Development of New Antimicrobial Peptides by Directional Selection

<b>Background/Objectives</b>: The global rise in antibiotic resistance necessitates the development of novel antimicrobial agents. Antimicrobial peptides (AMPs), key components of innate immunity, are promising candidates. This study aimed to develop novel therapeutic peptides with enhanced properties through the mutagenesis of natural AMPs and high-throughput screening. <b>Methods</b>: We constructed mutant libraries of three broad-spectrum AMPs-melittin, cecropin, and Hm-AMP2-using mutagenesis with partially degenerate oligonucleotides. Libraries were expressed in <i>Escherichia coli</i>, and antimicrobial activity was assessed through bacterial growth kinetics and droplet serial dilution assays. Candidate molecules were identified by DNA sequencing, and the most promising variants were chemically synthesized. Antimicrobial activity was determined by minimal inhibitory concentration (MIC) against <i>E. coli</i> and <i>Bacillus subtilis</i>, while cytotoxicity was evaluated in human Expi293F cells (IC₉₀) viability. The therapeutic index was calculated as the ratio of an AMP's cytotoxic concentration to its effective antimicrobial concentration. <b>Results</b>: Mutant forms of melittin (MR1P7, MR1P8) showed significantly reduced cytotoxicity while retaining antimicrobial activity. Cecropin mutants exhibited reduced efficacy against <i>E. coli</i>, but variants CR2P2, CR2P7, and CR2P8 gained activity against Gram-positive bacteria. Mutagenesis of Hm-AMP2 generally decreased activity against <i>E. coli</i>, though two variants (A2R1P5 and A2R3P6) showed retained or enhanced efficacy against <i>B. subtilis</i> while maintaining low cytotoxicity. <b>Conclusions</b>: The proposed strategy successfully generated peptides with improved therapeutic profiles, including reduced toxicity or a broader spectrum of antimicrobial activity, despite not improving all parameters. This approach enables the discovery of novel bioactive peptides to combat antibiotic-resistant pathogens.