A NOVEL MECHANISM-BASED MAMMALIAN-CELL ASSAY FOR THE IDENTIFICATION OF SH2-DOMAIN SPECIFIC PROTEIN-PROTEIN INHIBITORS

Background: Many intracellular signal-transduction pathways are regula ted by specific protein-protein interactions. These interactions are m ediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, prc -homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As pro domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets. Resu lts: We have developed a novel mechanism-based cellular assay to monit or specific SH2-domain-dependent protein-protein interactions. The ass ay is based on a two-hybrid system adapted to function in mammalian ce lls where the pro domain ligand is phosphorylated, and binding to a sp ecific pro domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to m onitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppres sive and anti-osteoclastic compounds, respectively, and demonstrate he re the utility of this system for the identification of small-molecule , cell-permeant pro domain inhibitors. Conclusions: A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2- domain-depnndent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for c ell-permeant, nontoxic inhibitors of protein-protein interactions.