RESUMO
Sonic hedgehog (Shh) is a morphogen with important roles in embryonic development and in the development of a number of cancers. Its activity is modulated by interactions with binding partners and co-receptors including heparin and heparin sulfate proteoglycans (HSPG). To identify antagonists of Shh/heparin binding, a diverse collection of 34,560 chemicals was screened in single point 384-well format. We identified and confirmed twenty six novel small molecule antagonists with diverse structures including four scaffolds that gave rise to multiple hits. Nineteen of the confirmed hits blocked binding of the N-terminal fragment of Shh (ShhN) to heparin with IC50 values <50⯵M. In the Shh-responsive C3H10T1/2 cell model, four of the compounds demonstrated the ability to block ShhN-induced alkaline phosphatase activity. To demonstrate a direct and selective effect on ShhN ligand mediated activity, two of the compounds were able to block induction of Gli1 mRNA, a primary downstream marker for Shh signaling activity, in Shh-mediated but not Smoothened agonist (SAG)-mediated C3H10T1/2 cells. Direct binding of the two compounds to ShhN was confirmed by thermal shift assay and molecular docking simulations, with both compounds docking with the N-terminal heparin binding domain of Shh. Overall, our findings indicate that small molecule compounds that block ShhN binding to heparin and act to inhibit Shh mediated activity in vitro can be identified. We propose that the interaction between Shh and HSPGs provides a novel target for identifying small molecules that bind Shh, potentially leading to novel tool compounds to probe Shh ligand function.
RESUMO
Obesity and insulin resistance are primary risk factors for Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD is generally exhibited by non-progressive simple steatosis. However, a significant subset of patient's progress to nonalcoholic steatohepatitis (NASH) that is defined by the presence of steatosis, inflammation and hepatocyte injury with fibrosis. Unfortunately, there are no approved therapies for NAFLD or NASH and therefore therapeutic approaches are urgently needed. Niclosamide is an U.S. Food and Drug Administration (FDA)-approved anthelmintic drug that mediates its effect by uncoupling oxidative phosphorylation. Niclosamide and its salt forms, Niclosamide Ethanolamine (NEN), and Niclosamide Piperazine (NPP) have shown efficacy in murine models of diet induced obesity characterized by attenuation of the prominent fatty liver disease phenotype and improved glucose metabolism. While the exact mechanism(s) underlying these changes remains unclear, the ability to uncouple oxidative phosphorylation leading to increased energy expenditure and lipid metabolism or attenuation of PKA mediated glucagon signaling in the liver have been proposed. Unfortunately, niclosamide has very poor water solubility, leading to low oral bioavailability. This, in addition to mitochondrial uncoupling activity and potential genotoxicity have reduced enthusiasm for its clinical use. More recently, salt forms of niclosamide, NEN and NPP, have demonstrated improved oral bioavailability while retaining activity. This suggests that development of safer more effective niclosamide derivatives for the treatment of NAFLD and Type 2 Diabetes may be possible. Herein we explored the ability of a series of N-substituted phenylbenzamide derivatives of the niclosamide salicylanilide chemotype to attenuate hepatic steatosis using a novel phenotypic in vitro model of fatty liver and the high fat diet-fed mouse model of diet induced obesity. These studies identified novel compounds with improved pre-clinical properties that attenuate hepatic steatosis in vitro and in vivo. These compounds with improved drug properties may be useful in alleviating symptoms and protection against disease progression in patients with metabolic syndrome and NAFLD.
Assuntos
Fármacos Antiobesidade/farmacologia , Benzamidas/farmacologia , Dieta Hiperlipídica/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Ratos Sprague-Dawley , Salmonella typhimurium/efeitos dos fármacosRESUMO
Starting from a weak omeprazole screening hit, replacement of the pyridine with a 1,3-benzodioxole moiety, modification of the thioether linkage, and substitution of the benzimidazole pharmacophore led to the discovery of nanomolar BRS-3 agonists.
Assuntos
Omeprazol/química , Receptores da Bombesina/agonistas , Baculoviridae/metabolismo , Benzimidazóis/química , Encéfalo/metabolismo , Química Farmacêutica/métodos , Clorofluorcarbonetos de Metano/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Modelos Químicos , Piridinas/química , Receptores da Bombesina/química , Sulfetos/químicaRESUMO
A novel series of potent substituted anilinoquinolines were discovered as c-fms inhibitors. The potency could be manipulated upon modification of the C4 aniline and C7 aryl functionality. Pharmacokinetic analysis identified a metabolically stable analog suitable for further investigative work.
Assuntos
Compostos de Anilina/química , Quinolinas/síntese química , Quinolinas/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Sítios de Ligação , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Concentração Inibidora 50 , Modelos Animais , Modelos Moleculares , Estrutura Molecular , Quinolinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Mapeamento de Interação de Proteínas/métodos , Proteínas ADAM , Proteína ADAM17 , Sítios de Ligação , Biotina/química , Cromatografia Líquida/métodos , Desenho de Fármacos , Processamento de Imagem Assistida por Computador , Espectrometria de Massas/métodos , Metaloendopeptidases/genética , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The synthesis of a novel series of 3-phenylprop-2-ynylamines as selective mammalian squalene epoxidase inhibitors is described. Structure activity relationship studies led to the discovery of compound 19, 1-[3-(3,5-dichlorophenyl)-prop-2-ynyl]-3- methylpiperidine hydrochloride with an IC50 of 2.8 +/- 0.6 microM against rat liver squalene epoxidase. Against 23 strains of fungal squalene epoxidase compound 19 was found to be inactive.
Assuntos
Aminas/química , Aminas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oxigenases/antagonistas & inibidores , Aminas/síntese química , Animais , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Inibidores Enzimáticos/síntese química , Feminino , Concentração Inibidora 50 , Ratos , Ratos Sprague-Dawley , Esqualeno Mono-Oxigenase , Relação Estrutura-AtividadeRESUMO
The design of rigid cyclic analogues derived from cinnamamide 1, (E)-N-cyclopropyl-3-(3-fluorophenyl)prop-2-enamide, and beta-methylcinnamamide 2, (E)-N-cyclopropyl-3-(3-fluorophenyl)but-2-enamide, has led to the discovery of the potent, centrally acting muscle relaxant (E)-2-(4,6-difluoro-1-indanylidene)acetamide, 17. Compound 17 also possesses potent antiinflammatory and analgesic activity. This paper describes the synthesis and the muscle relaxant, antiinflammatory, and analgesic structure-activity relationships of 17 and 67 of its analogues. Compound 17 has been taken into phase I clinical trials.
Assuntos
Acetamidas/síntese química , Analgésicos/síntese química , Anti-Inflamatórios não Esteroides/síntese química , Indanos/síntese química , Relaxantes Musculares Centrais/síntese química , Acetamidas/química , Acetamidas/farmacologia , Analgésicos/química , Analgésicos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Hipnóticos e Sedativos/síntese química , Hipnóticos e Sedativos/química , Hipnóticos e Sedativos/farmacologia , Indanos/química , Indanos/farmacologia , Camundongos , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/síntese química , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Relaxantes Musculares Centrais/química , Relaxantes Musculares Centrais/farmacologia , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Extension of the structure-activity relationship studies that led to the discovery of the nonsedating potent muscle relaxant, antiinflammatory, and analgesic agent (E)-2-(4,6-difluoro-1-indanylidene)acetamide, 1, has given rise to (E)-2-(4-chloro-6-fluoro-1-indanylidene)-N-methylacetamide, 2. Compound 2 is a potent antiinflammatory and analgesic agent without centrally acting muscle relaxant activity.