Structural dynamics influences the antibacterial activity of a cell-penetrating peptide (KFF) 3 K.

Given the widespread demand for novel antibacterial agents, we modified a cell-penetrating peptide (KFF) 3 K to transform it into an antibacterial peptide. Namely, we inserted a hydrocarbon staple into the (KFF) 3 K sequence to induce and stabilize its membrane-active secondary structure. The staples were introduced at two positions, (KFF) 3 K[5-9] and (KFF) 3 K[2-6], to retain the initial amphipathic character of the unstapled peptide. The stapled analogues are protease resistant contrary to (KFF) 3 K; 90% of the stapled (KFF) 3 K[5-9] peptide remained undigested after incubation in chymotrypsin solution. The stapled peptides showed antibacterial activity (with minimal inhibitory concentrations in the range of 2-16 µM) against various Gram-positive and Gram-negative strains, contrary to unmodified (KFF) 3 K, which had no antibacterial effect against any strain at concentrations up to 32 µM. Also, both stapled peptides adopted an α-helical structure in the buffer and micellar environment, contrary to a mostly undefined structure of the unstapled (KFF) 3 K in the buffer. We found that the antibacterial activity of (KFF) 3 K analogues is related to their disruptive effect on cell membranes and we showed that by stapling this cell-penetrating peptide, we can induce its antibacterial character.