PROJECT SUMMARY Antimicrobial drug resistance is an ongoing challenge for many serious diseases, including staphylococcal infections. Development of new antibiotics to combat methicillin-resistant Staphylococcus aureus—labeled a “serious threat” by the CDC—is a high priority. During drug development efforts, poor cellular penetration and drug-like features of compounds are a common roadblock. The studies in this proposal will advance a novel strategy to overcome this roadblock, by employing a prodrug approach, in which a bipartite molecule is activated intracellularly to release the active “warhead.” Central to our strategy is the determination of structure-activity relationships that define selective prodrug activation within S. aureus bacteria. We will advance this strategy by evaluating our preliminary prodrug SAR using two classes of inhibitors that are distinct in chemical structure and intracellular target. We will determine the enzymatic selectivity and evaluate how prodrugging alters biological properties of inhibitors, using in vitro and in vivo assays. In addition, we will use crystallographic approaches to delineate the structural features that define selective substrate recognition. Together, this project will establish and validate our approach for subsequent pre-clinical optimization of much-needed new antistaphylococcal therapies.