Serine ?-lactamases are the major resistance mechanism to ?-lactam antibiotics, such as penicillin. In the last 25 years, hundreds of mutant ?-lactamases have appeared in the clinic in response to newer ?-lactams. These enzymes are a pressing medical problem and a fascinating example of molecular evolution. One goal of this research program is to understand the molecular bases of this evolving activity. A second goal is to exploit this information to discover novel inhibitors. Such inhibitors may escape the current cycle of incremental antibiotic modification followed by rapid resistance response. The specific aims are: 1. To understand the molecular mechanisms of evolved mutant ?-lactamases. Initially introduced to confer stability to ?-lactamases, 3rd generation cephalosporins have promoted the evolution of ?-lactamase mutants. The range of substitutions, and the structural reorganization to which they lead, can be breathtaking. We consider the following questions: a. How do "extended spectrum" substitutions increase activity against once "?-lactamase-stable" cephalosporins? b. How do distant gain-of-function substitutions communicate their effects to the active site? c. Does the activity increase come at a cost to internal stability? We target mutants of the class A and class C ?-lactamases, which are the most widespread. Clinically isolated resistance mutants will be cloned, expressed, their kinetics and stability measured. Their x-ray structures will be determined in complex with substrates and transition-state analogs. These studies will not only inform our understanding of ?-lactamases, but also other resistance enzymes for which they are model systems. 2. To discover novel inhibitors of wild-type and mutant ?-lactamases. Guided by the structural studies of substrate recognition and evolution from aim 1, new ?-lactamase inhibitors will be designed. Both novel molecules, unrelated to ?-lactams, and transition-state analogs will be investigated, using a combination of structure-based design, synthetic elaboration, and crystallography. The following will be explored. a. What is the range of novel chemotypes that can be discovered for ?-lactamase; can these inhibitors be optimized for affinity? b. Optimization of leads from the last period: for transition-state analogs to the sub-nanomolar range, and for novel chemotypes to the sub-micromolar range. c. Will substrate-mimics be more susceptible to pre-evolved resistance mechanisms than the novel inhibitors?  ?-lactamases are the major resistance mechanism to penicillin and related antibiotics. In the last 25 years, hundreds of mutant ?-lactamases have appeared in the clinic in response to newer drugs. One goal of this research program is to understand the molecular bases of this evolving activity. A second is to exploit this information to discover novel inhibitors to escape the current cycle of incremental antibiotic modification followed by rapid resistance response.