Investigating beta-hairpin antimicrobial peptide membrane activity and toxicity.

Project Summary My immediate career goals are 1) to obtain a tenure track position as a principal investigator and 2) to improve our understanding of the structure and function of β-hairpin antimicrobial peptides to help combat antibiotic resistant pathogens. My previous expertise, along with the career development laid out in this proposal, will

allow me to complete the proposed research strategy and achieve these two immediate goals. New strategies and methods for antibiotic discovery are critically needed. Macrocyclic peptides are a promising class of antibiotic because they can target sites refractory to small molecule inhibition; however, their

large size limits their ability to bypass membranes and access intracellular targets. Macrocyclic cationic antimicrobial peptides (CAMPs) are capable of penetrating bacterial membranes, but often have associated toxicity, making them more difficult to use clinically. A small class of CAMPs with cyclic β-hairpin structure (β-

AMPs) access intracellular targets through bacterial membrane disruption and have a wide range of associated toxicity. Unfortunately, the rarity of this class prevents our understanding of how their amino acid sequence dictates their antibacterial potency and mammalian cell toxicity. I have helped developed a new synthetic

macrocyclic peptide screening technique which has identified thousands of synthetic β-AMPs, expanding sequence information and allowing us to begin analyzing how their amino acid sequence impacts their antibacterial potency, cell membrane specificity, and ultimately their therapeutic potential. During Phase I of this proposal, I plan to biochemically analyze a newly acquired dataset of putative β-

AMPs with diverse antibacterial potency and mammalian cell toxicity and use this data to train a machine learning algorithm to identify sequence features promoting antibacterial specificity. The in vivo activity of lead peptides will then be examined using the Galleria mellonella infection model. During Phase II, I will examine

how β-AMPs interact with and permeate cell membranes composed of different lipids using strains with genetically modified lipopolysaccharide, in vitro liposome disruption assays, and ultraviolet photodissociation mass spectrometry. This will help us understand how β-AMPs overcome traditional methods of CAMP

resistance and have an ability to selectively target bacterial cell membranes. Lastly, through modification of our screening technique, I have generated predictive data for how over 7,000 mutational variants of one promising natural β-AMP's amino acid sequence impacts its antimicrobial activity. I plan to evaluate the accuracy of these

predictions through biochemical evaluation of 48 of the 7,000 variants. Differences in the toxicity and membrane specificity of this group will also be evaluated and compared to the native sequence to better understand how certain residues impact therapeutic potential.