ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcɛRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcɛRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcɛRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcɛRI γ phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcɛRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.

Inverse agonism of histamine H2 antagonist accounts for upregulation of spontaneously active histamine H2 receptors.

Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time- and dose-dependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leu124Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonist-independent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. The displayed inverse agonism of H2 antagonists appears to be a mechanistic basis for the observed H2 antagonist-induced H2 receptor upregulation in transfected CHO cells. These observations shed new light on the pharmacological classification of the H2 antagonists and may offer a plausible explanation for the observed development of tolerance after prolonged clinical use.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.