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1.
Eur J Pharmacol ; 743: 106-16, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25261040

ABSTRACT

Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity of this antigen after direct application to the lungs of Brown Norway rats. Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and time dependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats. We established the temporal profile of eosinophilic infiltration and cytokine production, such as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production. Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic pulmonary inflammation.


Subject(s)
Alternaria/drug effects , Anti-Inflammatory Agents/pharmacology , Carbolines/pharmacology , Pneumonia/drug therapy , Receptors, Formyl Peptide/metabolism , Th2 Cells/drug effects , Allergens/immunology , Alternaria/immunology , Animals , Asthma/drug therapy , Asthma/immunology , Asthma/metabolism , Cytokines/immunology , Cytokines/metabolism , Eosinophils/drug effects , Eosinophils/immunology , Eosinophils/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Interleukin-13/immunology , Interleukin-13/metabolism , Interleukin-5/immunology , Interleukin-5/metabolism , Lung/drug effects , Lung/immunology , Lung/metabolism , Male , Ovalbumin/immunology , Ovalbumin/pharmacology , Pneumonia/immunology , Pneumonia/metabolism , Rats , Rats, Inbred BN , Receptors, Formyl Peptide/immunology , Th2 Cells/immunology
2.
Mol Pharmacol ; 79(1): 69-76, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20943773

ABSTRACT

The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor that has been reported to modulate inflammatory responses in various rodent models of asthma, allergic rhinitis and atopic dermatitis. In this study, we describe the biological and pharmacological properties of {(7R)-7-[[(4-fluorophenyl)sulfonyl](methyl)amino]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}acetic acid (MK-7246), a novel synthetic CRTH2 antagonist. We show that MK-7246 1) has high affinity for the human, monkey, dog, rat, and mouse CRTH2, 2) interacts with CRTH2 in a reversible manner, 3) exhibits high selectivity over all prostanoid receptors as well as 157 other receptors and enzymes, 4) acts as a full antagonist on recombinant and endogenously expressed CRTH2, 5) demonstrates good oral bioavailability and metabolic stability in various animal species, 6) yields ex vivo blockade of CRTH2 on eosinophils in monkeys and sheep, and 7) significantly blocks antigen-induced late-phase bronchoconstriction and airway hyper-responsiveness in sheep. MK-7246 represents a potent and selective tool to further investigate the in vivo function of CRTH2.


Subject(s)
Carbolines/chemistry , Carbolines/pharmacology , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/biosynthesis , Receptors, Prostaglandin/antagonists & inhibitors , Receptors, Prostaglandin/biosynthesis , Th2 Cells/metabolism , Animals , Dogs , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , HEK293 Cells , Humans , Macaca fascicularis , Mice , Platelet Aggregation Inhibitors/pharmacology , Protein Binding/immunology , Rats , Receptors, Immunologic/metabolism , Receptors, Immunologic/physiology , Receptors, Prostaglandin/metabolism , Receptors, Prostaglandin/physiology , Sheep , Species Specificity , Th2 Cells/drug effects
3.
Bioorg Med Chem Lett ; 21(1): 288-93, 2011 Jan 01.
Article in English | MEDLINE | ID: mdl-21106375

ABSTRACT

In this manuscript we wish to report the discovery of MK-7246 (4), a potent and selective CRTH2 (DP2) antagonist. SAR studies leading to MK-7246 along with two synthetic sequences enabling the preparation of this novel class of CRTH2 antagonist are reported. Finally, the pharmacokinetic and metabolic profile of MK-7246 is disclosed.


Subject(s)
Carbolines/chemistry , Lung Diseases/drug therapy , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Animals , Carbolines/pharmacokinetics , Carbolines/therapeutic use , Humans , Macaca mulatta , Microsomes, Liver/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Structure-Activity Relationship
4.
COPD ; 7(1): 51-8, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20214463

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is a prevalent pulmonary disease characterized by a progressive decline in lung function. The identification of biomarkers capable of predicting the rate of lung function decline or capable of giving an early read on drug efficacy in clinical trials would be very useful. The aim of this study was to identify plasma biomarkers capable of accurately distinguishing patients with COPD from healthy controls. Eighty-nine plasma markers in 40 COPD patients and 20 healthy smoker controls were analyzed. The COPD patients were divided into two subgroups, rapid and slow decliners based on their rate of lung function decline measured over 15 years. Univariate analysis revealed that 25 plasma markers were statistically different between rapid decliners and controls, 4 markers were different between slow decliners and controls, and 10 markers were different between rapid and slow decliners (p < 0.05). Multivariate analysis led to the identification of groups of plasma markers capable of distinguishing rapid decliners from controls (signature 1), slow decliners from controls (signature 2) and rapid from slow decliners (signature 3) with over 90% classification accuracy. Importantly, signature 1 was shown to be longitudinally stable using plasma samples taken a year later from a subset of patients. This study describes a novel set of plasma markers differentiating slow from rapid decline of lung function in COPD. If validated in distinct and larger cohorts, the signatures identified will have important implications in both disease diagnosis, as well as the clinical evaluation of new therapies.


Subject(s)
Biomarkers/blood , Pulmonary Disease, Chronic Obstructive/blood , Pulmonary Disease, Chronic Obstructive/physiopathology , Case-Control Studies , Female , Forced Expiratory Volume/physiology , Humans , Longitudinal Studies , Male , Middle Aged , Predictive Value of Tests , Prognosis , Pulmonary Disease, Chronic Obstructive/diagnosis , Severity of Illness Index , Time Factors
6.
Anal Chem ; 81(5): 1881-7, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19178285

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. The degradation of elastin gives rise to desmosine (DES) and isodesmosine (IDES), two major urinary products typified by a hydrophilic pyridinium-based cross-linker structure. A high sensitivity method based on nanoflow liquid chromatography tandem mass spectrometry with multiple reaction monitoring was developed for the analysis of urinary DES and IDES. The analytes were derivatized with propionic anhydride and deuterated DES (D(4)-DES) was used as an internal standard. This method enables the quantification of DES and IDES in as little as 50 microL of urine and provides a detection limit of 0.10 ng/mL (0.95 fmol on-column). We report the analysis of DES and IDES in a cohort of 40 urine specimens from four groups of individuals: (a) COPD rapid decliners (11.8 +/- 3.7 ng/mg creatine (crea)), (b) COPD slow decliners (16.0 +/- 3.1 ng/mg crea), (c) healthy smokers (13.2 +/- 1.9 ng/mg crea), and (d) healthy nonsmokers (14.9 +/- 2.9 ng/mg crea). Our analysis reveals a statistically significant decrease in the level of urinary DES and IDES in COPD rapid decliner patients compared to healthy nonsmoker controls and COPD slow decliner patients. This methodology may be useful for monitoring DES and IDES levels in well controlled animal models for COPD or for longitudinal studies in COPD patients.


Subject(s)
Chromatography, Liquid/methods , Desmosine/urine , Isodesmosine/urine , Limit of Detection , Mass Spectrometry/methods , Tandem Mass Spectrometry/methods , Elastin/analysis , Humans
7.
Apoptosis ; 11(11): 1863-76, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17013756

ABSTRACT

The formin homology (FH) proteins play a crucial role in cytoskeleton remodelling during many essential processes. In this study, we demonstrate for the first time that the formin-homology-domain-containing protein FHOD1 is cleaved by caspase-3 at the SVPD(616) site during apoptosis. Using confocal microscopy, we further demonstrate that while full length FHOD1 is mostly cytoplasmic, the FHOD1 N-terminal cleavage product is diffusely localized throughout the cytoplasm and the nucleoplasm, whereas the C-terminal cleavage product is almost exclusively nuclear with some nucleolar localization. Finally, using a run-on transcription assay we show that the C-terminal FHOD1 cleavage product has the ability to inhibit RNA polymerase I transcription when overexpressed in HeLa cells as shown by blockage of BrUTP incorporation.


Subject(s)
Apoptosis , Caspase 3/metabolism , Cell Nucleolus/metabolism , Fetal Proteins/metabolism , Nuclear Proteins/metabolism , RNA, Ribosomal/genetics , Amino Acid Motifs , Caspase 3/genetics , Cell Line, Transformed , DNA, Complementary , Fetal Proteins/chemistry , Formins , Gene Expression Regulation , HeLa Cells , Humans , Nuclear Proteins/chemistry , RNA Polymerase I/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Proteins , Transcription, Genetic , Transfection
8.
Immunol Lett ; 100(2): 139-45, 2005 Sep 15.
Article in English | MEDLINE | ID: mdl-16154494

ABSTRACT

The anaphylatoxin C3a is an important immune regulator with a number of distinct functions in both innate and adaptive immunity. Many of these roles have been ascribed to C3a based on studies in mice genetically modified to lack its precursor, C3, or its receptor, C3aR. However, other presumed functions of C3a are based on results obtained with a recently described small molecule ligand of C3aR, SB 290157. Although this compound was originally described as an antagonist and appears to act as such in some systems, it has recently been shown to have effects that cannot be explained by simple antagonism of C3aR. In the current study, SB 290157 is shown to have full agonist activity on C3aR in a variety of cell systems, including a calcium mobilization assay in transfected RBL cells, a beta-lactamase assay in CHO-NFAT-bla-Galpha(16) cells and an enzyme-release assay in differentiated U-937 cells. On the other hand, the compound lacks agonist activity in guinea pig platelets, cells known to express C3aR at very low levels. SB 290157 agonism of C3aR is consistent with recent discrepant data obtained using this molecule. These results caution against attributing novel roles to C3a based on data obtained with SB 290157 and highlight a continuing need for the identification of true small molecule C3aR antagonists.


Subject(s)
Arginine/analogs & derivatives , Benzhydryl Compounds/pharmacology , Calcium/metabolism , Membrane Proteins/agonists , Receptors, Complement/agonists , Animals , Arginine/pharmacology , Binding, Competitive , Blood Platelets/drug effects , Blood Platelets/metabolism , CHO Cells , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Complement C3a , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Humans , Macaca fascicularis , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Rats , Receptors, Complement/antagonists & inhibitors , Receptors, Complement/genetics , Transfection , U937 Cells , beta-Lactamases/genetics , beta-Lactamases/metabolism
9.
Mol Pharmacol ; 67(6): 1834-9, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15755909

ABSTRACT

The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor whose function in vivo has been incompletely characterized. One of the reasons is that its current known ligands, prostaglandin D(2) and some of its metabolites, have either poor selectivity for CRTH2 or are metabolically unstable in vivo. In this study, we describe the biological and pharmacological properties of L-888,607, the first synthetic potent and selective CRTH2 agonist. We show that L-888,607 exhibits 1) subnanomolar affinity for the human CRTH2 receptor, 2) high selectivity over all other prostanoid receptors and other receptors tested, 3) agonistic activity on recombinant and endogenously expressed CRTH2 receptor, and 4) relative stability in vivo. L-888,607 thus represents a suitable tool to investigate the in vivo function of CRTH2.


Subject(s)
Acetates/pharmacology , Heterocyclic Compounds, 3-Ring/pharmacology , Receptors, Immunologic/agonists , Receptors, Prostaglandin/agonists , Acetates/chemistry , Acetates/metabolism , Animals , Chemotaxis, Leukocyte/drug effects , Chemotaxis, Leukocyte/physiology , Eosinophils/drug effects , Eosinophils/metabolism , Eosinophils/physiology , Heterocyclic Compounds, 3-Ring/chemistry , Heterocyclic Compounds, 3-Ring/metabolism , Humans , Indomethacin/analogs & derivatives , Indomethacin/pharmacology , Ligands , Male , Mice , Mice, Inbred ICR , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism
10.
Prostaglandins Other Lipid Mediat ; 73(1-2): 87-101, 2004 Jan.
Article in English | MEDLINE | ID: mdl-15165034

ABSTRACT

BACKGROUND: Prostaglandin D2 (PGD2) is released from mast cells during the allergic response. OBJECTIVE: Since PGD2 has been shown to induce nasal congestion in humans, we investigated the distribution of hematopoietic prostaglandin D synthase (PGDS) and the two PGD2 receptors, DP and CRTH2 in human nasal mucosa from healthy subjects and subjects suffering from polyposis, a severe form of chronic rhinosinusitis. METHODS: DP mRNA expression was detected by in situ hybridization while PGDS, CRTH2 and various leukocyte markers expression were revealed by immunohistochemistry. RESULTS: In the normal mucosa, PGDS was only detected in few resident mast cells while CRTH2 was undetectable. In contrast, DP receptor mRNA was detected in epithelial goblet cells, serous glands and in the vasculature. In the nasal mucosa of subjects suffering from polyposis: (1) PGDS was detected in mast cells and other large infiltrating inflammatory cells, (2) both DP mRNA and CRTH2 were detected in eosinophils and (3) CRTH2 was detected on a subset of infiltrating T cells. Although DP mRNA could not be detected in the T cells invading the nasal mucosa, it was found to be expressed in the T cells present in the lymph node and the thymus from normal individuals. CONCLUSION: This study indicates that cells capable of producing PGD2 are present in the nasal mucosa and that both PGD2 receptors, DP and CRTH2, might play a role in inflammatory disease of the upper airways.


Subject(s)
Intramolecular Oxidoreductases/biosynthesis , Mast Cells/metabolism , Nasal Mucosa/metabolism , Nasal Polyps/metabolism , Receptors, Immunologic/biosynthesis , Receptors, Prostaglandin/biosynthesis , Adult , Aged , Eosinophils/metabolism , Female , Gene Expression Regulation , Humans , Hypersensitivity/metabolism , Hypersensitivity/pathology , In Situ Hybridization , Inflammation/metabolism , Inflammation/pathology , Lipocalins , Lymph Nodes/cytology , Lymph Nodes/metabolism , Male , Middle Aged , Nasal Mucosa/cytology , Nasal Mucosa/pathology , Nasal Polyps/pathology , RNA, Messenger/biosynthesis , T-Lymphocytes/metabolism , Thymus Gland/cytology , Thymus Gland/metabolism
11.
Br J Pharmacol ; 137(8): 1163-72, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12466225

ABSTRACT

1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)>13,14-dihydro-15-keto PGD(2)>15-deoxy-Delta(12,14)-PGJ(2)>PGJ(2)>Delta(12)-PGJ(2)>15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)>PGJ(2)>Delta(12)-PGJ(2)>15-deoxy-Delta(12,14)-PGJ(2) >>>13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.


Subject(s)
Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Binding, Competitive/drug effects , Binding, Competitive/physiology , Cell Line , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , HL-60 Cells , Humans , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , Receptors, Immunologic/agonists , Receptors, Immunologic/biosynthesis , Receptors, Prostaglandin/agonists , Receptors, Prostaglandin/biosynthesis , Receptors, Prostaglandin/physiology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Transfection
12.
Nat Cell Biol ; 4(2): 95-105, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11788820

ABSTRACT

In Huntington disease, polyglutamine expansion of the protein huntingtin (Htt) leads to selective neurodegenerative loss of medium spiny neurons throughout the striatum by an unknown apoptotic mechanism. Binding of Hip-1, a protein normally associated with Htt, is reduced by polyglutamine expansion. Free Hip-1 binds to a hitherto unknown polypeptide, Hippi (Hip-1 protein interactor), which has partial sequence homology to Hip-1 and similar tissue and subcellular distribution. The availability of free Hip-1 is modulated by polyglutamine length within Htt, with disease-associated polyglutamine expansion favouring the formation of pro-apoptotic Hippi-Hip-1 heterodimers. This heterodimer can recruit procaspase-8 into a complex of Hippi, Hip-1 and procaspase-8, and launch apoptosis through components of the 'extrinsic' cell-death pathway. We propose that Htt polyglutamine expansion liberates Hip-1 so that it can form a caspase-8 recruitment complex with Hippi. This novel non-receptor-mediated pathway for activating caspase-8 might contribute to neuronal death in Huntington disease.


Subject(s)
Carrier Proteins/metabolism , Caspases/metabolism , DNA-Binding Proteins , Huntington Disease/metabolism , Neurons/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Apoptosis/physiology , Carrier Proteins/genetics , Caspase 8 , Caspase 9 , Caspases/genetics , Cells, Cultured , Enzyme Activation , Humans , Huntingtin Protein , Huntington Disease/enzymology , Mice , Models, Molecular , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/ultrastructure , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Structure, Tertiary , Rats , Sequence Alignment , Tissue Distribution , Two-Hybrid System Techniques
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