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.