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

免疫抑制剂对于成功的器官移植是绝对必要的，并且在 自身免疫性疾病的治疗。分析它们的作用机制可以，就像环孢素一样 和钙调神经磷酸酶，对T细胞生物学的基本特征产生了重要的见解。我们最近开始了一项 雄心勃勃的三步计划，旨在使用化学生物学方法来识别未知的途径 参与淋巴细胞功能。该项目背后的最重要的理由是 通过未知的分子机制(MMOA)抑制T细胞的激活和工作可发展为化学 可以用来识别新的细胞目标的探测器，这将揭示目前未知的方面 基础T细胞生物学，并可能成为新型免疫抑制剂的基础。 该项目的第一步-筛选~375,000的NIH分子文库小分子文库 化合物和鉴定化合物与未知的MMOA-成功。我们监测了溶血颗粒 以人TALL-104细胞毒性T淋巴细胞为模型的胞吐作用，检测LAMP-1的外化 CD107a用流式细胞仪检测。在MMOA未知的目标化合物中有2-N-[(2-甲氧基苯基)甲基]-4-N-[(4- Propan-2-ylphenyl)methyl]thieno[3，2-d]pyrimidine-2，4-diamine，CID 49792547，这是这起案件的主题 申请。该化合物抑制溶解颗粒胞吐的效力在微摩尔范围内，但不起作用。 通过我们测试的任何一种机制工作。它抑制Jurkat人白血病T细胞产生IL-2， 证实其具有广泛的免疫抑制活性。这是一种易于合成的类药物分子。 以及不同类比的产生。 该项目计划的第二步是为结构-活动分析生成类似物，然后使用 设计用于第三步也是最后一步的化学探针的信息，应用基于亲和力的和/或光- 用交联法来确定化合物活性背后的未知目标。然而， 当NIH MLPCN计划结束时，化学中心的支持被撤回。这款R03的申请书是 旨在允许我们通过为目标创建探测来继续追求项目的总体目标 身份证明。我们将创建类似的CID 49792547，然后测试它们对溶解颗粒胞吐、IL-2的影响 分泌物和毒性。这一信息将使我们能够识别可能的最高亲和力类似物，并将 揭示可用于连接连接物的位点，该连接物将使化合物与生物素偶联以产生 亲和基质以及可用于标记的双官能光交联基/点击化学基团 靶蛋白。未来的努力将指向使用在这一领域开发的探测器识别目标 在之前提交的关于另一种化合物的配套申请中。

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.