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绝对至关重要，并且在 自身免疫性疾病的治疗。分析其作用机理可以，例如环孢霉素 和钙调神经酶，对T细胞生物学的基本特征产生重要的见解。我们最近开始了 雄心勃勃的三步项目旨在使用化学生物学方法来识别未知途径 参与淋巴细胞功能。该项目的基本原理是，化合物是 抑制T细胞激活和通过未知分子机制（MMOA）的工作可以发展为化学 可以用来识别新型细胞靶标的探针，这将揭示当前未知的方面 基本的T细胞生物学，可能成为新类免疫抑制剂的基础。 项目的第一步 - 筛选NIH的分子库小分子存储库约为375,000 具有未知MMOA-成功的化合物和识别化合物。我们监测了裂解颗粒 使用高104人类细胞毒性T淋巴细胞作为模型的胞吐作用，测量LAMP-1/的外部化 CD107A使用流式细胞仪。在未知MMOA的命中中为2-N - [（2-甲氧基苯基）甲基] -4-N - [（4--（4-） 丙烷-2-基苯基）甲基] Thieno [3,2-D]嘧啶-2,4-二胺，CID 49792547，这是该主题 应用。这种复合抑制裂解颗粒的胞吐作用，在微摩尔范围内抑制效力，但没有 通过我们测试的任何机制来工作。它抑制Jurkat人类白血病T细胞的IL-2产生， 确认具有广泛的免疫抑制活性。它是一种类似药物的分子，可与合成 以及不同的类似物的产生。 该项目的意图第二步是生成类似物进行结构激活分析，然后使用该类似物 在第三步和最后一步中设计用于使用的化学探针的信息，应用基于亲和力和/或照片 交联方法以识别基于该化合物活性的未知目标。然而， NIH MLPCN计划结束时，化学中心支持被撤回。 R03的此应用程序是 旨在让我们通过为目标创建探测来继续追求项目的整体目标 鉴别。我们将创建CID 49792547的类似物，然后测试其对裂解颗粒胞吐作用的影响，IL-2 分泌和毒性。这些信息将使我们能够确定最高的亲和力类似物，并将 揭示可用于连接接头的站点，该站点将允许化合物耦合到生物素进行创造 可用于标记的亲和力矩阵和双功能的照片连接/点击化学组 靶蛋白。未来的努力将用于使用此处开发的探针来实现目标识别 应用程序和同伴应用程序提交了以前的侧重于其他化合物。