In Project 2, we will develop three key areas of technology that will make it feasible to screen the million-member libraries developed in Project I and determine their mechanism of action. These are: 1) a high-density, nanoliter-volume assay format to enable extremely high-throughput screening, 2) a general screening method to enable assays to be performed as small-molecule-dependent genetic selections, and 3) a new expression cloning method to identify the protein targets of small molecules. No such general strategies exist and, once developed, these technologies will be generally useful for drug discovery in academia and industry. Conventional 96-well plate format assays are expensive, and are neither rapid nor sensitive enough to screen the small amounts of million-member libraries to be constructed in Project 1. We have recently developed a new nanoliter-volume assay format, called nanodroplets. We have already shown that yeast cell growth can be used as a useful readout in the nanodroplet format. We propose to adapt this format for assays in mammalian cells and extend its utility with a number of technical advances. We will initially use the nanodroplet format to identify compounds that are cytotoxic to yeast or mammalian cells. As the nanodroplet technology evolves, we will adapt the biological assays developed in Project 3 to this screening format. Because cell growth is such an effective readout in the nanodroplet format, we have genetically engineered yeast strains to make their growth dependent on the presence of a small molecule. This new assay takes two forms: in once case, the small molecule must bind a specific protein in order to yeast to grow; in the other it must interfere with a specific protein-protein interaction to allow growth. Here we propose to adapt these small-molecule-dependent genetic selection assays to identify new ligands that alter interactions between proteins know to be relevant to cancer. The targets of the drug leads discovered in cytotoxicity assays, or in the cytological assays proposed in Project 3, will be unknown. Here we propose to adapt a new in vitro expression cloning strategy to identify the protein targets of these small molecules. This will provide insight into the likely utility of the new agent, and set the stage for the determination of structure-activity relationships and optimization (to be performed by the Synthetic Chemistry Core).