ABSTRACT
1 Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell-mediated inflammation of the bronchial mucosa. 2 Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF-kappaB). 3 NF-kappaB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK-beta. 4 We identified COMPOUND A as a small-molecule, ATP-competitive inhibitor selectively targeting IKK-beta kinase activity with a K(i) value of 2 nM. 5 COMPOUND A inhibited stress-induced NF-kappaB transactivation, chemokine-, cytokine-, and adhesion molecule expression, and T- and B-cell proliferation. 6 COMPOUND A is orally bioavailable and inhibited the release of LPS-induced TNF-alpha in rodents. 7 In mice COMPOUND A inhibited cockroach allergen-induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. 8 COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. 9 Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed-type hypersensitivity. 10 These data suggest that IKK-beta inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Oxazines/pharmacology , Pneumonia/prevention & control , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/biosynthesis , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Edema/prevention & control , Female , Humans , I-kappa B Kinase , I-kappa B Proteins/metabolism , Leukocytes/cytology , Leukocytes/drug effects , Leukocytes/physiology , Male , Mice , Mice, Inbred BALB C , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Oxazines/adverse effects , Oxazines/pharmacokinetics , Phosphorylation , Pneumonia/immunology , Pyridines/adverse effects , Pyridines/pharmacokinetics , Rats , Rats, Wistar , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolismABSTRACT
AMP and adenosine are found in all cell types and can be released by cells or created extracellularly from the breakdown of ATP and ADP. We have identified an orphan G protein-coupled receptor with homology to the P2Y family of nucleotide receptors that can respond to both AMP and adenosine. Based on its ability to functionally bind the nucleotide AMP, we have named it P2Y15. Upon stimulation, P2Y15 induces both Ca2+ mobilization and cyclic AMP generation, suggesting coupling to at least two different G proteins. It is highly expressed in mast cells and is found predominantly in the tissues of the respiratory tract and kidneys, which are known to be affected by AMP, adenosine, and adenosine antagonists. Until now, the effects of AMP have been thought to depend on its dephosphorylation to adenosine but we demonstrate here that P2Y15 is a bona fide AMP receptor by showing that it binds [(32)P]AMP. Because AMP and adenosine have bronchoconstrictive effects that can be inhibited by theophylline, we tested whether theophylline and other adenosine receptor antagonists can block P2Y15. We found inhibition at a theophylline concentration well within the therapeutic dose range, indicating that P2Y15 may be a clinically important target of this drug.
Subject(s)
Adenosine Monophosphate/chemistry , Adenosine/chemistry , Calcium/metabolism , Cyclic AMP/metabolism , Receptors, Cell Surface/chemistry , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/physiology , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/physiology , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Cloning, Molecular , Cyclic AMP/chemistry , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , GTP-Binding Proteins/metabolism , Humans , Kinetics , Ligands , Mice , Molecular Sequence Data , Phosphorylation , Phylogeny , Protein Binding , RNA/chemistry , Rats , Receptors, Purinergic P1/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Signal Transduction , Theophylline/chemistry , Time Factors , Tissue DistributionABSTRACT
We cloned, expressed, and characterized in vitro and in vivo the gene encoding the rat ortholog of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a G protein-coupled receptor for prostaglandin D2 (PGD2). Quantitative reverse transcription-polymerase chain reaction analysis demonstrated highest CRTH2 expression in the lung, brain, ovary, and spleen. Pharmacologically, rat CRTH2 stably transfected in mouse preB lymphoma L1.2 cells behaved very similar compared with the mouse and human orthologs, showing a binding affinity for PGD2 of 11 nM, a functional calcium mobilization when exposed to agonist, and similar sensitivity to agonists and antagonists. In vivo, selective activation of CRTH2 by 13,14-dihydro-15-keto (DK)-PGD2 injection into rats led to a dose- and time-dependent increase of the number of leukocytes in the peripheral blood. Specifically, eosinophils, lymphocytes, and neutrophils were recruited with maximum effects seen 60 min after the injection of 300 microg of DK-PGD2 per rat. Pretreatment of the animals with the CRTH2/thromboxane A2 receptor antagonist, ramatroban, completely abrogated DK-PGD2-induced eosinophilia, suggesting that CRTH2 might have a physiological and/or pathophysiological role in controlling leukocyte migration.
Subject(s)
Bone Marrow/drug effects , Eosinophilia/chemically induced , Leukocyte Count , Leukocytes/drug effects , Prostaglandin D2/analogs & derivatives , Prostaglandin D2/pharmacology , Receptors, Immunologic/genetics , Receptors, Prostaglandin , Amino Acid Sequence , Animals , Base Sequence , Bone Marrow/physiology , Carbazoles/pharmacology , Cloning, Molecular , Humans , Leukocytes/physiology , Mice , Molecular Sequence Data , Prostaglandin D2/antagonists & inhibitors , Rats , Rats, Wistar , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/metabolism , Sequence Homology, Amino Acid , Sulfonamides/pharmacology , Th2 Cells/metabolismABSTRACT
IkappaB kinase beta (IKK-beta) is a serine-threonine protein kinase critically involved in the activation of the transcription factor Nuclear Factor kappa B (NF-kappaB) in response to various inflammatory stimuli. We have identified a small molecule inhibitor of IKK-beta. Optimization of the lead compound resulted in improvements in both in vitro and in vivo potency, and provided IKK-beta inhibitors exhibiting potent activity in an acute cytokine release model (LPS-induced TNFalpha).
