Creating a Chemical Probe to Identify the Target of a Novel Immune Suppressing Compound

创建化学探针来识别新型免疫抑制化合物的靶标

• 批准号: 9226933
• 负责人: ADAM ZWEIFACH
• 金额: $ 7.98万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-07 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9226933
• 关键词: Acetylation; Affinity; Alcohols; Allogenic; Amination; Amines; Antibodies; Applications Grants; Autoimmune Diseases; Award; Binding; Biological Assay; Biology; Biotin; CD3 Antigens; Calcineurin; Calcium Channel; Calmodulin; Cells; Cellular biology; Chemicals; Chemistry; Chlorides; Companions; Coupled; Cyclosporine; Cytoplasmic Granules; Cytotoxic T-Lymphocytes; Diamines; Exocytosis; Flow Cytometry; Funding; Future; Gel; Generations; Goals; Human; Immune; Immunosuppression; Immunosuppressive Agents; Interleukin-2; Jurkat Cells; LAMP-1; Label; Lead; Libraries; Lymphocyte Function; Lytic; Measures; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular; Molecular Bank; Molecular Mechanisms of Action; Monitor; Organ Transplantation; Pathway interactions; Pharmaceutical Preparations; Phase; Phenols; Positioning Attribute; Potassium Channel; Process; Production; Protein Kinase C; Proteins; Pyrimidine; Reagent; Site; Solid; Structure; Structure-Activity Relationship; T-Cell Activation; T-Lymphocyte; Testing; Toxic effect; Transplantation; United States National Institutes of Health; Work; analog; base; cellular targeting; crosslink; cytokine; design; direct application; esterase; insight; novel; programs; repository; screening; small molecule libraries

Immunosuppressants are absolutely essential for successful organ transplantation and are useful in the treatment of autoimmune disorders. Analyzing their mechanism of action can, as in the case of cyclosporine and calcineurin, yield important insights into basic features of T cell biology. We recently embarked upon an ambitious three-step project intended to use chemical biology approaches to identify unknown pathways involved in lymphocyte function. The over-arching rationale underlying the project was that compounds that inhibit T cell activation and work via unknown molecular mechanism (MMOA) could be developed into chemical probes that could be used to identify novel cellular targets, which would reveal currently-unknown aspects of basic T cell biology and might become the basis for new classes of immunosuppressant agents. The first step of the project- screening the NIH's Molecular Libraries Small Molecule Repository of ~375,000 compounds and identifying compounds with unknown MMOA- succeeded. We monitored lytic granule exocytosis using TALL-104 human cytotoxic T lymphocytes as a model, measuring externalization of LAMP-1/ CD107a using flow cytometry. Among hits with unknown MMOA was 2-N-[(2-methoxyphenyl)methyl]-4-N-[(4- propan-2-ylphenyl)methyl]thieno[3,2-d]pyrimidine-2,4-diamine, CID 49792547, which is the subject of this application. This compound inhibits lytic granule exocytosis with potency in the micromolar range, but does not work via any of the mechanisms we tested. It inhibits IL-2 production by Jurkat human leukemic T cells, confirming that has broad immunosuppressive activity. It is a drug-like molecule that is amenable to synthesis and the generation of diverse analogs. The project's intended second step was to generate analogs for structure-activity analysis, then use that information to design chemical probes to use in the third and final step, applying affinity-based and/or photo- crosslinking approaches to identify the unknown target that underlies the compound's activity. However, Chemistry Center support was withdrawn when the NIH MLPCN program ended. This application for an R03 is intended to allow us to continue to pursue the overall goals of the project by creating probes for target identification. We will create analogs of CID 49792547, then test their effects on lytic granule exocytosis, IL-2 secretion and toxicity. This information will allow us to identify the highest possible affinity analogs and will reveal sites that can be used to attach linkers which will allow the compound to be coupled to biotin for creation of an affinity matrix and to bifunctional photo-crosslinking/ click-chemistry groups that can be used to label target proteins. Future efforts will be directed towards target identification using probes developed in this application and in a companion application submitted previously focused on another compound.

免疫抑制剂对于成功的器官移植是绝对必要的， 自身免疫性疾病的治疗。分析它们的作用机制，就像环孢菌素一样， 和钙调神经磷酸酶，产生对T细胞生物学的基本特征的重要见解。我们最近开始了一项 一个雄心勃勃的三步计划，旨在利用化学生物学方法来确定未知的途径 参与淋巴细胞功能。该项目的基本原理是， 抑制T细胞活化并通过未知分子机制（MMOA）起作用可被开发成化学品 探针，可用于识别新的细胞靶点，这将揭示目前未知的方面， 基础的T细胞生物学，并可能成为新的免疫抑制剂的基础。 该项目的第一步-筛选NIH的分子库小分子库~ 375，000 化合物和鉴定未知MMOA的化合物-成功。我们监测了溶解颗粒 使用TALL-104人细胞毒性T淋巴细胞作为模型的胞吐作用，测量LAMP-1/ CD 107 a，使用流式细胞术。在具有未知MMOA的命中中是2-N-[（2-甲氧基苯基）甲基]-4-N-[（4-甲氧基苯基）甲基]-N-甲基-N- 丙-2-基苯基）甲基]噻吩并[3，2-d]嘧啶-2，4-二胺，CID 49792547，其是本申请的主题。 应用程序.该化合物抑制溶解性颗粒胞吐作用的效力在微摩尔范围内，但不 通过我们测试的任何机制工作。它抑制Jurkat人白血病T细胞产生IL-2， 证实它具有广泛的免疫抑制活性。它是一种类似药物的分子， 和产生不同的类似物。 该项目的第二步是产生类似物进行结构活性分析，然后使用 信息来设计化学探针以用于第三步和最后一步，应用基于亲和力的和/或光- 交联的方法来确定未知的目标，基础上的化合物的活性。然而，在这方面， 当NIH MLPCN项目结束时，化学中心的支持被撤回。R 03的应用程序是 目的是让我们继续追求项目的总体目标，为目标创建探测器， 识别.我们将创建CID 49792547的类似物，然后测试它们对溶解颗粒胞吐、IL-2 分泌和毒性。这些信息将使我们能够识别最高可能的亲和力类似物， 揭示了可用于连接接头的位点，所述接头将允许化合物偶联至生物素以用于产生 和双官能光交联/点击化学基团， 靶蛋白。未来的努力将针对使用本发明中开发的探针的目标识别。 在先前提交的一份针对另一种化合物的配套申请中，