ABSTRACT
A synthetic glucocorticoid receptor (GR) ligand with the efficacy of a glucocorticoid, but without the accompanying side effects, would meet an unmet medical need for the treatment of inflammatory diseases. It was hypothesized that a GR ligand that shifted helix 12 in a manner distinct from an agonist and an antagonist would confer a distinct GR conformation, resulting in differential gene expression and, ultimately, dissociation of antiinflammatory activity from side effects. A structural feature expected to interfere with helix 12 was incorporated into a nonsteroidal, tricyclic scaffold to create novel, high-affinity, and selective GR ligands that manifested a dual function in cellular assays, partial but robust agonist activity for inflammatory cytokine inhibition, and full antagonist activity for reporter gene activation. In contrast, analogs not likely to hinder helix 12 exhibited partial agonist activity for reporter gene activation. The requirement of full antagonist activity for substantial side effect dissociation was demonstrated in primary human preadipocytes, hepatocytes, and osteoblasts in which effects on adipogenesis, key genes involved in gluconeogenesis, and genes important for bone formation were examined, respectively. The dissociated GR ligands, despite lacking significant reporter gene activation, weakly recruit a limited number of coactivators such as peroxisomal proliferator-activated receptor-γ coactivator 1α. Transcriptional activation was sensitive to both peroxisomal proliferator-activated receptor-γ coactivator 1α and GR levels, providing a basis for cell-selective modulation of gene expression. The antiinflammatory activity of the dissociated ligands was further demonstrated in mouse models of inflammation. Together these results suggest that these ligands are promising candidates with robust antiinflammatory activity and likely dissociation against glucocorticoid-induced side effects.
Subject(s)
Glucocorticoids/adverse effects , Receptors, Glucocorticoid/drug effects , Adipocytes/cytology , Adipocytes/drug effects , Animals , Anti-Inflammatory Agents/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Female , Gluconeogenesis/drug effects , Heat-Shock Proteins/physiology , Humans , Mice , Mifepristone/pharmacology , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Receptors, Glucocorticoid/agonists , Receptors, Glucocorticoid/antagonists & inhibitors , Transcription Factors/physiologyABSTRACT
Series of aminopyridinecarboxamide-based inhibitors were synthesized and tested against human recombinant IKK-2 and in IL-1beta stimulated synovial fibroblasts. The 2-amino-5-chloropyridine-4-carboxamides were identified as the most potent inhibitors with improved cellular activity.
Subject(s)
Aminopyrine/chemistry , Aminopyrine/pharmacology , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , I-kappa B Kinase/antagonists & inhibitors , Interleukin-1beta/antagonists & inhibitors , Amides/chemistry , Amides/pharmacology , Amino Acid Sequence , Arthritis, Rheumatoid/drug therapy , Fibroblasts/drug effects , Fibroblasts/immunology , Humans , I-kappa B Kinase/metabolism , I-kappa B Proteins/chemistry , Interleukin-1beta/metabolism , Joint Capsule/cytology , Molecular Sequence Data , Structure-Activity RelationshipABSTRACT
A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of the production of inflammatory cytokine IL-8 in IL-1beta stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating their potential usefulness as NF-kappaB regulators.
Subject(s)
Protein Serine-Threonine Kinases/antagonists & inhibitors , Thiophenes/pharmacology , Cells, Cultured , Fibroblasts/drug effects , Humans , I-kappa B Kinase , Interleukin-8/antagonists & inhibitors , Interleukin-8/biosynthesis , Thiophenes/chemistryABSTRACT
NF-kappaB is sequestered in the cytoplasm by the inhibitory IkappaB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IkappaBs leading to their degradation that results in NF-kappaB activation. IKK-1 and IKK-2 are two direct IkappaB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IkappaBalpha peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rhIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions.
