Advances in screening technologies have made ligand discovery against biological targets routine, but converting binding ligands into specific enzyme inhibitors is extremely challenging, even for metalloproteinases and other enzymes with well-defined active sites. The lack of specific inhibitors prevents full elucidation of biological processes as basic as extracellular matrix remodeling. Proteins and small molecules each lack key features of inhibitors. Antibodies and other proteins rarely disrupt enzyme function, but usually exhibit high binding specificity. Small molecules frequently lack single-enzyme specificity, but interfere with enzymatic activity. Neither of these modalities is well-suited for generating potent, specific enzyme inhibitors. My long-term goals are to 1) establish general principles for discovering potent, specific inhibitors against medically relevant enzymes;; and 2) utilize the resulting inhibitors to understand the roles of enzymes such as metalloproteinases in normal physiology and pathological processes. I hypothesize that simultaneously leveraging the complementary strengths of proteins and small molecules will give rise to entirely new classes of potent, specific inhibitors. The goal during this proposal period is to convert yeast display, a powerful ligand discovery platform, into a comprehensive inhibitor discovery platform. My lab has already established strategies for expanding the chemical functionality that can be utilized in combination with yeast display. Here, we will enhance our platform further and use it to identify inhibitors against a test set of metalloproteinase targets. In the process, we will gain fundamental insights into how to generate inhibitors that are not accessible using any current inhibitor discovery approaches, setting the stage for 1) a greatly expanded toolkit for studying basic biology;; and 2) much broader inhibitor discovery efforts. The initial directions we will pursue are: Direction 1. Expand the range of chemistries that can be encoded in yeast-displayed proteins. Proteins containing canonical amino acids lack key groups found in enzyme inhibitors. We will utilize our quantitative reporter of ncAA incorporation to encode these functionalities in yeast-displayed proteins. Direction 2. Establish assays for quantitatively evaluating enzyme inhibition on the yeast surface. No existing display technologies support quantitative evaluations of enzyme inhibition during high throughput screening. We will utilize dual yeast display technology to establish these capabilities. Direction 3. Use chemically augmented antibody libraries to evolve potent, specific inhibitors. Antibodies rarely inhibit enzymes. We will generate and screen libraries of antibodies containing added chemical groups to establish general principles for inhibitor isolation in this unexplored discovery space. To focus our discovery efforts, we and our collaborators have identified metallo...