FRET (Forster resonance energy transfer)- is an energy transfer phenomenon which occurs between two spectrum-overlapping fluorophores in close proximity. The efficiency of FRET is highly dependent on the distance between the donor and acceptor fluorophores in a range of 100nm, which makes FRET very powerful in the detection of molecular interactions. FRET-based techniques have been extensively used in the studies of protein-protein interactions especially in living cells because of genetically encoding fluorescence proteins. Using fluorescent protein pairs to tag protein components involved in the SUMOylation process, we have successfully detect the interaction of SUMO with the E2 enzyme and one E3 ligase in living cells. Sumoylation (SUMO or small ubiquitin-like modifier) is a protein post-translational modification of proteins (e.g., p53, MDM2, STATs [signal transducer and activator of transcription], IB, and androgen and estrogen receptors) that are involved in many critical physiological processes, such as signal transduction, genome integrity, cell-cycle regulation, cell proliferation, and tumor genesis. Studies of these important processes would be greatly accelerated by both elucidating the specificity of interactions among different SUMO peptides, E2 ligase, E3 ligase and SUMO-specific proteases(SENPs) and identifying small-molecule inhibitors of sumoylation. We propose to develop new assay protocols to identify and study these inhibitors for the SUMO system. We have been using FRET-based method to analyze the interaction between SUMO and other components involved in the SUMOylation network. We will then further develop this method into a high-throughput screening assay to look for small chemical inhibitors which can specifically disrupt protein-protein interactions involved in this network. The small chemical inhibitors will not only contribute to the investigation of SUMOylation and improve our knowledge about this important process, but the proposed work will also provide a novel approach for high-throughput screening assays targeting protein-protein interactions.