RESUMO
We have explored a series of spirocyclic piperidine amide derivatives (5) as tryptase inhibitors. Thus, 4 (JNJ-27390467) was identified as a potent, selective tryptase inhibitor with oral efficacy in two animal models of airway inflammation (sheep and guinea pig asthma models). An X-ray co-crystal structure of 4 x tryptase revealed a hydrophobic pocket in the enzyme's active site, which is induced by the phenylethynyl group and is comprised of amino acid residues from two different monomers of the tetrameric protein.
Assuntos
Asma/tratamento farmacológico , Hipersensibilidade Respiratória/tratamento farmacológico , Inibidores de Serina Proteinase/farmacologia , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Triptases/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Inibidores das Enzimas do Citocromo P-450 , Modelos Animais de Doenças , Cães , Cobaias , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Ratos , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacocinética , Ovinos , Espectrometria de Massas por Ionização por Electrospray , Compostos de Espiro/farmacocinética , Tripsina/metabolismo , Triptases/metabolismoRESUMO
Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography-tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.
Assuntos
Diglicerídeos/análise , Inibidores Enzimáticos/análise , Ensaios de Triagem em Larga Escala , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Acil Coenzima A/metabolismo , Animais , Chlorocebus aethiops , Cromatografia Líquida/métodos , Diglicerídeos/antagonistas & inibidores , Diglicerídeos/biossíntese , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Haplorrinos , Humanos , Intestinos/efeitos dos fármacos , Intestinos/enzimologia , Cinética , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor membrane preparations that maintain the transmembrane signaling activity of the receptor and provide robust biophysical and biochemical assay systems. Here we report a technique for self-assembling functional beta2-adrenergic receptor (beta2AR) into a nanoscale phospholipid bilayer system (Nanodisc) that is highly soluble in aqueous solution. The approximately 10-nm nanobilayer particles contain beta2AR in a native-like phospholipid bilayer domain of approximately 100 phospholipid molecules circumferentially bound by a membrane scaffold protein (MSP). The resulting construct allows for access to the physiologically intracellular and extracellular faces of the receptor and thus allows unrestricted access of antagonists, agonists, and G proteins. These Nanodisc-solubilized GPCRs can be directly purified by normal chromatographic procedures. We define the resultant Nanodisc-embedded monomeric beta2AR by antagonist and agonist binding isotherms and demonstrate faithful G protein coupling.
Assuntos
Bicamadas Lipídicas/metabolismo , Nanoestruturas/química , Nanotecnologia , Receptores Adrenérgicos beta 2/isolamento & purificação , Receptores Adrenérgicos beta 2/metabolismo , Linhagem Celular , Cromatografia em Gel , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Bicamadas Lipídicas/química , Modelos Biológicos , Tamanho da Partícula , Fosfatidilcolinas/química , Ligação Proteica , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Água/químicaRESUMO
Sensory adaptation and chemotaxis by Escherichia coli require a specific pentapeptide at the chemoreceptor carboxyl terminus. This sequence binds the two enzymes of receptor adaptational modification, enhancing catalysis, but with different binding features and mechanisms. We investigated the relative importance of each pentapeptide side chain for the two enhancing interactions.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Metiltransferases/metabolismo , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Transdução de Sinais , Sítios de Ligação , Quimiotaxia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/fisiologia , Proteínas de Escherichia coli/análise , Metilação , Metiltransferases/análise , Oligopeptídeos/químicaRESUMO
Heat shock protein 90 (Hsp90) is critical for the maturation of numerous client proteins, many of which are involved in cellular transformation and oncogenesis. The ansamycins, geldanamycin (GA) and its derivative, 17-allylaminogeldanamycin (17-AAG), inhibit Hsp90. As such, the prototypical Hsp90 inhibitor, 17-AAG, has advanced into clinical oncology trials. GA and 17-AAG potently inhibit tumor cell proliferation and survival but have been reported to bind weakly to Hsp90 in vitro. Recent studies have suggested that the in vitro potency of ansamycins against Hsp90 may be enhanced in the presence of cochaperones. Here, we present evidence of an alternative explanation. Ansamycins reduced to their dihydroquinones in the presence of common reducing agents in vitro have approximately 40-fold greater affinity than the corresponding oxidized quinones. The dihydroquinone of 17-AAG is not generated in an aqueous environment in the absence of reducing agents but is produced in both tumor and normal quiescent epithelial cells. The reduced form of 17-AAG is differentiated from its oxidized form not only by the higher affinity for Hsp90 but also by a protracted K(off) rate. Therefore, the in vivo accumulation of the high-affinity dihydroquinone ansamycins in tumor cells contributes to the antitumor activity of these compounds and alters our understanding of the active species driving the efficacy of this class of compounds.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Quinonas/metabolismo , Quinonas/farmacologia , Antineoplásicos , Benzoquinonas , Linhagem Celular Tumoral/efeitos dos fármacos , Células Cultivadas , Estabilidade de Medicamentos , Humanos , Lactamas Macrocíclicas , Ligação Proteica/efeitos dos fármacos , Substâncias Redutoras/farmacologia , Rifabutina/análogos & derivados , Rifabutina/metabolismo , Rifabutina/farmacologia , SolubilidadeRESUMO
Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In Escherichia coli and Salmonella enterica serovar typhimurium, efficient adaptational modification by either enzyme is dependent on a conserved pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from the sites of modification. For CheR-catalyzed methylation, previous work demonstrated that this sequence acts as a high affinity docking site, enhancing methylation by increasing enzyme concentration near methyl-accepting glutamates. We investigated pentapeptide-mediated enhancement of CheB-catalyzed demethylation and found it occurred by a distinctly different mechanism. Assays of binding between CheB and the pentapeptide sequence showed that it was too weak to have a significant effect on local enzyme concentration. Kinetic analyses revealed that interaction of the sequence and the methylesterase enhanced the rate constant of demethylation not the Michaelis constant. This allosteric activation occurred if the sequence was attached to chemoreceptor, but hardly at all if it was present as an isolated peptide. In addition, free peptide inhibited demethylation of the native receptor carrying the pentapeptide sequence at its carboxyl terminus. These observations imply that the allosteric change is transmitted through the protein substrate, not the enzyme.