Title: Bacterial cell death: Atomistic simulations reveal pore formation as a mode of action of structurally nano engineered star peptide polymers

Abstract: Multidrug resistance (MDR) to conventional antibiotics is one of the most urgent global health threats, necessitating the development of effective and biocompatible antimicrobial agents that are less inclined to provoke resistance. Structurally Nanoengineered Antimicrobial Peptide Polymers (SNAPPs) are a novel and promising class of such alternatives. These star-shaped polymers are made of a dendritic core with multiple arms made of co-peptides with varying amino acid sequences. Through a comprehensive set of in vivo experiments, we (Nature Microbiology, 1, 16162, 2016) showed that SNAPPs with arms made of random blocks of lysine (K) and valine (V) residues exhibit sub-micron M efficacy against Gram-negative and Gram-positive bacteria tested. Cryo-TEM images suggested pore formation by SNAPP with random block co-peptide arms as one of their mode of actions. However, the molecular mechanisms responsible for this mode of action of SNAPP were not fully understood. To address this gap, we employed atomistic molecular dynamics simulation technique to investigate the influence of three different sequences of amino acids, namely 1) alternating block KKV 2) random block and 3) di-block motifs on secondary structure of their arms and SNAPP's overall configuration as well as their interactions with lipid bilayer. We, for the first time identified a step-by-step mechanism through which alternating block and random SNAPPs interact with lipid bilayer and leads to pore formation, hence cell death. These insights provide a strong foundation for further optimization of the chemical structure of SNAPPs for maximum performance against MDR bacteria, therefore offering a promising avenue for addressing antibiotic resistance and development of effective antibacterial agents.