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
The cytokine interleukin-10 (IL-10) potently inhibits macrophage function through activation of the transcription factor STAT3. The expression of SOCS3 (suppressor of cytokine signaling-3) has been shown to be induced by IL-10 in a STAT3-dependent manner. However, the relevance of SOCS3 expression to the anti-inflammatory effect of IL-10 on macrophages has been controversial. Through kinetic analysis of the requirement for SOCS3 in IL-10 inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNFalpha) transcription and translation, SOCS3 was found to be necessary for TNFalpha expression during the early phase, but not the late phase of IL-10 action. SOCS3 was essential for IL-10 inhibition of LPS-stimulated production of iNOS (inducible nitric-oxide synthase) protein and nitric oxide (NO). To determine the domains of SOCS3 protein important in mediating these effects, SOCS3-/- macrophages were reconstituted with SOCS3 mutated for the SH2, KIR, SOCS box domains, and tyrosines 204 (Tyr204) and 221 (Tyr221). The SH2 domain, SOCS box, and both Tyr204 and Tyr221 were required for IL-10 inhibition of TNFalpha mRNA and protein expression, but interestingly the KIR domain was necessary only for IL-10 inhibition of TNFalpha protein expression. In contrast, Tyr204 and Tyr221 were the only structural features of SOCS3 that were necessary in mediating IL-10 inhibition of iNOS protein expression and NO production. These data define SOCS3 as an important mediator of IL-10 inhibition of macrophage activation and that SOCS3 interferes with distinct LPS-stimulated signal transduction events through differing mechanisms.