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1.
J Med Chem ; 58(1): 517-21, 2015 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-24754609

RESUMO

Phosphoinositide 3-kinase γ (PI3Kγ) is an attractive target to potentially treat a range of disease states. Herein, we describe the evolution of a reported phenylthiazole pan-PI3K inhibitor into a family of potent and selective benzothiazole inhibitors. Using X-ray crystallography, we discovered that compound 22 occupies a previously unreported hydrophobic binding cleft adjacent to the ATP binding site of PI3Kγ, and achieves its selectivity by exploiting natural sequence differences among PI3K isoforms in this region.


Assuntos
Benzotiazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Benzotiazóis/química , Benzotiazóis/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
2.
Protein Expr Purif ; 104: 57-64, 2014 12.
Artigo em Inglês | MEDLINE | ID: mdl-25240855

RESUMO

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Pseudomonas aeruginosa/enzimologia , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Catálise , Cromatografia Líquida , Cristalografia por Raios X , Escherichia coli , Expressão Gênica , Espectrometria de Massas , Conformação Proteica , Zinco/química , Zinco/metabolismo
3.
Bioorg Med Chem ; 22(19): 5392-409, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25155913

RESUMO

Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa. Physicochemical properties (pKa and logD) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa, and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Animais , Físico-Química , Cães , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Cobaias , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Ratos , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química
4.
Arterioscler Thromb Vasc Biol ; 28(4): 665-71, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18202322

RESUMO

OBJECTIVE: TGF-beta plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. METHODS AND RESULTS: The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-beta and activin-induced Smad2/3 phosphorylation and TGF-beta-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle alpha-actin-positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. CONCLUSIONS: These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.


Assuntos
Compostos Azabicíclicos/farmacologia , Lesões das Artérias Carótidas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Ativinas Tipo I/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Administração Oral , Animais , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/metabolismo , Sítios de Ligação , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Humanos , Masculino , Mioblastos de Músculo Liso/efeitos dos fármacos , Mioblastos de Músculo Liso/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I , Fator de Crescimento Transformador beta/fisiologia
5.
Blood ; 102(13): 4464-71, 2003 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-12933585

RESUMO

Interaction of very late antigen-4 (VLA-4) with its ligand vascular cell adhesion molecule-1 (VCAM-1) is required for central nervous system (CNS) migration of encephalitogenic T cells in relapsing experimental autoimmune encephalomyelitis (R-EAE). Anti-VLA-4 monoclonal antibody (mAb) treatment prior to EAE onset inhibits disease induction; however, treatment initiated after the appearance of clinical symptoms increases relapse rates, augments Th1 responses, and enhances epitope spreading perhaps due to the activation of costimulatory signals. To negate the potential costimulatory activity of intact anti-VLA-4, we examined the ability of BIO 5192, a small-molecule VLA-4 antagonist, to regulate active proteolipid protein 139-151 (PLP139-151)-induced R-EAE. BIO 5192 administered one week after peptide priming (ie, before clinical disease onset) delayed the clinical disease onset but led to severe disease exacerbation upon treatment removal. BIO 5192 treatment initiated during disease remission moderately enhanced clinical disease while mice were on treatment and also resulted in posttreatment exacerbation. Interestingly, BIO 5192 treatment begun at the peak of acute disease accelerated entrance into disease remission and inhibited relapses, but treatment removal again exacerbated disease. Enhanced disease was caused by the release of encephalitogenic cells from the periphery and the rapid accumulation of T cells in the CNS. Collectively, these results further demonstrate the complexity of VLA-4/VCAM interactions, particularly in a relapsing-remitting autoimmune disease.


Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Integrina alfa4beta1/antagonistas & inibidores , Oligopeptídeos/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Células Th1/patologia , Sequência de Aminoácidos , Animais , Barreira Hematoencefálica/imunologia , Adesão Celular/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Modelos Animais de Doenças , Progressão da Doença , Esquema de Medicação , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Integrina alfa4beta1/fisiologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Esclerose Múltipla Recidivante-Remitente , Proteína Proteolipídica de Mielina/imunologia , Oligopeptídeos/administração & dosagem , Oligopeptídeos/toxicidade , Fragmentos de Peptídeos/imunologia , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/toxicidade , Recidiva , Células Th1/imunologia , Molécula 1 de Adesão de Célula Vascular/fisiologia
6.
J Pharmacol Exp Ther ; 306(3): 903-13, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12766251

RESUMO

An alpha4beta1/alpha4beta7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of alpha4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both alpha4beta1 and alpha4beta7, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino) pentanoylamino]-butyric acid (BIO7662), a specific alpha4beta1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that alpha4beta1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly alpha4beta7 with a KD of 1.9 nM in the presence of 1 mM Mn2+, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated alpha4beta7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily alpha4beta1 with a KD of 18 nM. In contrast, the binding of 35S-compound 1 to Mn2+-activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four alpha4beta1/alpha4beta7 antagonists to block binding of activated alpha4beta1 or alpha4beta7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to 35S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of 35S-compound 1 binding to alpha4beta1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that alpha4beta1 and alpha4beta7 have distinct binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.


Assuntos
Cátions Bivalentes/metabolismo , Dipeptídeos/farmacologia , Integrina alfa4beta1/antagonistas & inibidores , Integrinas/antagonistas & inibidores , Fenilalanina/farmacologia , Compostos de Fenilureia/farmacologia , Sítios de Ligação , Linhagem Celular , Dipeptídeos/química , Humanos , Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Células Jurkat , Células K562 , Cinética , Ligantes , Fenilalanina/análogos & derivados , Fenilalanina/química , Compostos de Fenilureia/química , Ligação Proteica , Ensaio Radioligante , Radioisótopos de Enxofre , Células Tumorais Cultivadas , Molécula 1 de Adesão de Célula Vascular/metabolismo
7.
J Med Chem ; 45(14): 2988-93, 2002 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-12086484

RESUMO

The antigen alpha4beta1 (very late antigen-4, VLA-4) plays an important role in the migration of white blood cells to sites of inflammation. It has been implicated in the pathology of a variety of diseases including asthma, multiple sclerosis, and rheumatoid arthritis. We describe a series of potent inhibitors of alpha4beta1 that were discovered using computational screening for replacements of the peptide region of an existing tetrapeptide-based alpha4beta1 inhibitor (1; 4-[N'-(2-methylphenyl)ureido]phenylacetyl-Leu-Asp-Val) derived from fibronectin. The search query was constructed using a model of 1 that was based upon the X-ray conformation of the related integrin-binding region of vascular cell adhesion molecule-1 (VCAM-1). The 3D search query consisted of the N-terminal cap and the carboxyl side chain of 1 because, upon the basis of existing structure-activity data on this series, these were known to be critical for high-affinity binding to alpha4beta1. The computational screen identified 12 reagents from a virtual library of 8624 molecules as satisfying the model and our synthetic filters. All of the synthesized compounds tested inhibit alpha4beta1 association with VCAM-1, with the most potent compound having an IC(50) of 1 nM, comparable to the starting compound. Using CATALYST, a 3D QSAR was generated that rationalizes the variation in activities of these alpha4beta1 antagonists. The most potent compound was evaluated in a sheep model of asthma, and a 30 mg nebulized dose was able to inhibit early and late airway responses in allergic sheep following antigen challenge and prevented the development of nonspecific airway hyperresponsiveness to carbachol. Our results demonstrate that it is possible to rapidly identify nonpeptidic replacements of integrin peptide antagonists. This approach should be useful in identification of nonpeptidic alpha4beta1 inhibitors with improved pharmacokinetic properties relative to their peptidic counterparts.


Assuntos
Integrinas/antagonistas & inibidores , Oligopeptídeos/química , Compostos de Fenilureia/química , Receptores de Retorno de Linfócitos/antagonistas & inibidores , Administração por Inalação , Aerossóis , Animais , Asma/tratamento farmacológico , Asma/imunologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/imunologia , Broncoconstrição/efeitos dos fármacos , Técnicas de Química Combinatória , Cristalografia por Raios X , Bases de Dados Factuais , Fibronectinas/química , Integrina alfa4beta1 , Modelos Moleculares , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacologia , Relação Quantitativa Estrutura-Atividade , Ovinos , Molécula 1 de Adesão de Célula Vascular/química
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