One of the greatest challenges presented by current genome sequencing projects is trying to understand the function of these many new gene products. In order to keep pace with this growing need for protein function analyses, we propose the development of a novel combinatorial protein: ligand binding assay, which will allow for the rapid identification of cell permeable, protein-specific probes on a large scale. These small organic binding ligands can be best described as 'cell-permeable monoclonal antibodies' insofar as they will be highly specific for a given gene product but yet be able to transverse the cell membrane and bind intracellular proteins. However, unlike monoclonal antibodies, derivatives of these compounds can be easily used in real time experiments to investigate the in vivo function of unknown gene products, since these ligands are cell membrane permeable. For example, the dynamic subcellular localization of a protein could be readily determined without the artificial gene overexpression, antibody injection or cell fixing/staining required by current protocols. The goal of this proposed research is to develop a methodology to identify cell membrane-permeable ligands using an in vivo binding assay and demonstrate the feasibility of its use in the large scale identification of novel protein-specific probes. Within the scope of this grant proposal, questions which will be addressed are the following: 1) Can a yeast-based assay derived from the Two Hybrid System be used with randomized crosslinking ligands from a chemical combinatorial library to identify cell membrane-permeable ligands capable of binding a desired protein sequence? 2) As a proof of concept, can the combinatorial Three Hybrid Ligand screen described above be used to identify isozyme-specific ligands able to distinguish between the related members of the Raf family of protein kinases? 3) Can this assay be generalized on large scale to rapidly identify ligands to multiple expressed cDNAs of unknown sequence simultaneously to generate a catalog of novel ligand:bound protein sequence pairs? By bridging the fields of chemistry, cell biology and the emerging field of genomics, the reagents necessary to explore protein function in the Post-Genomic Era may be developed.