ABSTRACT
Interleukin-12 (IL-12) and IL-23 are proinflammatory cytokines and therapeutic targets for inflammatory and autoimmune diseases, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, and multiple sclerosis. We describe the discovery of APY0201, a unique small molecular IL-12/23 production inhibitor, from activated macrophages and monocytes, and demonstrate ameliorated inflammation in an experimental model of colitis. Through a chemical proteomics approach using a highly sensitive direct nanoflow LC-MS/MS system and bait compounds equipped with the FLAG epitope associated regulator of PIKfyve (ArPIKfyve) was detected. Further study identified its associated protein phosphoinositide kinase, FYVE finger-containing (PIKfyve), as the target protein of APY0201, which was characterized as a potent, highly selective, ATP-competitive PIKfyve inhibitor that interrupts the conversion of phosphatidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P2. These results elucidate the function of PIKfyve kinase in the IL-12/23 production pathway and in IL-12/23-driven inflammatory disease pathologies to provide a compelling rationale for targeting PIKfyve kinase in inflammatory and autoimmune diseases.
Subject(s)
Interleukin-12/antagonists & inhibitors , Interleukin-23/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Small Molecule Libraries/pharmacology , Animals , Cell Line , Colitis/drug therapy , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Discovery , Female , Humans , Inflammation/drug therapy , Interleukin-10/deficiency , Leukocytes, Mononuclear/drug effects , Macrophages/drug effects , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, SCID , Models, Molecular , Molecular Structure , Pyrazoles/chemistry , Pyrimidines/chemistry , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
Endothelial cells participate in the inflammatory and immune reactions. Endothelial cell activation is a recurrent phenomenon linked to the pathogenesis of diverse human diseases, such as acute and chronic inflammation and cardiovascular disorders. Pro-inflammatory cytokines (e.g., IL-1, TNF) are well-known activators of endothelial cells, since they strongly induce production of chemokines (e.g., IL-8, MCP-1) and cell adhesion molecules, resulting in an activation of inflammatory transcription factors such as NF-kappaB. We have established a cell-based reporter assay for the NF-kappaB-dependent gene activation in HUVEC. Using this assay system, we have identified a novel synthetic small molecule, APC0576, 5-(((S)-2,2-dimethylcyclopropanecarbonyl)amino)-2-(4-(((S)-2,2-dimethylcyclopropanecarbonyl)amino)phenoxy)pyridine, as an inhibitor of IL-1-induced NF-kappaB-dependent gene activation without any adverse effects on the cell viability. APC0576 represses the IL-1-induced release of chemokines (e.g., IL-8, MCP-1) in HUVEC. This inhibitory effect occurred at the level of mRNA expression. Despite having a strong inhibitory effect on the NF-kappaB-dependent transcriptional activation, APC0576 does not inhibit the IL-1-induced DNA binding of NF-kappaB, degradation of I-kappaB-alpha, or phosphorylation of RelA (p65). Although its molecular mechanism of action is not yet clear, APC0576 is a promising therapeutic candidate for diverse diseases involved in the pathogenic endothelial activation.