PROJECT SUMMARY The emergence of resistance to almost every antibiotic underscores the urgent need to introduce new therapeutics and to understand antibiotic modes of action to help guide the development of new antimicrobials effective against drug-resistant organisms. S. aureus together with Enterobacter species, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecium, are categorized as the ESKAPE pathogens and are predominant causes of hospital-acquired infection worldwide. Among these, methicillin-resistant Staphylococcus aureus is the leading cause of mortality from antibiotic-resistant infections in the United States. Moreover, the propensity of these pathogens to form biofilms and persister cells is linked to recurrent and chronic infections leading to pneumonia, endocarditis, bacteremia, and sepsis. Biofilms consist of slow-growing bacterial cells surrounded by a protective extracellular matrix, while persister cells are dormant, highly antibiotic-tolerant bacteria that can persist in the host. Worldwide, tuberculosis is the second most common cause of death, following deaths from HIV/AIDS, and is caused by Mycobacterium tuberculosis. In the U.S., respiratory infections from non-tuberculosis mycobacteria (NTM) are increasing, notably prevalent among CF patients and individuals suffering from chronic lung disease. The treatment for NTM is complex, similar to that for TB, and requires prolonged combination drug therapy as monotherapy is highly associated with drug resistance. We have initiated an antibiotic discovery and mode-of-action activity program directed at the development of new therapeutics for these serious infectious diseases. In this proposed project, we leverage our recent success in designing a new vancomycin derivative, a vancomycin-D- octaarginine (V-r8) conjugate, that eradicates Gram-positive biofilm and persister cells and reduces pathogenesis in vivo. We propose to uncover new discoveries regarding V-r8’s unique mode of action, as compared to major high-value therapeutics that are now the drugs of last resort, e.g. oritavancin, towards its development and clinical potential and to inspire the generation of new antibacterial agents. The research design integrates interdisciplinary chemical and biochemical expertise and perspectives; mechanistic biochemistry; and integration of solid-state NMR approaches to measure compositional changes in whole cells and to determine distances between V-r8 and possible multiple binding sites. Furthermore, we will launch a new experimental solid-state NMR platform to enable us to evaluate drug modes of action in mycobacteria. This platform will be broadly applicable to investigations of complex mycobacterial cell walls and will be specifically directed here to interrogate the activity of CPZEN-45, an exciting therapeutic candidate for the treatment of both Mycobacterium tuberculosis and NTM.