Thymidylate Kinase-Targeted Antimicrobial Peptides via Phage Display: A Novel Strategy against Gram-Negative Bacteria

The rise of antimicrobial resistance (AMR) in Gram-negative bacteria, including Escherichia coli (E. coli), poses a major public health threat. This study aimed to address the limitations of existing antimicrobial peptides (AMPs) by designing hybrid peptides with enhanced targeting and antibacterial potency. Eight heptapeptide sequences were identified through phage display screening and hybridized with WP (WKKIWKPGIKKWIK), a peptide exhibiting weak antimicrobial activity against Gram-negative bacteria. The hybrid peptides were systematically evaluated for their antimicrobial activity, specificity, and biocompatibility. The hybrid peptide SWP exhibited superior antibacterial activity, particularly against E. coli K88 (TI = 2.378), and demonstrated specific binding to thymidylate kinase (TMK), a key bacterial enzyme. In vivo studies employing a mouse peritonitis model confirmed SWP’s ability to reduce bacterial loads and mitigate tissue damage while maintaining excellent biocompatibility. These findings underscore SWP as a promising candidate for the development of targeted antimicrobial agents with enhanced specificity and stability for Gram-negative pathogens.