Moriya K, Rivera J, Odom S, Sakuma Y, Muramato K, Yoshiuchi T, Miyamoto M, Yamada K
Department of Drug Discovery, Tsukuba Research Laboratories, Eisai Company, Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 30026, Japan.
Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12539-44. doi: 10.1073/pnas.94.23.12539.
Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcepsilonRI, 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 FcepsilonRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-gamma1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor alpha were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-gamma1 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 FcepsilonRI gamma 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 Igbeta ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcepsilonRI gamma phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcepsilonRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.
肥大细胞免疫球蛋白E（IgE）高亲和力受体FcepsilonRI的激活可诱导Syk（一种非受体酪氨酸激酶）的酪氨酸磷酸化，这已被证明对脱颗粒至关重要。在此我们描述一种合成化合物ER-27319，它是啮齿动物和人类肥大细胞抗原或抗IgE介导的脱颗粒的有效且选择性抑制剂。ER-27319既不影响Lyn激酶活性，也不影响抗原诱导的FcepsilonRI磷酸化，但能有效抑制Syk的酪氨酸磷酸化及其活性。结果，磷脂酶C-γ1的酪氨酸磷酸化、肌醇磷酸的生成、花生四烯酸的释放以及组胺和肿瘤坏死因子α的分泌也受到抑制。ER-27319不抑制Jurkat T细胞中抗CD3诱导的磷脂酶C-γ1酪氨酸磷酸化，表明其对Syk诱导信号具有特异性。相反，ER-27319可特异性抑制与FcepsilonRIγ亚基的磷酸化免疫受体酪氨酸基激活基序（ITAM）体外孵育或RBL-2H3细胞抗原激活所诱导的Syk酪氨酸磷酸化和激活。然而，当将ER-27319添加到来自活化细胞的免疫沉淀Syk中时，未观察到对Syk活性的影响。ER-27319不抑制在Igbeta ITAM存在下激活诱导的Syk酪氨酸磷酸化或人外周血B细胞中抗IgM诱导的Syk磷酸化。因此，ER-27319在体外和完整细胞中选择性干扰FcepsilonRIγ磷酸化ITAM对Syk的激活。这些结果证实了Syk在肥大细胞FcepsilonRI介导的反应中的重要性，并证明了ER-27319在治疗过敏性疾病中的肥大细胞选择性和治疗潜力。
