ABSTRACT
Because phosphoinositide 3-kinase (PI3K) plays a central role in cellular activation, proliferation, and survival, pharmacologic inhibitors targeting components of the PI3K pathway are actively being developed as therapeutics for the treatment of inflammatory disorders and cancer. These targeted drugs inhibit the activity of either PI3K itself or downstream protein kinases. However, a previously unexplored, alternate strategy is to activate the negative regulatory phosphatases in this pathway. The SH2-containing inositol-5'-phosphatase SHIP1 is a normal physiologic counter-regulator of PI3K in immune/hematopoietic cells that hydrolyzes the PI3K product phosphatidylinositiol-3,4,5-trisphosphate (PIP(3)). We now describe the identification and characterization of potent and specific small-molecule activators of SHIP1. These compounds represent the first small-molecule activators of a phosphatase, and are able to activate recombinant SHIP1 enzyme in vitro and stimulate SHIP1 activity in intact macrophage and mast cells. Mechanism of activation studies with these compounds suggest that they bind a previously undescribed, allosteric activation domain within SHIP1. Furthermore, in vivo administration of these compounds was protective in mouse models of endotoxemia and acute cutaneous anaphylaxis, suggesting that SHIP1 agonists could be used therapeutically to inhibit the PI3K pathway.
Subject(s)
Anaphylaxis/drug therapy , Endotoxemia/drug therapy , Enzyme Inhibitors/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/metabolism , Polycyclic Compounds/pharmacology , Sesquiterpenes/pharmacology , Allosteric Regulation , Anaphylaxis/metabolism , Anaphylaxis/pathology , Animals , Calcium/metabolism , Cells, Cultured , Endotoxemia/metabolism , Endotoxemia/pathology , Enzyme Activation/drug effects , Gene Expression Regulation, Enzymologic , Humans , Immunoprecipitation , Inositol Polyphosphate 5-Phosphatases , Kidney/cytology , Kidney/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mast Cells/drug effects , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Structure , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphorylation/drug effects , Polycyclic Compounds/chemistry , Porifera/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sesquiterpenes/chemistry , Signal Transduction/drug effects , Skin TestsABSTRACT
Pulmonary emphysema is a major cause of mortality and morbidity in chronic obstructive pulmonary disease (COPD). Cigarette smoking is a major risk factor in the development of pulmonary emphysema. In this study, we investigated the acute effect of cigarette smoke in vitro on the production of tumour necrosis factor-alpha (TNF-alpha) using differentiated U937 cells, a macrophage model system. We found that stimulation of the macrophages with cigarette smoke media (CSM) leads to a rapid activation of extracellular-regulated kinases 1 and 2 (erk1/2), p90RSK and a transient decrease in phosphorylation of PKB/akt. The CSM also caused the subsequent induction of TNF-alpha release. Our studies revealed that U0126, an inhibitor of the erk1/2 pathway, markedly suppressed CSM-induced TNF-alpha release. Consistent with this finding, U0126 blocked CSM-stimulated erk1/2 phosphorylation, as well as phosphorylation of the downstream kinase, p90RSK. On the other hand, the PI3-K inhibitor, LY294002, and epidermal growth factor receptor (EGFR)-specific inhibitor, AG1478, did not suppress the release of TNF-alpha. Thus, CSM induction of TNF-alpha production by differentiated macrophages is regulated primarily via the erk1/2 pathway.