Antimicrobial peptides (AMPs) represent an essential component of the innate immune system that provides resistance to a variety of pathogenic bacteria. Given their broad activity against Gram positive and Gram negative bacteria, AMPs are potential candidates for novel-mechanism iv antibiotics. However, they also have significant liabilities including problems associated with their in vivo toxicity and poor tissue distribution, as well as the difficulties and expense of producing these peptides. In the previous funding period we designed a number of small molecules, sequence-specific oligomers, and polymers that address many of these problems. These compounds have robust in vivo activity against S. aureus in mouse models, suggesting a novel approach to the development of therapeutics. These small mimics of antimicrobial peptides (SMAMPs) also provide excellent handles for biophysical, biochemical, genetic and computational investigations concerning antimicrobial mechanisms. In the coming period we propose to accomplish the following objectives: Aim 1: Synthesize analogues of previously designed SMAMPs to further improve their activities, and also design new structural classes of SMAMPs. Aim2: A wide range of computational approaches will be taken to assist the design and analysis of SMAMPs, including: quantum approaches for the design and parameterization of small molecules; all atom simulations of their interactions with bilayers; and coarse-grained simulations that allow one to study the interactions of AMPs and SMAMPs with bilayers on physiologically relevant scales and time frames. Aim 3: Evaluate the activities of SMAMPs using experimental systems of increasing complexity, including: 1) binding to phospholipid monolayers and bilayers; 2) measuring disruption of bilayer integrity and/or vesicle leakage; 3) determining the activity of the peptides against a panel of Gram negative and positive bacteria including Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Staphylococcus aureus; 4) measuring in vivo activity versus S. aureus and P. aeruginosa in mouse models. Aim 4: Compare the mechanisms by which E. coli, Salmonella enterica, and S. aureus respond to AMPs versus SMAMPs using gene microarrays and genetic analyses. Microarrays will be used to broadly screen for regulons that are activated by SMAMPs and AMPs, and specific regulatory circuits will be examined using fluorescent reporters. In particular, we will focus on known regulatory circuits such as the PhoP/PhoQ two- component system and the RcsB/RcsC system, which are known to be activated by AMPs and stress to the cell envelope. PUBLIC HEALTH RELEVANCE The incidence of antibiotic-resistant infections has increased significantly in recent years and has become an important cause of morbidity and mortality in hospitals in the US and worldwide. One promising approach to address this issue is to design drugs that mimic the activities of the antimicrobial peptides that form an important part of an animal's defense against bacteria. In this proposal we develop small, drug-like molecules that are more potent, less toxic, and easier to produce than natural antimicrobial peptides.