First-in-Class Cyclic Temporin L Analogue: Design, Synthesis, and Antimicrobial Assessment

Abstract

The pharmacodynamic and pharmacokinetic properties of bioactive peptides can be modulated by introducing conformational constraints such as intramolecular macrocyclizations, which can involve either the backbone and/or side chains. Herein, we aimed at increasing the α-helicity content of temporin L, an isoform of an intriguing class of linear antimicrobial peptides (AMPs), endowed with a wide antimicrobial spectrum, by the employment of diverse side-chain tethering strategies, including lactam, 1,4-substituted [1,2,3]-triazole, hydrocarbon, and disulfide linkers. Our approach resulted in a library of cyclic temporin L analogues that were biologically assessed for their antimicrobial, cytotoxic, and antibiofilm activities, leading to the development of the first-in-class cyclic peptide related to this AMP family. Our results allowed us to expand the knowledge regarding the relationship between the α-helical character of temporin derivatives and their biological activity, paving the way for the development of improved antibiotic cyclic AMP analogues.

Chart 1. Examples of Macrocyclic Peptide-Based Antibiotic Molecules (1–9), such as Lipopeptides (1 and 5), Glycopeptides (2–4), and Others, Including Temporin L (7) and Its Derivatives [Pro³]TL (8) and [Pro³,dLeu⁹]TL (9)a

Figure 1

(a) Three-dimensional (3D) structure of peptide 9 from previous NMR analysis taken in consideration for the design of cyclic peptide derivatives (PDB ID 7OS8). Peptide 9 was depicted as sticks and ribbons (green, hydrophobic residues; blue, basic residues; gray, backbone). (b) Sequences of the designed peptides 10–26 featuring diverse types of cyclization and distances. Lowercase letters in italic mean d isomers. Pra = propargylalanine; Az = azidolysine; S₅ = (S)-2-(4-pentenyl)alanine.

Scheme 1. Synthetic Strategy for Achieving Representative Compounds 12, 17, 25, and 26

(a) US–SPPS—Fmoc deprotection: 20% piperidine in DMF, 0.5 + 1 min; (b) US–SPPS—coupling: Fmoc-AA, COMU, Oxyma, DIEA, 5 min. See Scheme S14, for paths A (lactamization), B (CuAAC reaction), C (RCM reaction), and D (disulfide formation), embraced for the syntheses of 12, 17, 25, and 26, respectively, as well as for the entire peptide library.

Figure 2

Viability of peptide-treated HaCaT cells evaluated by MTT assay at 2 h (a) and 24 h (b). All data are expressed as a percentage with respect to the untreated control cells and are the mean of three independent experiments ± standard error of the mean (SEM).

Figure 3

(a) CD spectra of peptide 9 and its cyclic analogues 12, 17, 24, 25, and 26 in water. (b, c) CD spectra of peptides 9, 12, 17, 24, 25, and 26 in SDS and in DPC micelles, respectively.

Figure 4

(a, d) Percentage of aggregation as a function of peptide concentration by monitoring ThT emission and CD spectra of peptides 9, 12, and 17 in the presence of liposomes mimicking Gram-positive (a–c) and Gram-negative (d–f) membranes. (b, e), CD spectra of peptides 9, 12, and 17 in SUVs (9, black line; 12, blue line; 17, red line). (c, f) Table reports the ratio of the ellipticities at 222 and 208 nm, which discriminates between monomeric and oligomeric states of helices for peptides 9, 12, and 17.

Figure 5

Aggregate formation in the interaction between peptides 9 (a), 12 (b), and 17 (c) (20 μM) and LPS (1 mg/mL) monitored by ThT fluorescence (25 μM). Peptide alone and ThT alone were used as controls.

Figure 6

Ability of lactam-bridged peptide 12 to induce leakage of both LUVs mimicking Gram-positive (violet) and Gram-negative (blue) membranes.

Figure 7

In vitro time kill assay. S. aureus ATCC 43300 was exposed to 12.5 μM peptide 12 and 25 μM peptide 9, for 10 min, 30 min, 1 h, 2 h, 6 h, and 24 h. The inhibitory effect on bacterial growth was assessed by measuring the number of CFU obtained after the treatment.

Figure 8

Antibiofilm activity of 9 and 12 against preformed biofilm of S. aureus ATCC 25923 and A. baumannii ATCC 19606 after 2 h of peptide treatment. Biofilm viability was determined as indicated in the Materials and General Procedures section and expressed as a percentage compared to that of untreated samples (100%). Values are the mean of at least three replicates ± SEM. Data are considered to be statistically significant as follows: *p < 0.05; ***p < 0.001; ****p < 0.0001. Biofilm eradication concentration 90, BEC₉₀, was defined as the concentration able to reduce at least 90% of biofilm cells.

Figure 9

Percentage of intact lactam-stapled peptide (12) and its linear precursor (9) by an incubation with 90% human serum at 37 ± 1 °C at different time intervals (0.25, 0.75, 1.5, 2, 3, 4, 6, 7, 8, and 12 h).

Figure 10

(a) NMR-derived 10 lowest-energy structures of peptide 12 (PDB ID 7OSD). (b) Superimposition of the lowest-energy conformers of peptide 12 (gray cartoon) and 9 (purple cartoon). Helical regions are depicted as ribbon. Side chains of hydrophobic and hydrophilic residues in the C-terminal regions are reported in light (12) or dark (9) green and cyan (12) or blue (9), respectively. Bridge atoms are reported in orange.