Stapled β-Hairpin Antimicrobial Peptides with Improved Stability and Activity against Drug-Resistant Gram-Negative Bacteria

Abstract

Different stapling techniques have been used recently to address the subpar performance of antimicrobial peptides (AMPs) in clinical trials with ample focus on α-helical AMPs. In comparison, a systematic evaluation of such strategies on β-hairpin AMPs is lacking. Herein, we report the design, synthesis, and evaluation of a library of all-hydrocarbon-stapled β-hairpin AMPs with variation in key parameters intended as potent therapeutics against drug-resistant pathogens. We observed an interesting interplay between the activity, stability, and structural strength. Single-stapled peptides with a 6-carbon staple at peptide termini such as 5(c⁶) displayed the most potent activity against colistin-resistant clinical isolates. Using imaging techniques, we observed translocation of 5(c⁶) across bacterial membranes without causing extensive damage. Overall, we have engineered novel all-hydrocarbon-stapled β-hairpin AMPs with structural and functional proficiency that can effectively combat resistant pathogens, with findings from this study a point of reference for future interests in developing novel β-hairpin AMPs.

Figure 1

(A) Fmoc-based SPPS and on-resin RCM of BTT3 analogues. (B) Library of 12 stapled β-hairpin peptides synthesized using SPPS and RCM based on the unstapled parent peptide, BTT3.

Figure 2

Heat map depicting the MIC and HC₅₀ of the peptides against the ATCC strains (E. coli, S. aureus, and P. aeruginosa) and colistin-resistant clinical isolates of E. coli (R1, R2, and R3). The corresponding resistance mutation in each of these isolates are listed in Table S3. Colistin was used as a positive control for E. coli, P. aeruginosa, and the clinical strains. Vancomycin was used as a positive control for S. aureus.

Figure 3

(A) Minimum inhibitory concentration (MIC) of the peptides when treated with trypsin (1:2000), 10% serum, and 150 mM NaCl against E. coli. Stability of peptides treated with (B) trypsin (1:2000), (C) 10% serum, and (D) 150 mM NaCl (represented as fold change in MIC with respect to the original MIC). (E) The stability of peptides after treatment with trypsin at a 1:2000 (trypsin/peptide) molar ratio at 37 °C for different periods (15, 30, 60, and 90 min) was analyzed using a C18 RP-HPLC column. The AUC measured from the HPLC run is displayed as % intact peptide.

Figure 4

Circular dichroism (CD) spectra of the peptides measured at room temperature (25 °C) in (A) aqueous buffer (10 mM Tris–Cl buffer) and (B) in the presence of 25 mM sodium dodecyl sulfate (SDS), mimicking the negatively charged bacterial microenvironment. Each spectrum represents an average of three measurements. (C) Table showing θ_M signals at β-sheet-specific wavelengths and % anti-parallel β-sheet of the peptides when incubated with SDS, as estimated using the web-based tool BeStSel (http://bestsel.elte.hu/index.php).

Figure 5

Outer (A) and inner (B) membrane permeabilization profile of BTT3 and 5(c⁶) against E. coli evaluated using 1-N-phenylnaphthylamine (NPN) and SYTOX green uptake assay, respectively, at three different concentrations (4, 8, and 16 μM). The data is presented based on three repeats. Localization of stapled peptide in E. coli assessed using (C) confocal laser scanning microscopy (CLSM) (scale = 10 μm) and (D) three-dimensional (3D)-SIM (scale = 0.4 μm) upon treatment with FITC-tagged 5(c⁶) (green) at 1× MIC for 1 h. The bacterial membrane is stained with FM 4–64 dye (red). (E) Membrane permeabilization profile of different stapled peptides in E. coli studied using the degree of bacterial membrane damage upon treatment with 1× MIC of peptide 5(c⁶) assessed using transmission electron microscopy (TEM). (F) Killing kinetics of 5(c⁶) was determined against E. coli ATCC 25922. Black dotted line indicates the limit of detection (50 CFU/mL). Experiments were repeated two times.