Covalent Fluorescent Probes for Cancer Cell Detection

摘要：荧光成像是一个强大的工具，允许可视化特定的细胞状态的人口，然而，现有的荧光标记的方法是不实验上可用于许多生物系统。此外，细胞状态的荧光小分子传感器可以提供一种有价值的替代方案，相对于现有的荧光成像方法具有显着的优势。的总目标 本项目旨在研制一种荧光小分子ATP位点定向探针，它可以选择性地标记特定的激酶，并作为正常和病理细胞状态的成像探针。蛋白激酶在许多方面是用于开发癌症生物学中使用的选择性小分子成像探针的理想靶标。 这是因为蛋白激酶参与大多数细胞过程，并且其定位、可接近性和丰度的变化与细胞状态的变化相关。蛋白激酶已被用作癌症生物学中的生物标志物，因为点突变、基因缺失、基因扩增和染色体重排引起的内源性激酶调节机制的丧失已得到充分证实 是许多癌症的关键事件。本项目的具体目标是：1）合成能够与ATP位点半胱氨酸残基形成共价键的荧光标记激酶抑制剂; 2）使用目标1中制备的化合物的基于显微镜的筛选来鉴定作为正常和病理细胞状态的选择性探针的化合物; 3）鉴定目标2中鉴定的活性化合物的细胞内靶标。 这项提议是Nathanael Gray博士（Dana Farber癌症研究所）、Priscilla Yang（哈佛医学院）和Wei Zhang（麻省大学波士顿分校）之间正在进行的合作的延伸。这三个人也是达纳法伯/哈佛癌症中心的成员。该提案的一个优点是，每个Co-Pl负责三个具体目标之一，使项目受益于每个人及其后续机构为伙伴关系带来的平衡技能和专业知识。因为所有三 研究人员的职业生涯刚刚开始，格雷博士、杨博士和张博士将从职业发展活动和内部和外部导师中受益。此外，培训核心将大大有助于这一项目。

Abstract: Fluorescence imaging is a powerful tool that permits visualization of specific cell states within a population; however, existing methods for fluorescence labeling are not experimentally accessible for many biological systems. Furthermore, fluorescent small-molecule sensors of cell state may provide a valuable alternative with significant benefits relative to existing methods for fluorescence imaging. The overall goal of this project is to create fluorescent small molecule ATP-site directed probes that can selectively label particular kinases and serve as imaging probes of normal versus pathological cell state. Protein kinases are in many ways ideal targets for the development of selective small molecule imaging probes for use in cancer biology. This is because protein kinases are involved in most cellular processes and changes in their localization, accessibility, and abundance are associated with changes in cellular state. Protein kinases have been used as biomarkers in cancer biology because the loss of endogenous kinase regulatory mechanisms by point mutations, gene deletions, gene amplifications, and chromosomal rearrangements has been well-established as crucial events in many cancers. The specific aims of this project are to: 1) Synthesize fluorescently-tagged kinase inhibitors capable of forming covalent bonds with ATP-site cysteine residues; 2) Use microscopy-based screening ofthe compounds prepared in Aim 1 to identify compounds that are selective-probes of normal and pathological cellular states and 3) Identify the intracellular target(s) of active compounds identified in Aim 2. This proposed is the extension of an ongoing collaboration between Drs. Nathanael Gray (Dana Farber Cancer Institute), Priscilla Yang (Harvard Medical School) and Wei Zhang (UMass Boston). All three are also members of the Dana Farber/Harvard Cancer Center. One of the strong points of this proposal is that each Co-Pl is responsible for one of the three Specific Aims allowing the project to be benefited by the balanced skills and expertise that each person and their subsequent institution brings to the partnership. Because all three investigators are eariy in their careers, Drs. Gray, Yang and Zhang will benefit from career development activities and mentors who are intemal and extemal. Additionally, the Training Core will significantiy contribute to this project.