Reactivation of an inert 15-mer antimicrobial peptide by truncation-directed i, i + 7 lactam cyclization.

Abstract

The parent 15-mer peptide exhibited no activity against Pseudomonas aeruginosa. Previous studies demonstrated that neither all-hydrocarbon stapling (i, i + 4 octenyl bridge) nor divinylpyrimidine exogenous linker cyclization conferred anti-P. aeruginosa activity upon this sequence. In the present study, this peptide served as a template for computer-assisted truncation to a 13-mer (CSP0), followed by amino acid substitution and the introduction of an i, i + 7 lactam bridge on the predicted α-helical face to afford the cyclic peptide CSP2. CSP2 exhibited potent antibacterial activity, with MICs of 8 μg/mL against P. aeruginosa ATCC 27853 (>16-fold improvement over the linear precursor CSP1), 4 μg/mL against Acinetobacter baumannii BNCC 337173 (16-fold), and 4 μg/mL against Staphylococcus aureus (8-fold). Time-kill assays revealed rapid bactericidal kinetics against P. aeruginosa (bactericidal endpoint at 2× MIC within 2 h) and dose-dependent activity against S. aureus (up to 4 log10 reduction at 4× MIC within 4 h). The minimum hemolytic concentration (MHC) was 50 μg/mL, and the 50% hemolytic concentration (HC50) exceeded 100 μg/mL, affording a therapeutic index (TI = HC50/MIC) against A. baumannii of >25. N-terminal acylation of CSP2 further enhanced antibacterial activity, with CSP2-2 exhibiting particular potency against S. aureus ST9, achieving a MIC of 4 μg/mL-a 16-fold improvement over CSP2-and a MIC of 16 μg/mL against Listeria monocytogenes ATCC13932 (4-fold). However, this augmented efficacy was concomitant with significantly elevated hemolytic toxicity; consequently, the overall therapeutic indices of the acylated derivatives failed to surpass those of CSP2.