Abstract
Antimicrobial peptides (AMPs) have gained attention as potential alternatives to conventional antibiotics due to their broad-spectrum activity against multidrug-resistant pathogens. This project builds upon an ongoing in silico optimization strategy, which aims to obtain shortened peptide fragments with an improved drug profile from in-house AMPs from the black soldier fly. The in silico approach allows the construction and evaluation of large collection of short peptides, with the best AMP candidates being selected for further experiments. The selected AMPs will be synthesized using solid-phase peptide synthesis (SPPS), purified to high purity, and lyophilized for further testing. The antibacterial activity of the peptides will be evaluated against clinically relevant strains, including E. coli, P. aeruginosa, and S. aureus using standardized high-throughput screening methods to determine their minimum inhibitory concentration (MIC), IC50, and minimum bactericidal concentration (MBC). To assess therapeutic potential, cytotoxicity studies will be performed on human lung fibroblasts and erythrocytes to ensure selectivity. Stability assays will determine the peptides' resistance to salt and pH variations, stability in lung fluid and serum, and proteolytic degradation. The mechanism of action will be investigated through a propidium iodide (PI)-based fluorescence assay to monitor membrane permeabilization in real time, while scanning electron microscopy (SEM) will provide high-resolution images of bacterial membrane disruption. This comprehensive approach aims to deliver AMPs with improved efficacy, stability, and safety, serving as a solid foundation for further drug development projects.
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