RESUMO
Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides , Benzamidas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Benzamidas/farmacocinética , Disponibilidade Biológica , Western Blotting , Moléculas de Adesão Celular/biossíntese , Movimento Celular/efeitos dos fármacos , Quimiocinas/biossíntese , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Citometria de Fluxo , Humanos , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Interleucina-8/biossíntese , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Masculino , Cadeias Leves de Miosina/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores CCR2/biossínteseRESUMO
In the present study, capillary liquid chromatography (LC) nano electrospray ionization quadruple time-of-flight (nano-ESI-Q-TOF) mass spectrometry was utilized to identify the unique proteotypic peptides for liquid chromatography-tandem mass spectrometry (LC-MS/MS) mediated breast cancer resistance protein (BCRP/ABCG2) and bile salt export pump (BSEP/ABCG11) quantification, using insect membrane vesicles overexpressing the proteins. The lower limit of quantification was established to be 31.25 pM and 125 nM for BCRP/ABCG2 and BSEP/ABCG11, respectively. The linearity of standard curves was up to 5000 pM. The accuracy and precision of the LC-MS/MS method were evaluated by adding the known amount of synthetic proteotypic peptide or synthetic surrogate peptide substrates in the membrane protein extracts of livers or hepatocytes. The overall relative error (RE) and coefficient of variation (CV) were below 15.9% and 14.2% for BCRP/ABCG2 quantification or below 15.6% and 6.4% for BSEP/ABCG11, respectively. The absolute differences of BCRP/Bcrp and BSEP/Bsep proteins were determined in livers and isolated hepatocytes across species by the newly developed LC-MS/MS methods, with ranking order of dog > rat > monkey approximately = human and rat approximately = monkey > dog approximately = human, respectively (where the uppercase letters identify the human protein, i.e., BSEP and BCRP, and lowercase letters indicate that the transporter derives from a preclinical species, i.e., Bsep and Bcrp). The freshly isolated and cryopreserved hepatocytes conserved the protein levels of BSEP/Bsep and BCRP/Bcrp similarly to those found in liver tissue. We report, for the first time, an absolution quantification method for BCRP/Bcrp and BSEP/Bsep and the differences of the protein expressions across species. The results could serve as supportive information for extrapolation of hepatobiliary elimination from preclinical species to human.
Assuntos
Transportadores de Cassetes de Ligação de ATP/análise , Cromatografia Líquida de Alta Pressão/métodos , Hepatócitos/metabolismo , Fígado/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Cães , Feminino , Haplorrinos , Humanos , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/metabolismo , Ratos , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
The contribution of inducible nitric oxide synthase (iNOS) to oxidative/nitrative stress is well-documented in inflammation, but difficult to quantify. Using a novel, recently developed assay for 3-nitrotyrosine (3-NT), we characterized iNOS activity and its inhibition in preclinical models of inflammation. In particular, we utilized the 3-NT assay to assess the role of iNOS in the disease pathology as well as for proof of pharmacology of iNOS inhibitors in an acute endotoxin challenge model, in models of rheumatoid arthritis (RA) such as rat adjuvant- and collagen-induced arthritis (AIA and CIA) and a model of osteoarthritis (OA) such as rat sodium monoiodoacetate-induced arthritis (MIA). Quantification of nitrotyrosine was performed using immuno-affinity 2-D LC-MS/MS assay. This assay is a very specific and reproducible and is amenable to a number of biological fluids. Plasma levels of 3-NT were significantly elevated in an acute model of inflammation (rat LPS) and in models of rheumatoid arthritis (adjuvant- and collagen-induced arthritis), and osteoarthritis (monoiodoacetate-induced arthritis). Plasma 3-NT correlated with the severity of the inflammatory response; thus, a 20-fold increase was observed in the rat LPS model, a 10-fold increase in AIA, and only a 2.5-fold elevation in CIA. Pharmacological intervention with iNOS inhibitors decreased 3-NT levels and associated pathology. 3-NT determination allowed for better elucidation of the role of iNOS in RA and OA disease pathology and provided proof of pharmacology for NOS inhibitors in animal models of RA and OA.
Assuntos
Óxido Nítrico Sintase Tipo II/fisiologia , Tirosina/análogos & derivados , Animais , Artrite Experimental , Artrite Reumatoide , Biomarcadores/sangue , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inflamação , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite , Ratos , Índice de Gravidade de Doença , Tirosina/sangueRESUMO
Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.
Assuntos
Biomarcadores/análise , Biomarcadores/metabolismo , Líquidos Corporais/química , Cromatografia de Afinidade/métodos , Espectrometria de Massas em Tandem/métodos , Tirosina/análogos & derivados , Anticorpos/imunologia , Modelos Lineares , Pronase/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Líquido Sinovial/química , Tirosina/análise , Tirosina/imunologia , Tirosina/metabolismoRESUMO
The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25pM with the linearity of the standard curve spanned to 2500pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.
Assuntos
Cromatografia Líquida/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Calibragem , Linhagem Celular , Cães , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Nanotecnologia/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
The protonated molecules and radical cations of meso-tetraphenylporphyrins with beta-pyrrolic substituents, when formed by fast atom bombardment (FAB) and subjected to high-energy collisions, give rise to unexpected fragment ions. The reaction involves hydrogen migration from the ortho position of the phenyl ring to the a atom of the substituent, with formation of an intramolecular, six-membered ring. The process is analogous to condensed-phase cyclizations described for the same type of compounds. The fragmentation requires the presence of a double bond in the substituent group attached to the pyrrolic ring. A rearrangement process involving anchimeric assistance by the phenyl group (analogous to an ortho effect) is proposed for the formation of these ions.
Assuntos
Porfirinas/química , Acrilatos/química , Cátions , Ciclização , Hidrogênio/química , Nitrocompostos/química , Propionatos/química , Prótons , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Compostos de Vinila/químicaRESUMO
To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire(®) mass spectrometry system.
Assuntos
Inibidores Enzimáticos/sangue , Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Aminofenóis/metabolismo , Aminofenóis/farmacologia , Relação Dose-Resposta a Droga , Humanos , Cinética , Lisofosfolipídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Tiazóis/metabolismo , Tiazóis/farmacologiaRESUMO
Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E(2) (PGE(2)) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE(2) synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC(50)=16.5+/-3.8nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE(2) synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE(2) synthesis (IC(50) in the range of 0.5-5 microM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF(1alpha) (PGF(1alpha)) and PGF(2alpha). In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE(2), PGF(1alpha) and PGF(2alpha) synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.
Assuntos
Óxidos S-Cíclicos/antagonistas & inibidores , Inibidores de Ciclo-Oxigenase 2/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Tiazinas/antagonistas & inibidores , Animais , Artrite Reumatoide/metabolismo , Carragenina/farmacologia , Células Cultivadas , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Interleucina-1beta/farmacologia , Oxirredutases Intramoleculares/biossíntese , Oxirredutases Intramoleculares/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Prostaglandina-E Sintases , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: Fibronectin fragments are thought to play a critical role in the initiation and progression of cartilage degradation in arthritis. In a recent study, fibronectin neoepitopes resulting from cleavage of intact fibronectin at the Ala(271)/Val(272) scissile bond, generating an approximately 30-kd fragment with the new C-terminus VRAA(271) and an approximately 50-85-kd fragment with the new N-terminus (272)VYQP, were identified in osteoarthritis (OA) cartilage. The present study was undertaken to isolate the enzymes responsible for this cleavage from human OA chondrocytes. METHODS: Fibronectin-degrading activity in human OA chondrocyte-conditioned medium (OACCM) was purified using conventional chromatography. A fluorescent peptide was developed based on the fibronectin scissile bond (269)RAA downward arrowVal(272), and this peptide was used to track fibronectinase activity during purification. Western blotting with antibodies that detect the fibronectin neoepitopes VRAA(271) and (272)VYQP was used to confirm cleavage of intact fibronectin by the enzymatically active fractions. Mass spectrometry was used to identify the proteins found in the fibronectinase-enriched fractions, with further confirmation by Western blotting. In addition, a recombinant enzyme identified by mass spectrometry was tested by Western blotting and dimethylmethylene blue assay for its ability to produce fibronectin neoepitopes in OA cartilage. RESULTS: Purification of OACCM by chromatography resulted in isolation of a fibronectin-degrading enzyme, and mass spectrometry identified ADAM-8 as the fibronectinase present in these preparations. Furthermore, treatment of OA cartilage with recombinant human ADAM-8 promoted cartilage catabolism. CONCLUSION: The results of this study identify ADAM-8 as a fibronectinase in human OA chondrocytes. Because ADAM-8 is capable of producing the fibronectin neoepitopes VRAA(271) and (272)VYQP in human OA cartilage, this enzyme may be an important mediator of cartilage catabolism.