PROJECT SUMMARY Methicillin-resistant Staphylococcus aureus (MRSA) continues to gain resistance to existing antibiotics, and the lack of big pharma pipelines to develop new compounds that target superbugs means that serious attention is needed within the academic research community. Although a limited number of compounds have been put forth to address the MRSA resistance problem, there is still a critical need to develop novel compounds with improved antibacterial activity. Synthetic unsaturated fatty acids (uFAs) are attractive candidates to become next- generation antibacterial agents for treating MRSA infections because they appear to have multiple mechanisms of action, making it more difficult for bacteria to develop resistance. In addition, synthetic uFAs can directly kill multi-drug resistant bacteria at very low concentrations (i.e., at the micromolar and nanomolar level). Recent experimental data suggest that the cytotoxic activity of either 2-hexadecynoic acid (2- HDA, triple-bonded FA) or DAT-51(double-bonded FA) against MRSA is due to their ability to disrupt the cell membrane, possibly by pore formation. The long-term goal of this project is to define the mechanisms that explain the total antibacterial activity of uFAs and apply this knowledge to develop a next-generation of synthetic uFAs with even better efficacy as antibacterial agents. Our central hypothesis is that uFAs such as 2-HDA or DAT-51 can induce membrane disruption through direct pore formation. Two independent but related Specific Aims proposed here will test our central hypothesis. In the first aim, we will determine if 2-HDA and/or DAT- 51 disrupt the cell membrane of S. aureus using flow cytometry, DNA/RNA leakage assays, and electron microscopy. The second aim will define the inhibitory effects of 2-HDA or DAT-51 on S. aureus peptidoglycan biosynthesis by using Western blot and standard colorimetric assays. The knowledge acquired from this project will significantly impact the field because it will allow us to establish the chemical and biological foundations required for the targeted design of the next generation of synthetic uFAs with improved antibacterial activity. Therefore, the successful completion of this project is likely to lead to the development of new therapeutic options to treat devastating MRSA infections.