Abstract: Antimicrobial resistance (AMR) represents a global public health crisis, with an estimated 1 0 million deaths per year expected by 2050 without significant public health intervention and the development of new classes of antibiotics. Towards combating this crisis, the parent grant funding of this supplement proposal has facilitated the development of GmPcides, a novel class of synthetic antibiotics that target a wide range of Gram-positive pathogens including vancomycin-resistant Enterococci (VRE), erythromycin and clindamycin-resistant streptococci, methicillin-resistant and vancomycin-insensitive Staphylococcus aureus (MRSA, VISA; respectively). Second generation GmPcides, including the lead compound PS757, have bactericidal activity against stationary phase VRE as well as both exponential and stationary phase MRSA and have shown synergy when combined with several classes of existing antibiotics. Further optimization of GmPcides have led to fourth generation compounds with an sp3-rich 2-pyridone core with dramatically improved compound solubility with equal or greater potency against VRE and MRSA than PS757. Treatment with lead 4th generation compounds has also been shown to reduce ulcer size and time to healing in a murine skin and soft tissue infection model. While resistance mutants can be selected against second generation compounds, repeated attempts to generate resistance against fourth generation sp3-rich 2-pyridone GmPcides have proven unsuccessful. While good for prospects of clinical efficacy, the lack of detectable resistance mutants has complicated identification of the cellular targets of 4th generation compounds, which may be essential and difficult to assess with standard approaches. This proposal will elucidate essential targets in MRSA of GmPcides in vitro and virulence determinants that are important for survival to GmPcide challenge in the murine SSTI model. Understanding the molecular interactions between GmPcides and their essential targets in vitro and during in vivo infection will allow for further refinement of 4th generation GmPcides that improve their clinical effectiveness.