134 results on '"Sarau HM"'
Search Results
2. Pharmacological characterization of GSK573719 (umeclidinium): a novel, long-acting, inhaled antagonist of the muscarinic cholinergic receptors for treatment of pulmonary diseases.
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Salmon M, Luttmann MA, Foley JJ, Buckley PT, Schmidt DB, Burman M, Webb EF, DeHaas CJ, Kotzer CJ, Barrett VJ, Slack RJ, Sarau HM, Palovich MR, Lainé DI, Hay DW, and Rumsey WL
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- Administration, Inhalation, Animals, CHO Cells, Calcium metabolism, Carbachol pharmacology, Cholinergic Antagonists pharmacology, Cricetinae, Cricetulus, Guinea Pigs, Kinetics, Lung drug effects, Mice, Mice, Inbred BALB C, Muscarinic Agonists pharmacology, Muscarinic Antagonists administration & dosage, Plethysmography, Quinuclidines administration & dosage, Receptor, Muscarinic M3 drug effects, Receptors, Muscarinic, Scopolamine Derivatives pharmacology, Tiotropium Bromide, Lung Diseases drug therapy, Muscarinic Antagonists therapeutic use, Quinuclidines therapeutic use
- Abstract
Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 μg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 μg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.
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- 2013
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3. Tyrosine urea muscarinic acetylcholine receptor antagonists: achiral quaternary ammonium groups.
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Jin Q, Davis RS, Bullion AM, Jin J, Wang Y, Widdowson KL, Palovich MR, Foley JJ, Schmidt DB, Buckley PT, Webb EF, Salmon M, Belmonte KE, Sarau HM, and Busch-Petersen J
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- Animals, Bronchi drug effects, Mice, Muscarinic Antagonists chemical synthesis, Muscarinic Antagonists pharmacology, Receptors, Muscarinic metabolism, Structure-Activity Relationship, Urea chemical synthesis, Urea pharmacology, Muscarinic Antagonists chemistry, Quaternary Ammonium Compounds chemistry, Receptors, Muscarinic chemistry, Tyrosine chemistry, Urea analogs & derivatives
- Abstract
Tyrosine ureas had been identified as potent muscarinic receptor antagonists with promising in vivo activity. Controlling the stereochemistry of the chiral quaternary ammonium center had proved to be a serious issue for this series, however. Herein we describe the preparation and SAR of tyrosine urea antagonists containing achiral quaternary ammonium centers. The most successful such moiety was the 2-methylimidazo[2,1-b][1,3]thiazol-7-ium group which yielded highly potent antagonists with long duration of action in an inhaled animal model of bronchoconstriction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
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- 2012
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4. Design, synthesis and structure-activity relationship of N-substituted tropane muscarinic acetylcholine receptor antagonists.
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Lainé DI, Yan H, Xie H, Davis RS, Dufour J, Widdowson KL, Palovich MR, Wan Z, Foley JJ, Schmidt DB, Hunsberger GE, Burman M, Bacon AM, Webb EF, Luttmann MA, Salmon M, Sarau HM, Umbrecht ST, Landis PS, Peck BJ, and Busch-Petersen J
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- Animals, Bronchial Diseases drug therapy, Drug Design, Mice, Muscarinic Antagonists pharmacology, Receptors, Muscarinic drug effects, Structure-Activity Relationship, Tropanes pharmacology, Muscarinic Antagonists chemical synthesis, Tropanes chemical synthesis
- Abstract
A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24 displayed much slower reversibility at mAChRs than the methyl-substituted parent molecule darotropium. In addition, it was shown that this characteristic appeared to translate into enhanced which duration of action in a mouse model of bronchonstriction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
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- 2012
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5. Design, synthesis, and structure-activity relationship of tropane muscarinic acetylcholine receptor antagonists.
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Lainé DI, Wan Z, Yan H, Zhu C, Xie H, Fu W, Busch-Petersen J, Neipp C, Davis R, Widdowson KL, Blaney FE, Foley J, Bacon AM, Webb EF, Luttmann MA, Burman M, Sarau HM, Salmon M, Palovich MR, and Belmonte K
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- Animals, Biological Availability, Biphenyl Compounds chemistry, Biphenyl Compounds pharmacology, Bridged Bicyclo Compounds, Heterocyclic chemistry, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Bronchi drug effects, Bronchi physiology, Bronchoconstriction drug effects, CHO Cells, Calcium metabolism, Cricetinae, Cricetulus, Drug Design, Humans, In Vitro Techniques, Mice, Mice, Inbred BALB C, Muscarinic Antagonists chemistry, Muscarinic Antagonists pharmacology, Muscle Contraction, Muscle, Smooth drug effects, Muscle, Smooth physiology, Radioligand Assay, Rats, Receptor, Muscarinic M1 physiology, Receptor, Muscarinic M2 physiology, Receptor, Muscarinic M3 physiology, Stereoisomerism, Structure-Activity Relationship, Tropanes chemistry, Tropanes pharmacology, Biphenyl Compounds chemical synthesis, Bridged Bicyclo Compounds, Heterocyclic chemical synthesis, Muscarinic Antagonists chemical synthesis, Tropanes chemical synthesis
- Abstract
Novel tropane derivatives were characterized as muscarinic acetylcholine receptor antagonists (mAChRs). Through optimization of the structure-activity relationship around the tropane scaffold, the quaternary ammonium salt 34 was identified as a very potent M(3) mAChR antagonist. The compound was functionally active and displayed greater than 24 h duration of action in a mouse model of bronchoconstriction.
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- 2009
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6. Discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide as an efficacious inhaled muscarinic acetylcholine receptor antagonist for the treatment of COPD.
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Wan Z, Laine DI, Yan H, Zhu C, Widdowson KL, Buckley PT, Burman M, Foley JJ, Sarau HM, Schmidt DB, Webb EF, Belmonte KE, and Palovich M
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- Administration, Inhalation, Animals, Biphenyl Compounds chemical synthesis, Biphenyl Compounds pharmacokinetics, Bridged Bicyclo Compounds, Heterocyclic chemical synthesis, Bridged Bicyclo Compounds, Heterocyclic pharmacokinetics, Drug Discovery, Humans, Mice, Muscarinic Antagonists chemical synthesis, Muscarinic Antagonists pharmacokinetics, Rats, Receptors, Muscarinic metabolism, Structure-Activity Relationship, Biphenyl Compounds chemistry, Bridged Bicyclo Compounds, Heterocyclic chemistry, Muscarinic Antagonists chemistry, Pulmonary Disease, Chronic Obstructive drug therapy, Receptors, Muscarinic chemistry
- Abstract
Design and syntheses of a novel series of muscarinic antagonists are reported. These efforts have culminated in the discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide (4a) as a potent and pan-active muscarinic antagonist as well as a functionally active compound in a murine model of bronchoconstriction. The compound has also displayed pharmacokinetic characteristics suitable for inhaled delivery.
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- 2009
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7. M3 muscarinic acetylcholine receptor antagonists: SAR and optimization of bi-aryl amines.
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Budzik B, Wang Y, Shi D, Wang F, Xie H, Wan Z, Zhu C, Foley JJ, Nuthulaganti P, Kallal LA, Sarau HM, Morrow DM, Moore ML, Rivero RA, Palovich M, Salmon M, Belmonte KE, Laine DI, and Jin J
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- Amides chemistry, Amines pharmacology, Asthma drug therapy, Drug Design, Electrons, Humans, Inhibitory Concentration 50, Kinetics, Models, Chemical, Molecular Structure, Pulmonary Disease, Chronic Obstructive drug therapy, Receptor, Muscarinic M3 chemistry, Structure-Activity Relationship, Amines chemical synthesis, Chemistry, Pharmaceutical methods, Receptor, Muscarinic M3 antagonists & inhibitors
- Abstract
Exploration of multiple regions of a bi-aryl amine template led to the identification of highly potent M(3) muscarinic acetylcholine receptor antagonists such as 14 (pA(2)=11.0) possessing good sub-type selectivity for M(3) over M(2). The structure-activity relationships (SAR) and optimization of the bi-aryl amine series are described.
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- 2009
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8. Camphor sulfonamide derivatives as novel, potent and selective CXCR3 antagonists.
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Wang Y, Busch-Petersen J, Wang F, Kiesow TJ, Graybill TL, Jin J, Yang Z, Foley JJ, Hunsberger GE, Schmidt DB, Sarau HM, Capper-Spudich EA, Wu Z, Fisher LS, McQueney MS, Rivero RA, and Widdowson KL
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- Camphor chemical synthesis, Camphor pharmacology, Humans, Piperazines, Structure-Activity Relationship, Sulfonamides pharmacology, Camphor analogs & derivatives, Receptors, CXCR3 antagonists & inhibitors, Sulfonamides chemical synthesis
- Abstract
A series of N-arylpiperazine camphor sulfonamides was discovered as novel CXCR3 antagonists. The synthesis, structure-activity relationships, and optimization of the initial hit that resulted in the identification of potent and selective CXCR3 antagonists are described.
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- 2009
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9. Muscarinic acetylcholine receptor antagonists: SAR and optimization of tyrosine ureas.
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Jin J, Wang Y, Shi D, Wang F, Fu W, Davis RS, Jin Q, Foley JJ, Sarau HM, Morrow DM, Moore ML, Rivero RA, Palovich M, Salmon M, Belmonte KE, and Busch-Petersen J
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- Asthma drug therapy, Drug Design, Humans, Inhibitory Concentration 50, Models, Chemical, Molecular Structure, Muscarinic Antagonists pharmacology, Salts chemistry, Structure-Activity Relationship, Chemistry, Pharmaceutical methods, Muscarinic Antagonists chemical synthesis, Receptors, Muscarinic chemistry, Tyrosine chemistry, Urea chemistry
- Abstract
SAR exploration of multiple regions of a tyrosine urea template led to the identification of very potent muscarinic acetylcholine receptor antagonists such as 10b with good subtype selectivity for M(3) over M(1). The structure-activity relationships (SAR) and optimization of the tyrosine urea series are described.
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- 2008
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10. Discovery of biphenyl piperazines as novel and long acting muscarinic acetylcholine receptor antagonists.
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Jin J, Budzik B, Wang Y, Shi D, Wang F, Xie H, Wan Z, Zhu C, Foley JJ, Webb EF, Berlanga M, Burman M, Sarau HM, Morrow DM, Moore ML, Rivero RA, Palovich M, Salmon M, Belmonte KE, and Lainé DI
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- Administration, Intranasal, Animals, Bronchial Provocation Tests, Bronchoconstrictor Agents pharmacology, Bronchodilator Agents chemical synthesis, Bronchodilator Agents chemistry, Disease Models, Animal, Drug Evaluation, Preclinical, Methacholine Chloride pharmacology, Mice, Molecular Structure, Piperazines chemical synthesis, Piperazines chemistry, Stereoisomerism, Structure-Activity Relationship, Bronchoconstriction drug effects, Bronchodilator Agents pharmacology, Piperazines pharmacology, Receptors, Muscarinic drug effects
- Abstract
A series of novel biphenyl piperazines was discovered as highly potent muscarinic acetylcholine receptor antagonists via high throughput screening and subsequent optimization. Compound 5c with respective 500- and 20-fold subtype selectivity for M3 over M2 and M1 exhibited excellent inhibitory activity and long duration of action in a bronchoconstriction in vivo model in mice via intranasal administration. The novel inhaled mAChR antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease.
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- 2008
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11. Discovery of novel and long acting muscarinic acetylcholine receptor antagonists.
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Jin J, Wang Y, Shi D, Wang F, Davis RS, Jin Q, Fu W, Foley JJ, Webb EF, Dehaas CJ, Berlanga M, Burman M, Sarau HM, Morrow DM, Rao P, Kallal LA, Moore ML, Rivero RA, Palovich M, Salmon M, Belmonte KE, and Busch-Petersen J
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- Animals, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Bronchoconstriction drug effects, Drug Evaluation, Preclinical methods, Guinea Pigs, Mice, Rats, Tyrosine pharmacology, Muscarinic Antagonists pharmacology, Phenylurea Compounds pharmacology, Quaternary Ammonium Compounds pharmacology, Tyrosine analogs & derivatives
- Abstract
High throughput screening and subsequent optimization led to the discovery of novel quaternary ammonium salts as highly potent muscarinic acetylcholine receptor antagonists with excellent selectivity. Compounds 8a, 13a, and 13b showed excellent inhibitory activity and long duration of action in bronchoconstriction in vivo models in two species via intranasal or intratracheal administration. The novel inhaled muscarinic receptor antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease and other bronchoconstriction disorders.
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- 2008
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12. Urotensin-II receptor antagonists: synthesis and SAR of N-cyclic azaalkyl benzamides.
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Jin J, An M, Sapienza A, Aiyar N, Naselsky D, Sarau HM, Foley JJ, Salyers KL, Knight SD, Keenan RM, Rivero RA, Dhanak D, and Douglas SA
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- Binding Sites, Chemistry, Pharmaceutical methods, Diamines chemistry, Drug Design, Humans, Inhibitory Concentration 50, Kinetics, Models, Chemical, Structure-Activity Relationship, Benzamides chemistry, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
SAR exploration of the central diamine, benzyl, and terminal aminoalkoxy regions of the N-cyclic azaalkyl benzamide series led to the identification of very potent human urotensin-II receptor antagonists such as 1a with a K(i) of 4 nM. The synthesis and structure-activity relationships (SAR) of N-cyclic azaalkyl benzamides are described.
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- 2008
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13. 3-Arylamino-2H-1,2,4-benzothiadiazin-5-ol 1,1-dioxides as novel and selective CXCR2 antagonists.
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Wang Y, Busch-Petersen J, Wang F, Ma L, Fu W, Kerns JK, Jin J, Palovich MR, Shen JK, Burman M, Foley JJ, Schmidt DB, Hunsberger GE, Sarau HM, and Widdowson KL
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- Benzothiadiazines chemistry, Structure-Activity Relationship, Benzothiadiazines pharmacology, Receptors, Interleukin-8B antagonists & inhibitors
- Abstract
A series of 3-arylamino-2H-1,2,4-benzothiadiazin-5-ol 1,1-dioxides were prepared and shown to be novel and selective antagonists of the CXCR2 receptor. Synthesis, structure and activity relationships, selectivity, and some developability properties are described.
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- 2007
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14. Comparison of N,N'-diarylsquaramides and N,N'-diarylureas as antagonists of the CXCR2 chemokine receptor.
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McCleland BW, Davis RS, Palovich MR, Widdowson KL, Werner ML, Burman M, Foley JJ, Schmidt DB, Sarau HM, Rogers M, Salyers KL, Gorycki PD, Roethke TJ, Stelman GJ, Azzarano LM, Ward KW, and Busch-Petersen J
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- Animals, CHO Cells, Chemical Phenomena, Chemistry, Physical, Cricetinae, Cricetulus, Indicators and Reagents, Mass Spectrometry, Phenols chemistry, Rats, Rats, Sprague-Dawley, Structure-Activity Relationship, Sulfonamides chemistry, Cyclobutanes chemical synthesis, Cyclobutanes pharmacokinetics, Cyclobutanes pharmacology, Receptors, Interleukin-8B antagonists & inhibitors, Urea analogs & derivatives, Urea chemical synthesis, Urea pharmacokinetics, Urea pharmacology
- Abstract
N,N'-diarylsquaramides were prepared and evaluated as antagonists of CXCR2. The compounds were found to be potent and selective antagonists of CXCR2. Significant differences in SAR was observed relative to the previously described N,N'-diarylurea series. As was the case in the N,N'-diarylurea series, placing sulfonamide substituent adjacent to the acidic phenol significantly reduced the clearance in rat pharmacokinetic studies.
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- 2007
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15. Oxazolidinones as novel human CCR8 antagonists.
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Jin J, Wang Y, Wang F, Kerns JK, Vinader VM, Hancock AP, Lindon MJ, Stevenson GI, Morrow DM, Rao P, Nguyen C, Barrett VJ, Browning C, Hartmann G, Andrew DP, Sarau HM, Foley JJ, Jurewicz AJ, Fornwald JA, Harker AJ, Moore ML, Rivero RA, Belmonte KE, and Connor HE
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- Animals, CHO Cells, Chemotaxis, Leukocyte drug effects, Computer Simulation, Cricetinae, Cricetulus, Drug Evaluation, Preclinical, ERG1 Potassium Channel, Ether-A-Go-Go Potassium Channels antagonists & inhibitors, Humans, Indicators and Reagents, Myotonin-Protein Kinase, Protein Serine-Threonine Kinases drug effects, Receptors, CCR8, Structure-Activity Relationship, Th2 Cells drug effects, Oxazolidinones chemical synthesis, Oxazolidinones pharmacology, Receptors, Chemokine antagonists & inhibitors
- Abstract
High-throughput screening of the corporate compound collection led to the discovery of a novel series of N-substituted-5-aryl-oxazolidinones as potent human CCR8 antagonists. The synthesis, structure-activity relationships, and optimization of the series that led to the identification of SB-649701 (1a), are described.
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- 2007
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16. A novel flow cytometric assay of human whole blood neutrophil and monocyte CD11b levels: upregulation by chemokines is related to receptor expression, comparison with neutrophil shape change, and effects of a chemokine receptor (CXCR2) antagonist.
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Nicholson GC, Tennant RC, Carpenter DC, Sarau HM, Kon OM, Barnes PJ, Salmon M, Vessey RS, Tal-Singer R, and Hansel TT
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- Antigens, CD analysis, Cell Shape, Chemokine CXCL11, Chemokines, CXC metabolism, Dose-Response Relationship, Drug, GPI-Linked Proteins, Humans, Interleukin-8 metabolism, Lipopolysaccharide Receptors analysis, Monocytes cytology, Monocytes metabolism, Neutrophils cytology, Neutrophils metabolism, Pulmonary Disease, Chronic Obstructive blood, Pulmonary Disease, Chronic Obstructive diagnosis, Pulmonary Disease, Chronic Obstructive immunology, Receptors, IgG analysis, Receptors, Interleukin-8B antagonists & inhibitors, Receptors, Interleukin-8B metabolism, Reproducibility of Results, Up-Regulation drug effects, CD11b Antigen analysis, Flow Cytometry methods, Monocytes immunology, Neutrophils immunology
- Abstract
Rationale: Smokers who develop chronic obstructive pulmonary disease (COPD) have amplified inflammation within their lungs, involving selective tissue accumulation of neutrophils, macrophages and CD8+ T cells. CD11b (Mac-1, alphaMbeta(2)-integrin) is both a complement receptor (CR3) and a cell adhesion molecule present on the surface of peripheral blood leukocytes, and undergoes rapid surface upregulation from preformed cytoplasmic stores on activation. Cellular activation can also trigger chemotaxis and shape change, the activation itself being caused by the binding of chemokines to cell surface receptors., Methods: We developed a method of whole blood flow cytometry to measure neutrophil and monocyte CD11b upregulation on CD16+ and CD14+ cells, employing staining with the nuclear dye LDS-751 immediately before flow cytometry. In addition we assessed neutrophil shape change by modified gated autofluorescence with forward scatter (GAFS), this being correlated with chemotactic responses., Results: In smokers with COPD there was a lower maximal shape change for neutrophils in response to CXCL8 (IL-8) in comparison to healthy smokers (p=0.025), and a trend for lower expression of CD11b and shape change in response to CXCL1 (GRO-alpha). Neutrophils were found to predominantly express chemokine receptors CXCR1 and CXCR2 and respond to CXCL8 with CD11b upregulation, while monocytes express more CCR2 and upregulate CD11b preferentially to CCL2 (MCP-1). A CXCR2 antagonist (SB-656933) was found to inhibit neutrophil CD11b upregulation (IC50=260.7nM) and shape change (IC50=310.5nM) in COPD patients., Conclusions: Neutrophils and monocytes participate in inflammatory processes in a range of diseases. These whole blood assays can be employed to monitor activity in disease and perform in vitro and ex vivo assessment of chemokine receptor (CXCR) antagonists.
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- 2007
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17. N,N'-Diarylcyanoguanidines as antagonists of the CXCR2 and CXCR1 chemokine receptors.
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Nie H, Widdowson KL, Palovich MR, Fu W, Elliott JD, Bryan DL, Burman M, Schmidt DB, Foley JJ, Sarau HM, and Busch-Petersen J
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- Guanidines chemistry, Humans, Guanidines pharmacology, Receptors, Interleukin-8A antagonists & inhibitors, Receptors, Interleukin-8B antagonists & inhibitors
- Abstract
A series of N-(2-hydroxy-3-sulfonamidobenzene)-N'-arylcyanoguanidines was prepared. In general, these compounds proved to be potent antagonists of CXCR2 while the selectivity versus CXCR1 ranged from non-selective to >200-fold.
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- 2006
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18. The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems.
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Behm DJ, Stankus G, Doe CP, Willette RN, Sarau HM, Foley JJ, Schmidt DB, Nuthulaganti P, Fornwald JA, Ames RS, Lambert DG, Calo' G, Camarda V, Aiyar NV, and Douglas SA
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- Animals, Arteries drug effects, Arteries physiology, Binding, Competitive drug effects, Blood Pressure drug effects, Calcium metabolism, Cats, Cell Line, Dose-Response Relationship, Drug, Haplorhini, Humans, In Vitro Techniques, Male, Peptide Fragments metabolism, Peptides, Cyclic chemistry, Radioligand Assay, Rats, Rats, Sprague-Dawley, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins metabolism, Urotensins metabolism, Vasoconstriction drug effects, Vasoconstrictor Agents pharmacology, Peptide Fragments pharmacology, Peptides, Cyclic pharmacology, Receptors, G-Protein-Coupled antagonists & inhibitors, Urotensins pharmacology
- Abstract
Several peptidic urotensin-II (UT) receptor antagonists exert 'paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4-11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)>>GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human>>rat UT receptor). The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. In contrast to the 'low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5-fold less than seen with urantide). Since GSK248451 (1 mg kg(-1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.
- Published
- 2006
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19. Aminoalkoxybenzyl pyrrolidines as novel human urotensin-II receptor antagonists.
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Jin J, Dhanak D, Knight SD, Widdowson K, Aiyar N, Naselsky D, Sarau HM, Foley JJ, Schmidt DB, Bennett CD, Wang B, Warren GL, Moore ML, Keenan RM, Rivero RA, and Douglas SA
- Subjects
- Drug Evaluation, Preclinical, Humans, Pyrrolidines pharmacology, Stereoisomerism, Structure-Activity Relationship, Pyrrolidines chemical synthesis, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
High throughput screening of the corporate compound collection led to the discovery of a novel series of substituted aminoalkoxybenzyl pyrrolidines as human urotensin-II receptor antagonists. The synthesis, initial structure-activity relationships, and optimization of the initial hit that led to the identification of a truncated sub-series, represented by SB-436811 (1a), are described.
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- 2005
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20. Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375.
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Douglas SA, Behm DJ, Aiyar NV, Naselsky D, Disa J, Brooks DP, Ohlstein EH, Gleason JG, Sarau HM, Foley JJ, Buckley PT, Schmidt DB, Wixted WE, Widdowson K, Riley G, Jin J, Gallagher TF, Schmidt SJ, Ridgers L, Christmann LT, Keenan RM, Knight SD, and Dhanak D
- Subjects
- Algorithms, Animals, Aorta, Thoracic drug effects, Binding, Competitive drug effects, Cats, Cell Line, Tumor, Cell Membrane metabolism, Haplorhini, Humans, In Vitro Techniques, Mice, Muscle Contraction drug effects, Muscle, Smooth, Vascular drug effects, Radioligand Assay, Rats, Recombinant Proteins metabolism, Rhabdomyosarcoma metabolism, Species Specificity, Pyrrolidines pharmacology, Receptors, G-Protein-Coupled antagonists & inhibitors, Receptors, G-Protein-Coupled genetics, Sulfonamides pharmacology
- Abstract
1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and 'native' UT receptors (K(i) 4.7+/-1.5 to 20.7+/-3.6 nM at rodent, feline and primate recombinant UT receptors and K(i) 5.4+/-0.4 nM at the endogenous UT receptor in SJRH30 cells). 2. Prior exposure to SB-706375 (1 microM, 30 min) did not alter [(125)I]hU-II binding affinity or density in recombinant cells (K(D) 3.1+/-0.4 vs 5.8+/-0.9 nM and B(max) 3.1+/-1.0 vs 2.8+/-0.8 pmol mg(-1)) consistent with a reversible mode of action. 3. The novel, nonpeptidic radioligand [(3)H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (K(D) 2.6+/-0.4 nM, B(max) 0.86+/-0.12 pmol mg(-1)) in a manner that was inhibited by both U-II isopeptides and SB-706375 (K(i) 4.6+/-1.4 to 17.6+/-5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. 4. SB-706375 was a potent, competitive hU-II antagonist across species with pK(b) 7.29-8.00 in HEK293-UT receptor cells (inhibition of [Ca(2+)](i)-mobilization) and pK(b) 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (K(app) approximately 20 nM). 5. SB-706375 was a selective U-II antagonist with >/=100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (K(i)/IC(50)>1 microM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 microM). 6. In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals.
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- 2005
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21. Cloning and pharmacological characterization of the cat urotensin-II receptor (UT).
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Aiyar N, Johns DG, Ao Z, Disa J, Behm DJ, Foley JJ, Buckley PT, Sarau HM, van-der-Keyl HK, Elshourbagy NA, and Douglas SA
- Subjects
- Amino Acid Sequence, Animals, Calcium Signaling physiology, Cats, Cell Line, Cell Membrane physiology, Conserved Sequence, DNA Primers, Humans, Inositol Phosphates metabolism, Mice, Molecular Sequence Data, Organ Specificity, Polymerase Chain Reaction, RNA, Messenger genetics, Rats, Receptors, G-Protein-Coupled drug effects, Receptors, G-Protein-Coupled metabolism, Recombinant Proteins drug effects, Recombinant Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Transfection, Cloning, Molecular, Receptors, G-Protein-Coupled genetics
- Abstract
Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds. The purpose of this study was to clone and pharmacologically characterize cat recombinant UT to determine whether this system represents a model for characterizing UT antagonists. Cloned cat UT displayed 74% identity to primate UT, and 77% identity to rodent UT. [(125)I] hU-II bound in a saturable manner to a single site on recombinant cat UT with high affinity (K(D) 288+/-13pM) and high density (B(max) 747+/-66fmol/mg protein). U-II isopeptides displayed equipotent, high affinity binding to cat UT (K(i) 1.8-5.3nM). Cat UT was coupled to intracellular [Ca(2+)] release (EC(50) 0.6+/-0.2nM) and total inositol phosphate (IP) formation (EC(50) 0.4+/-0.1nM). Protein kinase C activation desensitized cat, but not human, UT-mediated IP formation. UT mRNA expression was detected in cat blood vessels, trachea, lung, and kidney, where the medulla (K(D) 815+/-34) and cortex and (K(D) 316+/-39pM) displayed high affinity binding for human U-II (hU-II). The cat urotensin-II receptor represents a suitable in vitro model to examine the role of the U-II/UT system in the etiology of hypertension, assisting in the evaluation of the UT antagonists to help treat cardiovascular disease.
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- 2005
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22. Neuromedin U elicits cytokine release in murine Th2-type T cell clone D10.G4.1.
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Johnson EN, Appelbaum ER, Carpenter DC, Cox RF, Disa J, Foley JJ, Ghosh SK, Naselsky DP, Pullen MA, Sarau HM, Scheff SR, Steplewski KM, Zaks-Zilberman M, and Aiyar N
- Subjects
- Animals, Calcineurin physiology, Calcium metabolism, Cell Line, Clone Cells, Cytokines antagonists & inhibitors, Dogs, Estrenes pharmacology, Humans, Interleukins antagonists & inhibitors, Interleukins metabolism, MAP Kinase Signaling System immunology, Membrane Proteins biosynthesis, Membrane Proteins genetics, Mice, Mitogen-Activated Protein Kinases metabolism, Neuropeptides metabolism, Phosphatidylinositol 3-Kinases physiology, Pyrrolidinones pharmacology, Rats, Receptors, Interleukin-4 physiology, Receptors, Neurotransmitter biosynthesis, Receptors, Neurotransmitter genetics, Signal Transduction immunology, Swine, Type C Phospholipases antagonists & inhibitors, Type C Phospholipases physiology, Cytokines metabolism, Membrane Proteins physiology, Neuropeptides physiology, Receptors, Neurotransmitter physiology, Th2 Cells immunology, Th2 Cells metabolism
- Abstract
Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for (125)I-labeled NmU (K(D) 364 pM and B(max) 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca(2+) concentration (EC(50) 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.
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- 2004
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23. Localisation of NMU1R and NMU2R in human and rat central nervous system and effects of neuromedin-U following central administration in rats.
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Gartlon J, Szekeres P, Pullen M, Sarau HM, Aiyar N, Shabon U, Michalovich D, Steplewski K, Ellis C, Elshourbagy N, Duxon M, Ashmeade TE, Harrison DC, Murdock P, Wilson S, Ennaceur A, Atkins A, Heidbreder C, Hagan JJ, Hunter AJ, and Jones DN
- Subjects
- Animals, Cell Line, Dose-Response Relationship, Drug, Humans, Injections, Intraventricular, Rats, Rats, Sprague-Dawley, Swine, Central Nervous System drug effects, Central Nervous System metabolism, Membrane Proteins agonists, Membrane Proteins metabolism, Neuropeptides administration & dosage, Receptors, Neurotransmitter agonists, Receptors, Neurotransmitter metabolism
- Abstract
Rationale: Neuromedin-U (NmU) is an agonist at NMU1R and NMU2R. The brain distribution of NmU and its receptors, in particular NMU2R, suggests widespread central roles for NmU. In agreement, centrally administered NmU affects feeding behaviour, energy expenditure and pituitary output. Further central nervous system (CNS) roles for NmU warrant investigation., Objectives: To investigate the CNS role of NmU by mapping NMU1R and NMU2R mRNA and measuring the behavioural, endocrine, neurochemical and c-fos response to intracerebroventricular (i.c.v.) NmU., Methods: Binding affinity and functional potency of rat NmU was determined at human NMU1R and NMU2R. Expression of NMU1R and NMU2R mRNA in rat and human tissue was determined using semi-quantitative reverse-transcription polymerase chain reaction. In in-vivo studies, NmU was administered i.c.v. to male Sprague-Dawley rats, and changes in grooming, motor activity and pre-pulse inhibition (PPI) were assessed. In further studies, plasma endocrine hormones, [DOPAC + HVA]/[dopamine] and [5-HIAA]/[5-HT] ratios and levels of Fos-like immunoreactivity (FLI) were measured 20 min post-NmU (i.c.v.)., Results: NmU bound to NMU1R ( K(I), 0.11+/-0.02 nM) and NMU2R ( K(I), 0.21+/-0.05 nM) with equal affinity and was equally active at NMU1R (EC(50), 1.25+/-0.05 nM) and NMU2R (EC(50), 1.10+/-0.20 nM) in a functional assay. NMU2R mRNA expression was found at the highest levels in the CNS regions of both rat and human tissues. NMU1R mRNA expression was restricted to the periphery of both species with the exception of the rat amygdala. NmU caused a marked increase in grooming and motor activity but did not affect PPI. Further, NmU decreased plasma prolactin but did not affect levels of corticosterone, luteinising hormone or thyroid stimulating hormone. NmU elevated levels of 5-HT in the frontal cortex and hypothalamus, with decreased levels of its metabolites in the hippocampus and hypothalamus, but did not affect dopamine function. NmU markedly increased FLI in the nucleus accumbens, frontal cortex and central amygdala., Conclusions: These data provide further evidence for widespread roles for NmU and its receptors in the brain.
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- 2004
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24. Discovery of potent and orally bioavailable N,N'-diarylurea antagonists for the CXCR2 chemokine receptor.
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Jin Q, Nie H, McCleland BW, Widdowson KL, Palovich MR, Elliott JD, Goodman RM, Burman M, Sarau HM, Ward KW, Nord M, Orr BM, Gorycki PD, and Busch-Petersen J
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- Administration, Oral, Animals, Biological Availability, Humans, Microsomes, Liver drug effects, Microsomes, Liver metabolism, Protein Binding drug effects, Protein Binding physiology, Rats, Rats, Sprague-Dawley, Receptors, Interleukin-8B metabolism, Sulfonylurea Compounds metabolism, Sulfonylurea Compounds pharmacology, Receptors, Interleukin-8B antagonists & inhibitors, Sulfonylurea Compounds chemistry
- Abstract
A series of 3-substituted N,N'-diarylureas was prepared and the structure-activity relationship relative to CXCR2 receptor affinity as well as their pharmacokinetic properties were examined. In vitro microsomal metabolism studies indicated that the lower clearance rates of the 3-sulfonamido-substituted compounds were most likely due to the suppression of glucuronidation.
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- 2004
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25. A coding polymorphism in the CYSLT2 receptor with reduced affinity to LTD4 is associated with asthma.
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Pillai SG, Cousens DJ, Barnes AA, Buckley PT, Chiano MN, Hosking LK, Cameron LA, Fling ME, Foley JJ, Green A, Sarau HM, Schmidt DB, Sprankle CS, Blumenthal MN, Vestbo J, Kennedy-Wilson K, Wixted WE, Wagner MJ, Anderson WH, and Ignar DM
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- Adolescent, Adult, Alleles, Cell Line, Child, Child, Preschool, Cloning, Molecular, Family Health, Genetic Variation, Genotype, Humans, Leukotrienes metabolism, Linkage Disequilibrium, Middle Aged, Phenotype, Asthma genetics, Leukotriene D4 genetics, Membrane Proteins genetics, Polymorphism, Genetic, Receptors, Leukotriene genetics
- Abstract
Background: Cysteinyl leukotrienes (CYSLTR) are potent biological mediators in the pathophysiology of asthma for which two receptors have been characterized, CYSLTR1 and CYSLTR2. The leukotriene modifying agents currently used to control bronchoconstriction and inflammation in asthmatic patients are CYSLTR1-specific leukotriene receptor antagonists. In this report, we investigated a possible role for therapeutic modulation of CYSLTR2 in asthma by investigating genetic association with asthma and further characterization of the pharmacology of a coding polymorphism., Methods: The association of CYSLTR2 polymorphisms with asthma was assessed by transmission disequilibrium test in two family-based collections (359 families from Denmark and Minnesota, USA and 384 families from the Genetics of Asthma International Network)., Results: A significant association of the coding polymorphism, 601A>G, with asthma was observed (P = 0.003). We replicated these findings in a collection of 384 families from the Genetics of Asthma International Network (P = 0.04). The G allele is significantly under-transmitted to asthmatics, indicating a possible role for this receptor in resistance to asthma. The potency of cysteinyl leukotrienes at the wild-type CYSLTR2 and the coding polymorphism 601A>G were assessed using a calcium mobilization assay. The potency of LTC4 and LTE4 was similar for both forms of the receptor and LTB4 was inactive, however, LTD4 was approximately five-fold less potent on 601A>G compared to wild-type CYSLTR2., Conclusions: Since 601A>G alters the potency of LTD4 and this variant allele may be associated with resistance to asthma, it is possible that modulation of the CYSLTR2 may be useful in asthma pharmacotherapy.
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- 2004
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26. Radioligand binding and functional characterization of recombinant human NmU1 and NmU2 receptors stably expressed in clonal human embryonic kidney-293 cells.
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Aiyar N, Disa J, Foley JJ, Buckley PT, Wixted WE, Pullen M, Shabon U, Dul E, Szekeres PG, Elshourbagy NA, Sarau HM, Appelbaum E, and Bolaky J
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- Binding Sites, Cells, Cultured, Clone Cells, Humans, Kidney metabolism, Membrane Proteins genetics, Membrane Proteins physiology, Oligonucleotide Array Sequence Analysis, Radioligand Assay, Receptors, Neurotransmitter genetics, Receptors, Neurotransmitter physiology, Signal Transduction, Membrane Proteins metabolism, Receptors, Neurotransmitter metabolism
- Abstract
Neuromedin U (NmU) is a smooth muscle contracting peptide. Recently, two G-protein-coupled receptors for NmU (NmU1R and NmU2R) have been cloned having approximately 50% homology. They have distinct patterns of expression suggesting they may have different biological functions. This study provides a comprehensive characterization of both NmU receptors expressed in human embryonic kidney 293 cells. [125I]hNmU binding to the recombinant NmU receptors was rapid, saturable, of high affinity and to a single population of binding sites. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular [Ca2+]i release (EC50 value of 0.50 +/- 0.10 nmol/l) and inositol phosphate formation (EC50 1.6 +/- 0.2 and 1.50 +/- 0.4 nmol/l for NmU1R and NmU2R respectively). Furthermore, hNmU inhibited forskolin (3 micromol/l)-stimulated accumulation of cAMP in intact HEK-293 cells expressing either NmU1R or NmU2R. The inhibitory effect was significant for the cells expressing NmU2R with IC50 value of 0.80 +/- 0.21 nmol/l. In summary, both NmU1R and NmU2R in HEK-293 cells have similar signaling capability., (Copyright 2004 S. Karger AG, Basel)
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- 2004
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27. Evaluation of potent and selective small-molecule antagonists for the CXCR2 chemokine receptor.
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Widdowson KL, Elliott JD, Veber DF, Nie H, Rutledge MC, McCleland BW, Xiang JN, Jurewicz AJ, Hertzberg RP, Foley JJ, Griswold DE, Martin L, Lee JM, White JR, and Sarau HM
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- Animals, Anti-Inflammatory Agents chemistry, Anti-Inflammatory Agents pharmacology, Binding, Competitive, CHO Cells, Calcium metabolism, Chemokine CXCL1, Chemokines, Chemotactic Factors, Chemotaxis, Cricetinae, Humans, Hypersensitivity, Delayed chemically induced, Hypersensitivity, Delayed drug therapy, In Vitro Techniques, Intercellular Signaling Peptides and Proteins, Interleukin-8, Neutropenia chemically induced, Neutropenia drug therapy, Neutrophils drug effects, Neutrophils metabolism, Neutrophils physiology, Phenylurea Compounds chemistry, Phenylurea Compounds pharmacology, Rabbits, Radioligand Assay, Receptors, Interleukin-8A antagonists & inhibitors, Receptors, Interleukin-8A metabolism, Receptors, Interleukin-8B metabolism, Structure-Activity Relationship, Anti-Inflammatory Agents chemical synthesis, Phenylurea Compounds chemical synthesis, Receptors, Interleukin-8B antagonists & inhibitors
- Abstract
N,N'-Diarylureas were prepared, and the structure-activity relationship relative to the CXCR2 receptor was examined. This led to the identification of a potent and highly selective CXCR2 antagonist, which in addition was shown to be functionally active both in vitro against human neutrophils and in vivo in rabbit models of ear edema and neutropenia.
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- 2004
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28. A synthetic, non-peptide CXCR2 antagonist blocks MIP-2-induced neutrophil migration in mice.
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Matzer SP, Zombou J, Sarau HM, Röllinghoff M, and Beuscher HU
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- Animals, Bone Marrow Cells, Cell Migration Inhibition, Chemokine CXCL2, Chemotaxis, Leukocyte drug effects, Mice, Mice, Inbred BALB C, Neutrophils drug effects, Phenylurea Compounds administration & dosage, Phenylurea Compounds chemical synthesis, Zymosan administration & dosage, Zymosan pharmacology, Chemokines pharmacology, Neutrophil Infiltration drug effects, Neutrophils immunology, Phenylurea Compounds pharmacology, Receptors, Interleukin-8B antagonists & inhibitors
- Abstract
Non-peptide antagonists of chemokine receptors are considered an intriguing alternative for the treatment of acute and chronic diseases. Particularly the recruitment of neutrophils to inflammatory sites often causes harmful side effects and is mediated by chemokine ligands of the CXC chemokine receptor 2 (CXCR2). Hence, this receptor has been proposed as an important target for novel drugs. This study investigates the potential of the non-peptide CXCR2 antagonist SB 455821 to block neutrophil migration in mice. By using bone marrow derived neutrophils we established a migration assay which revealed SB 455821 as a potent inhibitor of macrophage inflammatory protein 2 (MIP-2)-induced neutrophil migration in vitro (IC50-20 nM). In vivo, injection of MIP-2 into the peritoneal cavities of mice markedly increased neutrophil numbers in peritoneal lavages which were reduced to control levels by co-administration of SB 455821 indicating that the compound effectively binds to the receptor under physiological conditions and exhibits biological activity in vivo. Nevertheless, using intraperitoneal injection of zymosan as a complex inflammatory stimulus, SB 455821 was unable to block neutrophil recruitment to the peritoneal cavity of mice possibly due to other chemotactic mediators overruling signals derived from CXCR2 ligands. Our data show that SB 455821 blocks MIP-2-induced neutrophil migration in vitro and after injection in mice suggesting that selective CXCR2 antagonists may be useful drugs in diseases where neutrophil accumulation plays a major role and leads to exacerbation of acute or chronic inflammations.
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- 2004
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29. Regulation of TNF-alpha- and IFN-gamma-induced CXCL10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease.
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Hardaker EL, Bacon AM, Carlson K, Roshak AK, Foley JJ, Schmidt DB, Buckley PT, Comegys M, Panettieri RA Jr, Sarau HM, and Belmonte KE
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- Cells, Cultured, Chemokine CXCL10, Drug Synergism, Humans, Inflammation immunology, Muscle, Smooth cytology, Muscle, Smooth drug effects, Pulmonary Disease, Chronic Obstructive pathology, Receptors, CXCR3, Receptors, Chemokine metabolism, Respiratory System anatomy & histology, Chemokines, CXC biosynthesis, Interferon-gamma pharmacology, Lung immunology, Muscle, Smooth immunology, Pulmonary Disease, Chronic Obstructive immunology, Tumor Necrosis Factor-alpha pharmacology
- Abstract
The chemokine CXCL10 is produced by many inflammatory cells found in the diseased lung and has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). The present study demonstrates elevated CXCL10 protein in the lungs of COPD patients, which appears histologically in airway smooth muscle (hASM). In primary cultured hASM cells taken from normal donors, CXCL10 protein expression was induced by IFN-gamma and TNF-alpha, cytokines reported as elevated in COPD, and a synergistic response was obtained when they were combined. TNF-alpha stimulation of hASM enhanced accumulation of CXCL10 mRNA, indicating regulation at the transcriptional level, while IFN-gamma stimulation resulted in a smaller accumulation of CXCL10 mRNA. When these cytokines were applied simultaneously, an additive effect was obtained. TNF-alpha-induced CXCL10 expression in hASM was dependent on NFkappaB activation, and a salicylanilide NFkappaB inhibitor blocked the CXCL10 expression. In contrast, IFN-gamma stimulation resulted in transient NFkappaB activation, and the inhibitor had little effect on CXCL10 expression. When these cytokines were added simultaneously, NFkappaB was activated earlier and lasted longer, and the effect was blocked by the inhibitor. These data demonstrate a potential active role for hASM in pulmonary inflammatory diseases such as COPD by producing CXCL10.
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- 2004
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30. The neuromedin B receptor antagonist, BIM-23127, is a potent antagonist at human and rat urotensin-II receptors.
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Herold CL, Behm DJ, Buckley PT, Foley JJ, Wixted WE, Sarau HM, and Douglas SA
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- Animals, Aorta, Thoracic drug effects, Binding, Competitive, Calcium metabolism, Cell Line, Dose-Response Relationship, Drug, Humans, In Vitro Techniques, Male, Rats, Rats, Sprague-Dawley, Urotensins pharmacology, Peptides, Cyclic pharmacology, Receptors, Bombesin antagonists & inhibitors, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
The functional activity of the peptidic neuromedin B receptor antagonist BIM-23127 was investigated at recombinant and native urotensin-II receptors (UT receptors). Human urotensin-II (hU-II) promoted intracellular calcium mobilization in HEK293 cells expressing the human UT (hUT) or rat UT (rUT) receptors with pEC(50) values of 9.80+/-0.34 (n=6) and 9.06+/-0.32 (n=4), respectively. While BIM-23127 alone had no effect on calcium responses in either cell line, it was a potent and competitive antagonist at both hUT (pA(2)=7.54+/-0.14; n=3) and rUT (pA(2)=7.70+/-0.05; n=3) receptors. Furthermore, BIM-23127 reversed hU-II-induced contractile tone in the rat-isolated aorta with a pIC(50) of 6.66+/-0.04 (n=4). In conclusion, BIM- 23127 is the first hUT receptor antagonist identified to date and should not be considered as a selective neuromedin B receptor antagonist.
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- 2003
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31. Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.
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Chandrasekar B, Melby PC, Sarau HM, Raveendran M, Perla RP, Marelli-Berg FM, Dulin NO, and Singh IS
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- Animals, Cells, Cultured, Chemokine CXCL1, Chemokine CXCL2, Chemokine CXCL5, Chemokines metabolism, Chemokines pharmacology, Chemokines, CXC pharmacology, Chemotactic Factors metabolism, Chemotactic Factors pharmacology, Intercellular Signaling Peptides and Proteins metabolism, Intercellular Signaling Peptides and Proteins pharmacology, Mice, Rats, Receptor Cross-Talk, Receptors, Interleukin-8A metabolism, Receptors, Interleukin-8B metabolism, Chemokines, CXC metabolism, Endothelium metabolism, NF-kappa B metabolism, Phosphatidylinositol 3-Kinases metabolism, Signal Transduction drug effects
- Abstract
It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-kappaB and induced kappaB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1beta and tumor necrosis factor-alpha promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK), and dominant negative IkappaB significantly inhibited LIX-mediated NF-kappaB activation in rat CDEC. Inhibition of G(i) protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-kappaB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated kappaB activation and kappaB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-kappaB and induces kappaB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IkappaB. Thus, in addition to attracting and activating neutrophils, the ELR(+) CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.
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- 2003
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32. A potent and selective nonpeptide antagonist of CXCR2 inhibits acute and chronic models of arthritis in the rabbit.
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Podolin PL, Bolognese BJ, Foley JJ, Schmidt DB, Buckley PT, Widdowson KL, Jin Q, White JR, Lee JM, Goodman RB, Hagen TR, Kajikawa O, Marshall LA, Hay DW, and Sarau HM
- Subjects
- Acute Disease, Animals, Arthritis, Experimental etiology, Arthritis, Rheumatoid etiology, Arthritis, Rheumatoid immunology, Chemotaxis, Leukocyte drug effects, Chronic Disease, Female, Humans, In Vitro Techniques, Interleukin-8 administration & dosage, Interleukin-8 immunology, Interleukin-8 metabolism, Lipopolysaccharides toxicity, Neutrophils drug effects, Neutrophils immunology, Ovalbumin administration & dosage, Ovalbumin immunology, Rabbits, Receptors, Interleukin-8B genetics, Receptors, Interleukin-8B metabolism, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins genetics, Recombinant Proteins metabolism, Urea analogs & derivatives, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Arthritis, Experimental immunology, Arthritis, Experimental prevention & control, Receptors, Interleukin-8B antagonists & inhibitors, Urea pharmacology
- Abstract
Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits (125)I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC(50) = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC(50) = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-alpha, IL-8, PGE(2), leukotriene B(4), and leukotriene C(4) levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-alpha and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC(50) = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.
- Published
- 2002
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33. Nonpeptide tachykinin receptor antagonists. III. SB 235375, a low central nervous system-penetrant, potent and selective neurokinin-3 receptor antagonist, inhibits citric acid-induced cough and airways hyper-reactivity in guinea pigs.
- Author
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Hay DW, Giardina GA, Griswold DE, Underwood DC, Kotzer CJ, Bush B, Potts W, Sandhu P, Lundberg D, Foley JJ, Schmidt DB, Martin LD, Kilian D, Legos JJ, Barone FC, Luttmann MA, Grugni M, Raveglia LF, and Sarau HM
- Subjects
- Animals, Behavior, Animal, Bronchial Hyperreactivity chemically induced, Calcium metabolism, Central Nervous System drug effects, Central Nervous System metabolism, Citric Acid, Cloning, Molecular, Cough chemically induced, Guinea Pigs, In Vitro Techniques, Iris drug effects, Male, Mice, Mice, Inbred BALB C, Muscle Contraction drug effects, Muscle, Smooth drug effects, Neurokinin A pharmacology, Peptide Fragments pharmacology, Pupil drug effects, Rabbits, Radioligand Assay, Rats, Recombinant Proteins metabolism, Substance P pharmacology, Acetates pharmacology, Antitussive Agents pharmacology, Bronchial Hyperreactivity prevention & control, Cough prevention & control, Quinolines pharmacology, Receptors, Neurokinin-3 antagonists & inhibitors, Receptors, Tachykinin antagonists & inhibitors, Substance P analogs & derivatives
- Abstract
In this report the in vitro and in vivo pharmacological and pharmacokinetic profile of (-)-(S)-N-(alpha-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB 235375), a low central nervous system (CNS)-penetrant, human neurokinin-3 (NK-3) receptor (hNK-3R) antagonist, is described. SB 235375 inhibited (125)I-[MePhe(7)]-neurokinin B (NKB) binding to membranes of Chinese hamster ovary (CHO) cells expressing the hNK-3R (CHO-hNK-3R) with a K(i) = 2.2 nM and antagonized competitively NKB-induced Ca(2+) mobilization in human embryonic kidney (HEK) 293 cells expressing the hNK-3R (HEK 293-hNK-3R) with a K(b) = 12 nM. SB 235375 antagonized senktide (NK-3R)-induced contractions in rabbit isolated iris sphincter (pA(2) = 8.1) and guinea pig ileal circular smooth muscles (pA(2) = 8.3). SB 235375 was selective for the hNK-3R compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 209 nM), and was without effect, at 1 microM, in 68 other receptor, enzyme, and ion channel assays. Intravenous SB 235375 produced a dose-related inhibition of miosis induced by i.v. senktide in the rabbit (ED(50) of 0.56 mg/kg). Intraperitoneal SB 235375 (10-30 mg/kg) inhibited citric acid-induced cough and airways hyper-reactivity in guinea pigs. In mice oral SB 235375 (3-30 mg/kg) was without significant effect on the behavioral responses induced by intracerebral ventricular administration of senktide. Pharmacokinetic evaluation in the mouse and rat revealed that oral SB 235375 was well absorbed systemically but did not effectively cross the blood-brain barrier. The preclinical profile of SB 235375, encompassing high affinity, selectivity, oral activity, and low CNS penetration, suggests that it is an appropriate tool compound to define the pathophysiological roles of the NK-3Rs in the peripheral nervous system.
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- 2002
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34. Cloning, pharmacology, and tissue distribution of G-protein-coupled receptor GPR105 (KIAA0001) rodent orthologs.
- Author
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Freeman K, Tsui P, Moore D, Emson PC, Vawter L, Naheed S, Lane P, Bawagan H, Herrity N, Murphy K, Sarau HM, Ames RS, Wilson S, Livi GP, and Chambers JK
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Southern, Cloning, Molecular, Humans, Mice, Molecular Sequence Data, Open Reading Frames, Rats, Receptors, Cell Surface agonists, Receptors, Cell Surface chemistry, Receptors, Cell Surface metabolism, Receptors, Purinergic P2Y, Sequence Homology, Amino Acid, Uridine Diphosphate Glucose pharmacology, GTP-Binding Proteins metabolism, Receptors, Cell Surface genetics, Receptors, G-Protein-Coupled, Receptors, Purinergic P2
- Abstract
It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.
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- 2001
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35. EDG1 receptor stimulation leads to cardiac hypertrophy in rat neonatal myocytes.
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Robert P, Tsui P, Laville MP, Livi GP, Sarau HM, Bril A, and Berrebi-Bertrand I
- Subjects
- Adrenergic alpha-Agonists pharmacology, Animals, Animals, Newborn, Cardiomegaly chemically induced, Cardiomegaly metabolism, Cells, Cultured, Culture Media, Serum-Free, Dose-Response Relationship, Drug, Humans, Microscopy, Fluorescence, Mitogen-Activated Protein Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinases metabolism, Myocardium cytology, Myocardium metabolism, Peptides immunology, Peptides metabolism, Phenylephrine pharmacology, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Protein Structure, Tertiary, Rats, Rats, Wistar, Receptors, Cell Surface metabolism, Receptors, Lysophospholipid, Sphingosine chemistry, Sphingosine metabolism, Stress Fibers metabolism, Tissue Extracts chemistry, Virulence Factors, Bordetella pharmacology, Cardiomegaly pathology, Heart drug effects, Immediate-Early Proteins metabolism, Lysophospholipids, Receptors, G-Protein-Coupled, Sphingosine analogs & derivatives, Sphingosine pharmacology
- Abstract
Sphingosine 1 phosphate (S1P), an aminophospholipid, acts extracellularly as a ligand via the specific G protein-coupled receptors of the endothelial differentiation gene (EDG) 1, 3, 5, 6 and 8 receptors family and intracellularly as a second messenger in various cellular types. The aim of this work was to investigate biological activity of S1P in cardiomyocytes with respect to related sphingolipids. S1P was applied for 48 h on rat neonatal cardiomyocytes at 10 nM, 100 nM and 1 microM. S1P induced a concentration-dependent cellular hypertrophy evidenced by an increase in cell size, [3H]-phenylalanine incorporation, protein content and Brain Natriuretic Peptide (BNP) secretion. Among the lipids tested S1P exhibits the lower EC50 (67 nM) followed by dihydro-S1P (107 nM) and sphingosylphosphorylcholine (1.6 microM). The effect of S1P could be related to a stimulation of the EDG1 receptor since we showed that the EDG1 receptor is predominantly expressed at the mRNA and protein levels in rat cardiomyocytes and that specific anti-EDG1 antibodies inhibited the hypertrophic effect induced by S1P. Furthermore the expression level of most other EDG receptors for S1P appeared very low in cardiac myocytes. S1P (100 nM) increased the phosphorylation of p42/44MAPK, p38MAPK, JNK, Akt and p70(S6K), this effect being reversed by inhibitors of their respective phosphorylation which also rescue the hypertrophic phenotype. Finally, S1P stimulated actin stress fibre formation reverted by the Rho inhibitor, the C3 exoenzyme. Altogether, our results show that S1P induces cardiomyocyte hypertrophy mainly via the EDG1 receptor and subsequently via Gi through ERKs, p38 MAPK, JNK, PI3K and via Rho pathway., (Copyright 2001 Academic Press.)
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- 2001
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36. AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1.
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Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P, Steplewski K, Shabon U, Miller JE, Middleton SE, Darker JG, Larminie CG, Wilson S, Bergsma DJ, Emson P, Faull R, Philpott KL, and Harrison DC
- Subjects
- Amino Acid Sequence, Animals, Brain metabolism, CHO Cells, Cricetinae, Female, Genes, Tumor Suppressor, Humans, Kinetics, Kisspeptins, Ligands, Melanoma genetics, Molecular Sequence Data, Nephropidae, Neurons metabolism, Organ Specificity, Peptide Fragments pharmacology, Pituitary Gland metabolism, Placenta metabolism, Pregnancy, Proteins chemistry, Rats, Receptors, Cell Surface chemistry, Receptors, G-Protein-Coupled, Receptors, Kisspeptin-1, Receptors, Neuropeptide chemistry, Receptors, Neuropeptide genetics, Recombinant Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sea Anemones, Sequence Alignment, Sequence Homology, Amino Acid, Transfection, Tumor Suppressor Proteins, Heterotrimeric GTP-Binding Proteins metabolism, Proteins genetics, Proteins metabolism, Receptors, Cell Surface genetics
- Abstract
A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.
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- 2001
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37. Discovery of potent and selective phenylalanine derived CCR3 antagonists. Part 1.
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Dhanak D, Christmann LT, Darcy MG, Jurewicz AJ, Keenan RM, Lee J, Sarau HM, Widdowson KL, and White JR
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- Humans, Receptors, CCR3, Receptors, Chemokine metabolism, Structure-Activity Relationship, Phenylalanine chemistry, Phenylalanine pharmacology, Receptors, Chemokine antagonists & inhibitors
- Abstract
The discovery of a series of phenylalanine derived CCR3 antagonists is reported. Parallel, solution-phase library synthesis has been utilized to delineate the structure-activity relationship leading to the synthesis of highly potent, CCR3-selective antagonists.
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- 2001
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38. Discovery of potent and selective phenylalanine derived CCR3 receptor antagonists. Part 2.
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Dhanak D, Christmann LT, Darcy MG, Keenan RM, Knight SD, Lee J, Ridgers LH, Sarau HM, Shah DH, White JR, and Zhang L
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- Humans, Phenylalanine chemical synthesis, Phenylalanine chemistry, Receptors, CCR3, Receptors, Chemokine metabolism, Structure-Activity Relationship, Phenylalanine pharmacology, Receptors, Chemokine antagonists & inhibitors
- Abstract
Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists.
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- 2001
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39. Interleukin-8 receptor antagonists in pulmonary diseases.
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Hay DW and Sarau HM
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- Animals, Humans, Interleukin-8 metabolism, Receptors, Interleukin-8A physiology, Interleukin-8 physiology, Lung Diseases drug therapy, Receptors, Interleukin-8A antagonists & inhibitors
- Abstract
Inflammatory cells are thought to be instrumental in the pathophysiology of pulmonary diseases, and control of their recruitment and activation in the lung would appear to be an attractive strategy for therapeutic intervention. Interleukin-8 and related CXC chemokines are involved in the function of neutrophils and T cells, and have been implicated in several lung diseases. Small-molecule antagonists of the interleukin-8 receptors have been identified, which may help elucidate the role of interleukin-8 and related chemokines in the pathophysiology of lung diseases.
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- 2001
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40. Stepwise modulation of neurokinin-3 and neurokinin-2 receptor affinity and selectivity in quinoline tachykinin receptor antagonists.
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Blaney FE, Raveglia LF, Artico M, Cavagnera S, Dartois C, Farina C, Grugni M, Gagliardi S, Luttmann MA, Martinelli M, Nadler GM, Parini C, Petrillo P, Sarau HM, Scheideler MA, Hay DW, and Giardina GA
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- Amino Acid Sequence, Animals, CHO Cells, Cloning, Molecular, Cricetinae, Humans, Models, Molecular, Molecular Sequence Data, Morpholines chemistry, Morpholines metabolism, Piperidines chemistry, Piperidines metabolism, Quinolines chemistry, Quinolines metabolism, Radioligand Assay, Receptors, Neurokinin-2 chemistry, Receptors, Neurokinin-2 metabolism, Receptors, Neurokinin-3 chemistry, Receptors, Neurokinin-3 metabolism, Receptors, Opioid, mu chemistry, Receptors, Opioid, mu metabolism, Structure-Activity Relationship, Morpholines chemical synthesis, Piperidines chemical synthesis, Quinolines chemical synthesis, Receptors, Neurokinin-2 antagonists & inhibitors, Receptors, Neurokinin-3 antagonists & inhibitors
- Abstract
A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.
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- 2001
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41. Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.
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Ames RS, Lee D, Foley JJ, Jurewicz AJ, Tornetta MA, Bautsch W, Settmacher B, Klos A, Erhard KF, Cousins RD, Sulpizio AC, Hieble JP, McCafferty G, Ward KW, Adams JL, Bondinell WE, Underwood DC, Osborn RR, Badger AM, and Sarau HM
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- Animals, Anti-Inflammatory Agents, Non-Steroidal metabolism, Anti-Inflammatory Agents, Non-Steroidal pharmacokinetics, Arginine analogs & derivatives, Arginine metabolism, Arginine pharmacokinetics, Arthritis, Experimental immunology, Arthritis, Experimental pathology, Benzhydryl Compounds metabolism, Benzhydryl Compounds pharmacokinetics, Binding, Competitive, Cell Line, Complement Inactivator Proteins metabolism, Complement Inactivator Proteins pharmacokinetics, Disease Models, Animal, Edema pathology, Edema prevention & control, Guinea Pigs, Hindlimb, Humans, Injections, Intraperitoneal, Leukocytosis immunology, Leukocytosis pathology, Male, Mice, Muscle Contraction drug effects, Neutrophil Infiltration drug effects, Rats, Rats, Inbred Lew, Rats, Sprague-Dawley, Receptors, Complement metabolism, Tumor Cells, Cultured, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Arginine pharmacology, Benzhydryl Compounds pharmacology, Complement C3a metabolism, Complement Inactivator Proteins pharmacology, Membrane Proteins, Receptors, Complement antagonists & inhibitors
- Abstract
The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.
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- 2001
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42. Cloning, expression, and pharmacological characterization of a novel human histamine receptor.
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Zhu Y, Michalovich D, Wu H, Tan KB, Dytko GM, Mannan IJ, Boyce R, Alston J, Tierney LA, Li X, Herrity NC, Vawter L, Sarau HM, Ames RS, Davenport CM, Hieble JP, Wilson S, Bergsma DJ, and Fitzgerald LR
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- Amino Acid Sequence, Animals, Calcium metabolism, Cloning, Molecular, Dose-Response Relationship, Drug, Gene Expression, Genes, Reporter, Humans, Luciferases, Mice, Molecular Sequence Data, Radioligand Assay, Receptors, Histamine metabolism, Receptors, Histamine H3 chemistry, Receptors, Histamine H3 metabolism, Sequence Homology, Amino Acid, Tissue Distribution, Tritium, Histamine metabolism, Receptors, Histamine genetics
- Abstract
Using a genomics-based reverse pharmacological approach for screening orphan G-protein coupled receptors, we have identified and cloned a novel high-affinity histamine receptor. This receptor, termed AXOR35, is most closely related to the H3 histamine receptor, sharing 37% protein sequence identity. A multiple responsive element/cyclic AMP-responsive element-luciferase reporter assay was used to identify histamine as a ligand for AXOR35. When transfected into human embryonic kidney 293 cells, the AXOR35 receptor showed a strong, dose-dependent calcium mobilization response to histamine and H3 receptor agonists including imetit and immepip. Radioligand binding confirmed that the AXOR35 receptor was a high-affinity histamine receptor. The pharmacology of the AXOR35 receptor was found to closely resemble that of the H3 receptor; the major difference was that (R)-alpha-methylhistamine was a low potency agonist of the AXOR35 receptor. Thioperamide is an antagonist at AXOR 35. Expression of AXOR35 mRNA in human tissues is highest in peripheral blood mononuclear cells and in tissues likely to contain high concentrations of blood cells, such as bone marrow and lung. In situ hybridization analysis of a wide survey of mouse tissues showed that mouse AXOR35 mRNA is selectively expressed in hippocampus. The identification and localization of this new histamine receptor will expand our understanding of the physiological and pathological roles of histamine and may provide additional opportunities for pharmacological modification of these actions.
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- 2001
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43. Molecular and pharmacological characterization of the murine tachykinin NK(3) receptor.
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Sarau HM, Feild JA, Ames RS, Foley JJ, Nuthulaganti P, Schmidt DB, Buckley PT, Elshourbagy NA, Brawner ME, Luttmann MA, Giardina GA, and Hay DW
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- Amino Acid Sequence physiology, Animals, Cloning, Molecular methods, Humans, Mice, Molecular Sequence Data, Neurokinin A metabolism, Neurokinin A pharmacology, Quinolines chemistry, Quinolines metabolism, Quinolines pharmacology, Receptors, Neurokinin-3 drug effects, Calcium metabolism, Receptors, Neurokinin-3 physiology
- Abstract
Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.
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- 2001
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44. Differential vasoconstrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey.
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Douglas SA, Sulpizio AC, Piercy V, Sarau HM, Ames RS, Aiyar NV, Ohlstein EH, and Willette RN
- Subjects
- Animals, Aorta, Abdominal drug effects, Aorta, Abdominal physiology, Aorta, Thoracic drug effects, Aorta, Thoracic physiology, Arteries drug effects, Arteries physiology, Blood Vessels physiology, Callithrix, Carotid Arteries drug effects, Carotid Arteries physiology, Coronary Vessels drug effects, Coronary Vessels physiology, Dogs, Dose-Response Relationship, Drug, Femoral Artery drug effects, Femoral Artery physiology, Humans, In Vitro Techniques, Macaca fascicularis, Mice, Muscle, Smooth, Vascular drug effects, Muscle, Smooth, Vascular physiology, Pulmonary Artery drug effects, Pulmonary Artery physiology, Pulmonary Veins drug effects, Pulmonary Veins physiology, Rats, Swine, Trachea drug effects, Trachea physiology, Veins drug effects, Veins physiology, Blood Vessels drug effects, Urotensins pharmacology, Vasoconstriction drug effects, Vasoconstrictor Agents pharmacology
- Abstract
1. Urotensin-II (U-II) and its G-protein-coupled receptor, GPR14, are expressed within mammalian cardiac and peripheral vascular tissue and, as such, may regulate mammalian cardiovascular function. The present study details the vasoconstrictor profile of this cyclic undecapeptide in different vascular tissues isolated from a diverse range of mammalian species (rats, mice, dogs, pigs, marmosets and cynomolgus monkeys). 2. The vasoconstrictor activity of human U-II was dependent upon the anatomical origin of the vessel studied and the species from which it was isolated. In the rat, constrictor responses were most pronounced in thoracic aortae and carotid arteries: -log[EC(50)]s 9.09+/-0.19 and 8.84+/-0.21, R(max)s 143+/-21 and 67+/-26% 60 mM KCl, respectively (compared, for example, to -log[EC(50)] 7.90+/-0.11 and R(max) 142+/-12% 60 mM KCl for endothelin-1 [ET-1] in thoracic aortae). Responses were, however, absent in mice aortae (-log[EC(50)] <6.50). These findings were further contrasted by the observation that U-II was a 'coronary-selective' spasmogen in the dog (-log[EC(50)] 9.46+/-0.11, R(max) 109+/-23% 60 mM KCl in LCX coronary artery), yet exhibited a broad spectrum of vasoconstrictor activity in arterial tissue from Old World monkeys (-log[EC(50)]s range from 8.96+/-0.15 to 9.92+/-0.13, R(max)s from 43+/-16 to 527+/-135% 60 mM KCl). Interestingly, significant differences in reproducibility and vasoconstrictor efficacy were seen in tissue from pigs and New World primates (vessels which responded to noradrenaline, phenylephrine, KCl or ET-1 consistently). 3. Thus, human U-II is a potent, efficacious vasoconstrictor of a variety of mammalian vascular tissues. Although significant species/anatomical variations exist, the data support the hypothesis that U-II influences the physiological regulation of mammalian cardiovascular function.
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- 2000
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45. Identification of potent, selective non-peptide CC chemokine receptor-3 antagonist that inhibits eotaxin-, eotaxin-2-, and monocyte chemotactic protein-4-induced eosinophil migration.
- Author
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White JR, Lee JM, Dede K, Imburgia CS, Jurewicz AJ, Chan G, Fornwald JA, Dhanak D, Christmann LT, Darcy MG, Widdowson KL, Foley JJ, Schmidt DB, and Sarau HM
- Subjects
- Asthma physiopathology, Binding, Competitive, Calcium metabolism, Cell Line, Chemokine CCL11, Chemokine CCL24, Humans, Phenylalanine pharmacology, Receptors, CCR3, Receptors, Chemokine physiology, Benzamides pharmacology, Cell Movement drug effects, Chemokines, CC antagonists & inhibitors, Cytokines antagonists & inhibitors, Eosinophils drug effects, Monocyte Chemoattractant Proteins antagonists & inhibitors, Naphthalenes pharmacology, Phenylalanine analogs & derivatives, Receptors, Chemokine antagonists & inhibitors, Receptors, HIV antagonists & inhibitors
- Abstract
Eosinophils have been implicated in the pathogenesis of asthma and other allergic diseases. Several CC chemokines including eotaxin (CCL-11), eotaxin-2 (CCL-24), RANTES (CCL-5), and monocyte chemotactic protein-3 (MCP-3, CCL-7) and 4 (MCP-4, CCL-13) are potent eosinophil chemotactic and activating peptides acting through CC chemokine receptor-3 (CCR3). Thus, antagonism of CCR3 could have a therapeutic role in asthma and other eosinophil-mediated diseases. A high throughput, cellular functional screen was configured using RBL-2H3 cells stably expressing CCR3 (RBL-2H3-CCR3) to identify non-peptide receptor antagonists. A small molecule CCR3 antagonist was identified, SK&F 45523, and chemical optimization led to the generation of a number of highly potent, selective CCR3 antagonists including SB-297006 and SB-328437. These compounds were further characterized in vitro and demonstrated high affinity, competitive inhibition of (125)I-eotaxin and (125)I-MCP-4 binding to human eosinophils. The compounds were potent inhibitors of eotaxin- and MCP-4-induced Ca(2+) mobilization in RBL-2H3-CCR3 cells and eosinophils. Additionally, SB-328437 inhibited eosinophil chemotaxis induced by three ligands that activate CCR3 with similar potencies. Selectivity was affirmed using a panel of 10 seven-transmembrane receptors. This is the first description of a non-peptide CCR3 antagonist, which should be useful in further elucidating the pathophysiological role of CCR3 in allergic inflammatory diseases.
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- 2000
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46. Nonpeptide tachykinin receptor antagonists. II. Pharmacological and pharmacokinetic profile of SB-222200, a central nervous system penetrant, potent and selective NK-3 receptor antagonist.
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Sarau HM, Griswold DE, Bush B, Potts W, Sandhu P, Lundberg D, Foley JJ, Schmidt DB, Webb EF, Martin LD, Legos JJ, Whitmore RG, Barone FC, Medhurst AD, Luttmann MA, Giardina GA, and Hay DW
- Subjects
- Animals, Brain metabolism, CHO Cells, Calcium metabolism, Cricetinae, Dose-Response Relationship, Drug, Humans, In Vitro Techniques, Iris drug effects, Iris physiology, Male, Mice, Mice, Inbred BALB C, Peptide Fragments pharmacology, Quinolines pharmacokinetics, Rabbits, Rats, Rats, Sprague-Dawley, Substance P analogs & derivatives, Substance P pharmacology, Brain drug effects, Quinolines pharmacology, Receptors, Neurokinin-3 antagonists & inhibitors
- Abstract
The pharmacological and pharmacokinetic profile of SB-222200 [(S)-(-)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-car boxami de], a human NK-3 receptor (hNK-3R) antagonist, was determined. SB-222200 inhibited (125)I-[MePhe(7)]neurokinin B (NKB) binding to Chinese hamster ovary (CHO) cell membranes stably expressing the hNK-3 receptor (CHO-hNK-3R) with a K(i) = 4.4 nM and antagonized NKB-induced Ca(2+) mobilization in HEK 293 cells stably expressing the hNK-3 receptor (HEK 293-hNK-3R) with an IC(50) = 18.4 nM. SB-222200 was selective for hNK-3 receptors compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 250 nM). In HEK 293 cells transiently expressing murine NK-3 receptors (HEK 293-mNK-3R), SB-222200 inhibited binding of (125)I-[MePhe(7)]NKB (K(i) = 174 nM) and antagonized NKB (1 nM)-induced calcium mobilization (IC(50) = 265 nM). In mice oral administration of SB-222200 produced dose-dependent inhibition of behavioral responses induced by i.p. or intracerebral ventricular administration of the NK-3 receptor-selective agonist, senktide, with ED(50) values of approximately 5 mg/kg. SB-222200 effectively crossed the blood-brain barrier in the mouse and rat. The inhibitory effect of SB-222200 against senktide-induced behavioral responses in the mouse correlated significantly with brain, but not plasma, concentrations of the compound. Pharmacokinetic evaluation of SB-222200 in rat after oral administration (8 mg/kg) indicated sustained plasma concentrations (C(max) = about 400 ng/ml) and bioavailability of 46%. The preclinical profile of SB-222200, demonstrating high affinity, selectivity, reversibility, oral activity, and central nervous system penetration, suggests that it will be a useful tool compound to define the physiological and pathophysiological roles of NK-3 receptors, in particular in the central nervous system.
- Published
- 2000
47. Evidence that the proposed novel human "neurokinin-4" receptor is pharmacologically similar to the human neurokinin-3 receptor but is not of human origin.
- Author
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Sarau HM, Mooney JL, Schmidt DB, Foley JJ, Buckley PT, Giardina GA, Wang DY, Lee JA, and Hay DW
- Subjects
- Binding, Competitive, Biological Transport, Calcium metabolism, Cells, Cultured, DNA, Complementary analysis, Humans, Polymerase Chain Reaction, Radioligand Assay, Receptors, Neurokinin-3 drug effects, Receptors, Neurokinin-3 genetics, Receptors, Tachykinin drug effects, Receptors, Tachykinin genetics, Receptors, Tachykinin isolation & purification, Restriction Mapping, Tachykinins metabolism, Receptors, Neurokinin-3 metabolism, Receptors, Tachykinin metabolism
- Abstract
There have been proposals that the tachykinin receptor classification should be extended to include a novel receptor, the "neurokinin-4" receptor (NK-4R), which has a close homology with the human NK-3 receptor (hNK-3R). We compared the pharmacological and molecular biological characteristics of the hNK-3R and NK-4R. Binding experiments, with (125)I-[MePhe(7)]-NKB binding to HEK 293 cell membranes transiently expressing the hNK-3R (HEK 293-hNK-3R) or NK-4R (HEK 293-NK-4R), and functional studies (Ca(2+) mobilization in the same cells) revealed a similar profile of sensitivity to tachykinin agonists and antagonists for both receptors; i.e., in binding studies with the hNK-3R, MePhe(7)-NKB > NKB > senktide >> NKA = Substance P; with the NK-4R, MePhe(7)-NKB > NKB = senktide >> Substance P = NKA; and with antagonists, SB 223412 = SR 142801 > SB 222200 >> SR 48968 >> CP 99994 for both hNK-3R and NK-4R. Thus, the pharmacology of the two receptors was nearly identical. However, attempts to isolate or identify the NK-4R gene by using various molecular biological techniques were unsuccessful. Procedures, including nested polymerase chain reaction studies, that used products with restriction endonuclease sites specific for either hNK-3R or NK-4R, failed to demonstrate the presence of NK-4R in genomic DNA from human, monkey, mouse, rat, hamster, or guinea pig, and in cDNA libraries from human lung, brain, or heart, whereas the hNK-3R was detectable in the latter libraries. In view of the failure to demonstrate the presence of the putative NK-4R it is thought to be premature to extend the current tachykinin receptor classification.
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- 2000
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48. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor.
- Author
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Elshourbagy NA, Ames RS, Fitzgerald LR, Foley JJ, Chambers JK, Szekeres PG, Evans NA, Schmidt DB, Buckley PT, Dytko GM, Murdock PR, Milligan G, Groarke DA, Tan KB, Shabon U, Nuthulaganti P, Wang DY, Wilson S, Bergsma DJ, and Sarau HM
- Subjects
- Amino Acid Sequence, Arrestins metabolism, Base Sequence, Calcium metabolism, Cell Line, FMRFamide pharmacology, Humans, Ligands, Molecular Sequence Data, Receptors, Neuropeptide genetics, beta-Arrestins, Neuropeptides metabolism, Oligopeptides metabolism, Receptors, Neuropeptide metabolism
- Abstract
Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.
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- 2000
- Full Text
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49. Identification of an EDG7 variant, HOFNH30, a G-protein-coupled receptor for lysophosphatidic acid.
- Author
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Fitzgerald LR, Dytko GM, Sarau HM, Mannan IJ, Ellis C, Lane PA, Tan KB, Murdock PR, Wilson S, Bergsma DJ, Ames RS, Foley JJ, Campbell DA, McMillan L, Evans N, Elshourbagy NA, Minehart H, and Tsui P
- Subjects
- Amino Acid Sequence, Base Sequence, Calcium metabolism, Chromosome Mapping, Chromosomes, Human, Pair 1, Cloning, Molecular, Enzyme Activation, Humans, Jurkat Cells, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases metabolism, Molecular Sequence Data, Phosphorylation, Receptors, Cell Surface chemistry, Receptors, Cell Surface metabolism, Receptors, Lysophosphatidic Acid, Sequence Homology, Amino Acid, Tumor Cells, Cultured, GTP-Binding Proteins metabolism, Lysophospholipids metabolism, Receptors, Cell Surface genetics, Receptors, G-Protein-Coupled
- Abstract
We have identified a cDNA, designated HOFNH30, which encodes a 354 amino acid G-protein-coupled receptor (GPCR). This receptor has 96% amino acid identity to the Jurkat-T cell-derived EDG7 and could be a splice variant. RT-PCR analysis demonstrated that HOFNH30 mRNA is expressed in placenta whereas EDG7 mRNA shows highest expression in prostate. The HOFNH30 gene is localized to human chromosome 1p22. 3-1p31.1. When HOFNH30 was expressed in RBL-2H3 cells, LPA and phosphatidic acid (PA) induced a calcium mobilization response with EC(50) values of 13 nM and 3 microM, respectively. LPA also induced phosphorylation of mitogen-activated protein kinase (p42(MAPK) and p44(MAPK)) in HOFNH30-transfected but not vector-transfected RBL-2H3 cells. In the present study, we have identified a novel variant from the EDG receptor family, a GPCR for which LPA is a high-affinity endogenous ligand., (Copyright 2000 Academic Press.)
- Published
- 2000
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50. Identification and molecular characterization of rat CXCR3: receptor expression and interferon-inducible protein-10 binding are increased in focal stroke.
- Author
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Wang X, Li X, Schmidt DB, Foley JJ, Barone FC, Ames RS, and Sarau HM
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Brain Ischemia metabolism, Cells, Cultured, Cerebral Arterial Diseases metabolism, Cerebral Cortex metabolism, Chemokine CXCL10, Cloning, Molecular, DNA, Complementary analysis, Humans, Iodine Radioisotopes, Male, Molecular Sequence Data, RNA, Messenger biosynthesis, Radioligand Assay, Rats, Rats, Inbred SHR, Receptors, CXCR3, Receptors, Chemokine biosynthesis, Sequence Homology, Amino Acid, Transfection, Chemokines, CXC metabolism, Receptors, Chemokine genetics, Stroke metabolism
- Abstract
We describe here the cloning and characterization of a rat homolog of the chemokine receptor CXCR3. The predicted amino acid sequence of rat CXCR3 contains 367 amino acid residues, sharing 96 and 87% amino acid sequence identity to the murine and human CXCR3, respectively. Among a large panel of chemokines tested, only interferon-inducible protein-10 (IP-10), interferon-gamma-induced monokine, and interferon-inducible T cell alpha-chemoattractant demonstrated specific abilities to induce an intracellular calcium mobilization response in human embryonic kidney 293 cells transfected with rat CXCR3 expression vector. (125)I-IP-10 competition binding studies to the CXCR3-transfected human embryonic kidney 293 cells demonstrated that human IP-10 and interferon-inducible T cell alpha-chemoattractant are more potent ligands than human interferon-gamma-induced monokine. Following our previous observation for the induced expression of IP-10 in focal stroke, we demonstrate here the time-dependent up-regulation of CXCR3 mRNA in the rat ischemic cortex after permanent occlusion of the middle cerebral artery. A significant increase in (125)I-IP-10-specific binding to ischemic cerebral cortical samples was obtained and paralleled the increase in CXCR3 mRNA expression. The changes in receptor expression and ligand binding correlate highly with known changes in leukocyte accumulation, and gliosis occurred after focal stroke. These data suggest that CXCR3/IP-10 may be a potential novel therapeutic target in focal stroke. In addition, the cloning of rat CXCR3 provides an important tool for the investigation of the pathophysiological role of CXCR3 in other rodent disease models.
- Published
- 2000
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