RESUMEN
Mimics of the benzimidazolone nucleus found in inhibitors of p38 kinase are proposed, and their theoretical potential as bioisosteres is described. A set of calculated descriptors relevant to the anticipated binding interaction for the fragments 1-methyl-1H-benzotriazole 5, 3-methyl-benzo[d]isoxazole 3, and 3-methyl-[1,2,4]triazolo[4,3-a]pyridine 4, pyridine 1, and 1,3-dimethyl-1,3-dihydro-benzoimidazol-2-one 2 are reported. The design considerations and synthesis of p38 inhibitors based on these H-bond acceptor fragments is detailed. Comparative evaluation of the pyridine-, benzimidazolone-, benzotriazole-, and triazolopyridine-based inhibitors shows the triazoles 20 and 25 to be significantly more potent experimentally than the benzimidazolone after which they were modeled. An X-ray crystal structure of 25 bound to the active site shows that the triazole group serves as the H-bond acceptor but unexpectedly as a dual acceptor, inducing movement of the crossover connection of p38alpha. The computed descriptors for the hydrophobic and pi-pi interaction capacities were the most useful in ranking potency.
Asunto(s)
Bencimidazoles/química , Piridinas/química , Triazoles/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/química , Bencimidazoles/síntesis química , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Imitación Molecular , Estructura Molecular , Unión Proteica , Piridinas/síntesis química , Relación Estructura-Actividad Cuantitativa , Electricidad Estática , Triazoles/síntesis químicaRESUMEN
The metabolism of arachidonic acid through the cyclooxygenase pathway is a highly regulated cellular process that results in the formation of PGH2. This unstable intermediate can be enzymatically metabolized to PGE2 by the actions of a microsomal 17 kDa PGE synthase (mPGES1). Treatment of A549 cells with IL-1beta for 24 h resulted in a twofold increase in mPGES1 mRNA, protein expression, and PGES specific activity. To understand the relationship between expression of mPGES1 and PGE2 formation, IL-1beta treated cells were incubated with increasing concentrations of antisense oligonucleotides (ASO) and their effects compared to cells treated with reverse sense oligonucleotides (RSO) designed against the ATG translation initiation codon of mPGES1. Incubation with ASO resulted in a 44% reduction in mRNA expression level as compared to RSO-treated cells. Microsomal preparations isolated from ASO- and RSO-treated cells were analyzed for their ability to convert PGH2 to PGE2 in the presence 2.5 mM reduced glutathione. An approximate 50% reduction (ASO: 1.8 nmol/min/mg, RSO: 3.7 nmol/min/mg) in PGES activity, protein expression by immunodetection, and extracellular PGE2 release was detected in these samples. As a control in these studies, the protein levels of COX2 and secreted IL-8 were quantified; no change in these levels was observed. These results demonstrate the direct association between mPGES1 expression, its enzymatic activity, and total PGE2 production following an inflammatory stimulus.
Asunto(s)
Dinoprostona/biosíntesis , Interleucina-1/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/genética , Oligonucleótidos Antisentido/genética , Prostaglandina-Endoperóxido Sintasas/genética , Animales , Secuencia de Bases , Dinoprostona/genética , Expresión Génica , Ratones , Microsomas/enzimología , Datos de Secuencia Molecular , Prostaglandina-E Sintasas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The synthesis, structure-activity relationship, in vivo activity, and metabolic profile for a series of triazolopyridine-oxazole based p38 inhibitors are described. The deficiencies of the lead structure in the series, CP-808844, were overcome by changes to the C4 aryl group and the triazole side-chain culminating in the identification of several potential clinical candidates.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Oxazoles/química , Piridinas/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/química , Química Farmacéutica , Diseño de Fármacos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Cinética , Modelos Químicos , Solubilidad , Relación Estructura-Actividad , Triazoles/químicaRESUMEN
Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.
Asunto(s)
Eicosanoides/metabolismo , Oxidorreductasas Intramoleculares/biosíntesis , Oxidorreductasas Intramoleculares/fisiología , Macrófagos/metabolismo , Animales , Ácido Araquidónico/metabolismo , Western Blotting , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Ácidos Grasos Insaturados/metabolismo , Genotipo , Imidazoles/farmacología , Inflamación , Oxidorreductasas Intramoleculares/metabolismo , Cinética , Lipopolisacáridos/metabolismo , Macrófagos/enzimología , Ratones , Ratones Transgénicos , Microsomas/metabolismo , Prostaglandina-E Sintasas , Prostaglandinas/metabolismo , Tioglicolatos/farmacología , Tromboxano B2/farmacología , Factores de TiempoRESUMEN
A series of pipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was discovered based on screening known inhibitors of TNF-alpha converting enzyme (TACE). Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group. Incorporation of geminal alkyl substitution at the 3-position of the piperidine ring improved metabolic stability, presumably by increasing steric hindrance around the metabolically labile hydroxamic acid. This modification also resulted in dramatic improvement of aggrecanase activity with a slight reduction in selectivity versus MMP-1. Synthesis, structure activity relationships, and strategies to reduce metabolic clearance are described.
Asunto(s)
Endopeptidasas/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/farmacología , Ácidos Hidroxámicos/química , Metaloproteinasa 13 de la Matriz , Inhibidores de Proteasas/químicaRESUMEN
A series of 3-hydroxy-3-methylpipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was designed based on the observation of increased aggrecanase activity with substitution at the 3-position of the piperidine ring. Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group that binds in the S1' pocket. These compounds also possess markedly improved bioavailability and lower metabolic clearance compared to analogous 3,3-dimethyl-5-hydroxypipecolic hydroxamates. These improvements are attributed to lowered lipophilicity proximal to the metabolically labile hydroxamic acid. Synthesis, structure activity relationships, and in vivo efficacy data are described.
Asunto(s)
Endopeptidasas/efectos de los fármacos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacocinética , Inhibidores de la Metaloproteinasa de la Matriz , Ácidos Pipecólicos/síntesis química , Ácidos Pipecólicos/farmacocinética , Administración Oral , Animales , Colagenasas/metabolismo , Diseño de Fármacos , Endopeptidasas/metabolismo , Humanos , Ácidos Hidroxámicos/química , Metaloproteinasa 13 de la Matriz , Estructura Molecular , Ácidos Pipecólicos/química , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Relación Estructura-ActividadRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) is implicated in inflammatory processes and much effort is being directed at inhibiting the release of TNF-alpha for treatment of inflammatory conditions. In this context, the drug CP-661,631 has been developed to inhibit the TNF-alpha converting enzyme (TACE). However, TACE is also implicated in amyloid precursor protein secretion. Amyloid precursor protein (APP) undergoes constitutive and regulated secretion by alpha-secretase endoproteolytic cleavage within the amyloid beta peptide (Abeta) domain. Alternative cleavage at the N- and C-terminus of the Abeta domain by beta- and gamma-secretases results in the production of Abeta. In many cellular and in vivo animal models, increased secretion of APP results in a concomitant decrease in the production of Abeta suggesting that the two pathways are intricately linked. However, in human primary neuron cultures, increased APP secretion is not associated with a decrease in total Abeta production. To determine if the use of CP-661,631 may enhance amyloidogenic processing in human brain, we have assessed the effect of CP-661,631 on APP metabolism in primary cultures of human neurons. Our results show that CP-661,631 effectively prevents regulated APP secretion but does not increase total Abeta levels in human primary neuron cultures.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/metabolismo , Neuronas/efectos de los fármacos , Piperidinas/farmacología , Sulfonas/farmacología , Proteínas ADAM , Proteína ADAM17 , Péptidos beta-Amiloides/biosíntesis , Péptidos beta-Amiloides/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Neuronas/citología , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Piperidinas/toxicidad , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Sulfonas/toxicidad , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The synthesis and in vitro p38 alpha activity of a novel series of benzimidazolone inhibitors is described. The p38 alpha SAR is consistent with a mode of binding wherein the benzimidazolone carbonyl serves as the H-bond acceptor to Met109 of p38 alpha in a manner analogous to the pyridine nitrogen of prototypical pyridylimidazole p38 inhibitors. Potent p38 alpha activity comparable to that of several previously reported p38 inhibitors is observed for this novel chemotype.
Asunto(s)
Bencimidazoles/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Bencimidazoles/síntesis química , Inhibidores Enzimáticos/síntesis química , Imidazoles/farmacología , Proteína Quinasa 14 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estructura Molecular , Piridinas/farmacología , Relación Estructura-ActividadRESUMEN
A series of novel MMP-13 and TNF-alpha converting enzyme inhibitors based on piperazine 2-hydroxamic acid scaffolds are described. The TACE, MMP-1 and MMP-13 activity of these inhibitors as well as the effect of substitution of the piperazine nitrogen and the P-1' benzyloxy tailpiece is discussed. Moderate in vivo activity is observed with several members of this group.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Piperazinas/síntesis química , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animales , Colagenasas/metabolismo , Inhibidores Enzimáticos/farmacología , Metaloproteinasa 13 de la Matriz , Metaloendopeptidasas/metabolismo , Piperazinas/farmacología , RatasRESUMEN
Prostaglandin (PG)E2 is a potent mediator of pain and inflammation, and high levels of this lipid mediator are observed in numerous disease states. The inhibition of PGE2 production to control pain and to treat diseases such as rheumatoid arthritis to date has depended on nonsteroidal antiinflammatory agents such as aspirin. However, these agents inhibit the synthesis of all prostanoids. To produce biologically active PGE2, PGE synthases catalyze the isomerization of PGH2 into PGE2. Recently, several PGE synthases have been identified and cloned, but their role in inflammation is not clear. To study the physiological role of the individual PGE synthases, we have generated by targeted homologous recombination a mouse line deficient in microsomal PGE synthase 1 (mPGES1) on the inbred DBA/1lacJ background. mPGES1-deficient (mPGES1-/-) mice are viable and fertile and develop normally compared with wild-type controls. However, mPGES1-/- mice displayed a marked reduction in inflammatory responses compared with mPGES1+/+ mice in multiple assays. Here, we identify mPGES1 as the PGE synthase that contributes to the pathogenesis of collagen-induced arthritis, a disease model of human rheumatoid arthritis. We also show that mPGES1 is responsible for the production of PGE2 that mediates acute pain during an inflammatory response. These findings suggest that mPGES1 provides a target for the treatment of inflammatory diseases and pain associated with inflammatory states.
Asunto(s)
Inflamación/fisiopatología , Oxidorreductasas Intramoleculares/deficiencia , Dolor/fisiopatología , Animales , Artritis Experimental/etiología , Artritis Experimental/patología , Artritis Experimental/fisiopatología , Artritis Reumatoide/etiología , Artritis Reumatoide/patología , Artritis Reumatoide/fisiopatología , Dinoprostona/biosíntesis , Femenino , Humanos , Hipersensibilidad Tardía , Mediadores de Inflamación/metabolismo , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/fisiología , Macrófagos/enzimología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Dolor/tratamiento farmacológico , Prostaglandina-E SintasasRESUMEN
A series of novel, selective TNF-alpha converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydroxamic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the sulfonamide group which interacts with the S1' site of the enzyme. Substituted 4-benzyloxybenzenesulfonamides exhibit excellent TACE potency with >100x selectivity over inhibition of matrix metalloprotease-1 (MMP-1). Alkyl substituents on the ortho position of the benzyl ether moiety give the most potent inhibition of TNF-alpha release in LPS-treated human whole blood.