New classes of antibacterials are required to combat the spread of antimicrobial resistance (AMR). AMR is an underlying factor in ~5 million deaths worldwide each year and deaths due to resistant microbes are predicted to overtake cancer by 2050. Despite this grave forecast, the pipeline of new antimicrobials with new targets is surprisingly limited, with most compounds in clinical trials being modified versions of existing antimicrobials (e.g., fluoroquinolones, beta-lactams, aminoglycosides, etc.). Novel classes of antibacterials have the potential to combat AMR because existing resistance to these new agents is not predicted to be widespread. In this proposal, we will explore the use of ozonide synthetic peroxides to kill diverse species of bacterial pathogens. Ozonides are currently being developed as antimalarials and have pharmacokinetic (PK) and safety properties that make them attractive as potential antibacterials, including oral bioavailability, excellent tissue distribution, long half-life enabling daily dosing, and proven safety in humans. We have screened ozonide analogs for activity against Mycobacterium tuberculosis (Mtb) and discovered that some have antimycobacterial activity. For example, against whole cell Mtb, the ozonides OZ539 and OZ707 have growth inhibition half- maximal effective concentrations (EC50) of 5 µM and 2 µM, respectively. Given the antibacterial properties of ozonides against Mtb, we tested OZ539 against a panel of bacterial pathogens including M. abscessus (Mab) and M. smegmatis, and selected Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram- negative (E. coli and Proteus vulgaris) pathogens. We observed that OZ539 inhibited growth of mycobacteria and the Gram-positive pathogens, with EC50 from 2-8 µM, but had no activity against the Gram-negative strains. Transcriptional profiling studies of Mtb treated with OZ539 identified significant differential regulation of iron- acquisition and storage genes, suggesting that ozonides may function by targeting iron-dependent pathways. The goal of this R03 is to determine the potential utility of ozonides as antibacterials. Towards this goal, we will test ozonides against a diverse panel of bacterial pathogens to determine their spectrum of activity (Aim 1.1). Next, we will screen a structurally diverse collection of ozonides for activity against a panel of prioritized pathogens (Aim 1.2). Based on this initial screen, we will conduct a more focused screen driven by the identified structure–activity relationships (SAR). We will then prioritize analogs for studies examining their ability to inhibit growth in different in vitro conditions associated with pathogenesis (Aim 1.3). We will conclude the study by exploring the potential mechanism of action of the ozonides, testing the hypothesis that ozonides target iron- dependent pathways (Aim 1.4). Overall, this proposal will provide the key data needed to determine if ozonides hold promise for future development as antibacterials.

PROJECT NARRATIVE New classes of antibacterials are required to combat the spread of antimicrobial resistance (AMR). AMR is an underlying factor in ~5 million deaths worldwide each year and deaths due to resistant microbes are predicted to overtake cancer by 2050. Novel classes of antibacterials have the potential to combat AMR because existing resistance to these new agents is not predicted to be widespread.