Our results show that introduction of appropriately-sized substituents at the 4- and 5-position of the 2-aminothiazole ring improves the inhibitory activity and selectivity for iNOS. We also found that the selectivity of 5a [5-(1-methyl)ethyl-4-methylthiazol-2-ylamine] and 5b [5-(1,1-dimethyl)ethyl-4-methylthiazol-2-ylamine] for iNOS was similar to that of oxazolidine derivative 1b (4-methyl-5-propyl-2-imino-1,3-oxazolidine) and much higher than that of L-NAME. However, we could not enhance the inhibitory activity against iNOS by introducing an alkyl substituent into the 2-aminothiazole ring as we could in the case of oxazolidine one. On the other hand, introduction of bulky or hydrophilic substituent at any position of the 2-aminothiazole ring remarkably decreased or even abolished the inhibitory activity against NOS.
Assuntos
Inibidores Enzimáticos/química , Óxido Nítrico Sintase/antagonistas & inibidores , Tiazóis/química , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Tiazóis/farmacologiaRESUMO
In the course of our search for selective iNOS inhibitors, we have previously reported that 2-imino-1,3-oxazolidine derivatives (1) and 2-aminothiazole derivatives (2) are selective iNOS inhibitors. In order to find more potent iNOS inhibitors, we focused our efforts on the synthesis and evaluation of the inhibitory activity against iNOS and selectivity for iNOS both in vitro and in vivo of a series of 2-imino-1,3-thiazolidine derivatives (3), which are analogues of 1 and 2. Our results show that among the compounds synthesized (4R,5R)-5-ethyl-2-imino-4-methyl-1,3-thiazolidine [(4R,5R)-14a: ES-1537] exhibited potent inhibitory activity and selectivity for iNOS. In addition, ES-1537 had good pharmacokinetic profile in rats with BA value of 80%. It is therefore expected that ES-1537 may be therapeutically useful for the treatment of diseases related to excess production of NO.