Sulfotyrosine-guided discovery of small molecule chemokine inhibitors

摘要 此行政补充请求的目标是提供一种微型热电泳仪，用于 为R01 GM097381的趋化因子药物发现测量蛋白质-配体和蛋白质-蛋白质相互作用。 趋化因子主要通过结合和激活G蛋白偶联受体家族来指导细胞迁移 翻译后被酪氨酸硫化修饰。趋化因子的功能需要硫酸盐化，建议 硫代酪氨酸受体直接参与趋化因子配体的特异性结合。我们正在确认 三种主要的促转移趋化因子(CXCL12、CCL19和CCL21)的小分子配体和 将它们优化为受体结合的竞争性抑制剂，从而阻止癌细胞迁移。建议数 该仪器将允许测定小分子与趋化因子以及趋化因子之间的亲和力- 趋化因子和趋化因子-受体的相互作用。微尺度热浸疗法(MST)提供了一种诱人的 对目前使用的工具的补充，因为它能够使用少一到五个数量级的工具 采样，在几分钟而不是几小时内收集数据，并能够对膜和细胞使用MST 裂解物。该仪器将安装在PIS实验室，作为化学生物学(PCB)计划的一部分 在威斯康星医学院。印刷电路板已经配备了必要的基础设施 该仪器已成功安装并投入使用。新仪器将由博士级别的人员进行维护 具有生物物理仪器操作和维护经验的员工。与它的记录一致 在生物物理研究基础设施和设施方面的重大投资中，MCW承诺以空间换房屋 所要求的仪器和维护所需的部分薪金。

ABSTRACT The goal of this administrative supplement request is to provide a microscale thermophoresis instrument for measuring protein-ligand and protein-protein interactions for chemokine drug discovery for R01 GM097381. Chemokines direct cell migration primarily by binding and activating a family of G protein-coupled receptors that are post- translationally modified by tyrosine sulfation. Sulfation is required for chemokine function, suggesting that receptor sulfotyrosines participate directly in specific binding of the chemokine ligand. We are identifying small molecule ligands of the three main pro-metastatic chemokines (CXCL12, CCL19 and CCL21) and optimizing them as competitive inhibitors of receptor binding that block cancer cell migration. The proposed instrument will allow determination of affinities between small molecules and chemokines as well as chemokine- chemokine and chemokine-receptor interactions. Microscale thermophoresis (MST) offers an attractive complement to the tools currently employed because of its ability to use one to five orders of magnitude less sample, collect data in minutes rather than hours, and the ability to use MST with membranes and cell lysates. This instrument will be installed in the PIs laboratory as part of the Program in Chemical Biology (PCB) at the Medical College of Wisconsin. The PCB is already equipped with the necessary infrastructure for successful installation and operation of this instrument. The new instrument will be maintained by PhD-level staff with experience in the operation and maintenance of biophysical instruments. Consistent with its record of major investments in biophysical research infrastructure and facilities, MCW has committed space to house the requested instrument and partial salary for its maintenance.