Screening for inhibitors of the T cell Tec kinase, ltk

筛选 T 细胞 Tec 激酶 ltk 抑制剂

• 批准号: 7993303
• 负责人: AMY H ANDREOTTI
• 金额: $ 17.56万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993303
• 关键词: Actins; Active Sites; Affinity; Animal Model; Animals; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Binding; Biochemical; Biological Assay; Bypass; C57BL/6 Mouse; CD28 gene; CD4 Positive T Lymphocytes; Cells; Communities; Disease; Disease Progression; Docking; Dose; Drug Kinetics; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Escherichia coli; Exhibits; Failure; Family; Goals; Human; Immune system; In Vitro; Individual; Infiltration; Insecta; Intervention; Investigation; Ionomycin; Lead; Length; Luminescent Measurements; Lymphocyte; Lymphoid Tissue; Measures; Monoclonal Antibody HuM291; Mus; Noise; Organ; PH Domain; Pathogenesis; Patients; Peptides; Phospholipase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Phosphotyrosine; Play; Process; Protein Tyrosine Kinase; Proteins; Reaction; Relative (related person); Reporting; Reproducibility; Role; Screening procedure; Self Tolerance; Serum; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; T cell response; T-Cell Activation; T-Lymphocyte; TEC Protein Tyrosine Kinase; Technology; Testing; Time; Tissues; Tyrosine; autoreactive T cell; base; cell motility; chemokine receptor; counterscreen; cytokine; high standard; high throughput screening; improved; in vivo; in vivo Model; inhibitor/antagonist; interest; mouse model; novel; polymerization; prevent; public health relevance; small molecule; src Homology Region 2 Domain; stem

DESCRIPTION (provided by applicant): Autoimmune diseases result from a breakdown of self-tolerance. Disease progression involves several key steps, including the inappropriate activation of autoreactive lymphocytes followed by the infiltration of pathogenic effectors T cells into the target tissues. Our recent studies have focused on a mouse model of multi- organ autoimmune disease that results from the absence of the costimulatory molecule, CTLA-4 (i.e., Ctla4-/- mice). In this model, mice succumb to a rapid and fatal disease that results from massive T cell activation, infiltration into vital organs, and the subsequent failure of those organs. We have found that the Tec family tyrosine kinase ITK plays a critical role in the process of autoreactive T cell migration into tissues in this multi- organ autoimmune disease. Thus, Itk-/-Ctla4-/- mice show unprecedented T cell activation and proliferation, but are protected from the lethal autoimmunity of Ctla4-/- mice due to a failure of these activated effectors T cells to accumulate in non-lymphoid tissues. In addition, we can prevent the onset of severe autoimmune disease in Ctla4-/- mice by treatment with a small molecule ITK inhibitor. ITK is a well-characterized signaling protein that is activated by TCR, CD28, and chemokine receptor stimulation; in turn, ITK activates phospholipase-Cg and induces actin polymerization. These findings indicate that ITK signaling in effectors T cells is critical in regulating T cell infiltration into tissues, and more importantly, that ITK is essential for the pathogenesis of autoimmune T cells. In this project, we propose to establish and validate a biochemical assay to screen for novel small molecule inhibitors of ITK. Although two groups have previously reported the identification of ITK inhibitors, these inhibitors are of relatively low potency and have poor pharmacokinetics in vivo. Moreover, the screens that produced these compounds used the isolated ITK kinase domain and so yielded only ATP-site directed inhibitors. Based on our in depth biochemical investigations of full length ITK and our recent description of a remote ITK substrate docking mechanism, we have begun to establish a greatly improved ITK in vitro kinase assay, in which the Km of ITK for its substrate is >15-fold higher than in previously-established assays. Use of this novel assay will provide a platform for identifying inhibitors that are efficacious at lower concentrations and with higher selectivity for ITK. In the first aim, we will optimize the ITK in vitro kinase assay to maximize reproducibility and to determine assay conditions amenable to HTS. In the second aim, we will establish cell-based and whole animal assays for ITK inhibitors as secondary and tertiary screens, including counter-screens for ITK inhibition. Our overall goal is to establish a robust high-throughput screen to identify novel, selective, and high affinity small molecule inhibitors of ITK, and ultimately, to assess the efficacy of these inhibitors in animal models of organ-specific autoimmune diseases, as well as in the inhibition of human T cell migration and diapedesis. PUBLIC HEALTH RELEVANCE: Autoimmune diseases occur when an individual's immune system attacks his/her own cells and organs. We have identified an enzyme that plays an essential role in this disease process. This proposal aims to develop a screen for inhibitors of this enzyme, which could be used to prevent the onset of autoimmune processes in susceptible individuals and to treat and cure patients with these diseases.

描述（由申请人提供）：自身免疫性疾病是由于自身耐受性的破坏。疾病进展涉及几个关键步骤，包括自身反应性淋巴细胞的不适当激活，随后是致病性效应T细胞浸润到靶组织中。我们最近的研究集中在多器官自身免疫性疾病的小鼠模型上，所述多器官自身免疫性疾病是由于缺乏共刺激分子CTLA-4（即，Ctla 4-/-小鼠）。在这个模型中，小鼠死于一种快速致命的疾病，这种疾病是由大量T细胞激活、浸润到重要器官以及随后这些器官的衰竭引起的。我们已经发现Tec家族酪氨酸激酶ITK在这种多器官自身免疫性疾病中的自身反应性T细胞迁移到组织中的过程中起关键作用。因此，Itk-/-Ctla 4-/-小鼠显示出前所未有的T细胞活化和增殖，但由于这些活化的效应T细胞不能在非淋巴组织中积累，因此受到保护免于Ctla 4-/-小鼠的致死性自身免疫。此外，我们可以通过用小分子ITK抑制剂治疗来预防Ctla 4-/-小鼠中严重自身免疫性疾病的发作。ITK是一种充分表征的信号传导蛋白，其通过TCR、CD 28和趋化因子受体刺激激活;反过来，ITK激活磷脂酶-Cg并诱导肌动蛋白聚合。这些发现表明，效应T细胞中的ITK信号传导在调节T细胞浸润到组织中是至关重要的，更重要的是，ITK对于自身免疫T细胞的发病机制是必不可少的。在这个项目中，我们建议建立和验证一个生化检测来筛选新的ITK小分子抑制剂。尽管两个研究小组先前已经报道了ITK抑制剂的鉴定，但这些抑制剂的效力相对较低，并且在体内的药代动力学较差。此外，产生这些化合物的筛选使用分离的ITK激酶结构域，因此仅产生ATP位点定向抑制剂。基于我们对全长ITK的深入生物化学研究和我们最近对远程ITK底物对接机制的描述，我们已经开始建立大大改进的ITK体外激酶测定，其中ITK对其底物的Km比先前建立的测定高>15倍。使用这种新的测定法将提供一个平台，用于鉴定在较低浓度下有效且对ITK具有较高选择性的抑制剂。在第一个目标中，我们将优化ITK体外激酶测定，以最大限度地提高重现性，并确定适合HTS的测定条件。在第二个目标中，我们将建立用于ITK抑制剂的基于细胞的和整体动物测定作为二级和三级筛选，包括用于ITK抑制的反筛选。我们的总体目标是建立一个强大的高通量筛选，以确定新的，选择性的，高亲和力的ITK小分子抑制剂，并最终评估这些抑制剂在器官特异性自身免疫性疾病的动物模型中的疗效，以及在抑制人类T细胞迁移和渗出。 公共卫生相关性：当个体的免疫系统攻击他/她自己的细胞和器官时，就会发生自身免疫性疾病。我们已经确定了一种在这种疾病过程中起重要作用的酶。该提案旨在开发这种酶的抑制剂的筛选，其可用于预防易感个体的自身免疫过程的发生，并治疗和治愈患有这些疾病的患者。