To Fold or Not to Fold: Diastereomeric Optimization of an α-Helical Antimicrobial Peptide

Abstract

Membrane disruptive α-helical antimicrobial peptides (AMPs) offer an opportunity to address multidrug resistance; however, most AMPs are toxic and unstable in serum. These limitations can be partly overcome by introducing D-residues, which often confers protease resistance and reduces toxicity without affecting antibacterial activity, presumably due to lowered α-helicity. Here, we investigated 31 diastereomers of the α-helical AMP KKLLKLLKLLL. Three diastereomers containing two, three, and four D-residues showed increased antibacterial effects, comparable hemolysis, reduced toxicity against HEK293 cells, and excellent serum stability, while another diastereomer with four D-residues additionally displayed lower hemolysis. X-ray crystallography confirmed that high or low α-helicity as measured by circular dichroism indicated α-helical or disordered structures independently of the number of chirality switched residues. In contrast to previous reports, α-helicity across diastereomers correlated with both antibacterial activity and hemolysis and revealed a complex relationship between stereochemistry, activity, and toxicity, highlighting the potential of diastereomers for property optimization.

Figure 1

(a) Chemical structure of ln65. (b) Helix wheel of ln65 sequence predicted by HeliQuest. Blue and yellow indicate, respectively, cationic and hydrophobic residues. The arrow inside the helix wheel indicates the magnitude and direction of the hydrophobic moment. (c) Structure of ln65 (PDB 7NEF, chain I) obtained by X-ray crystallography of a fucosylated analogue in complex with the bacterial lectin LecB. Cationic side chains are colored in blue, and hydrophobic side chains are colored in red.

Figure 2

CD-spectra of ln65, dln65, ln69, dln69, sr-ln65, sr-ln65L⁶, HP5, HP7, and HP9, measured with 0.1 mg/mL peptide in phosphate buffer at pH 7.4 with 10 and 20% v/v 2,2,2-TFE and with 5 mM DPC of (a) ln65 (full lines) and dln65 (dashed lines), (b) ln69 (full lines) and dln69 (dashed lines), (c) sr-ln65 (full lines) and sr-ln65L⁶ (dashed lines), (d) HP5, (e) HP7, and (f) HP9.

Figure 3

(a) Percentage of undegraded peptide after 24 h incubation in 12.5% human serum in TRIS buffer (pH 7.4) at 37 °C. Data are presented in mean ± SD, n = 3. See the Supporting Information for full curves. (b) Toxicity on HEK293 and A549 cells represented as the IC₅₀ measured by Alamar blue assay after 24 h treatments with concentrations of peptide ranging from 0 to 200 μM. Data are presented as the mean ± SD, n = 3. See the Supporting Information for all data and procedure.

Figure 4

X-ray crystallography of mixed-chirality AMPs. (a) X-ray crystal structure of the FHP5·LecB complex (PDB 8AN9, chain E). Left panel: peptide is represented in stick, Ca²⁺ atoms in magenta spheres, and LecB in green cartoon. Blue mesh represents electron density (0.5σ level). Right panel: Stick model of the FHP5 crystal structure, lysine side chains shown in blue, and leucine side chains shown in red. (b) Superposition of the two complete non-equivalent peptides in the unit cell of PDB 8AN9. Fucose is omitted for more clarity. (c) Same as (a) for X-ray structure of the FHP8· LecB complex (PDB 8ANO, chain H). Electron density is shown for a 0.7σ level. (d) Same as (b) for the two complete non-equivalent peptides in the unit cell of PDB 8ANO. (e) X-ray structures of the two different asymmetric peptides in the FHP30·LecB complex (PDB 8ANR). Same color code as shown in (a). Electron densities are shown for a 1.0σ level. (f) Left panel: H-bonds between two symmetrical FHP30 chains. Right panel: full bundle of four symmetrical FHP30 chains. Lectin monomers and calcium atoms were omitted for clarity in the right panel. Same color code as shown in (a).

Figure 5

MD simulations of HP5 with and without DPC micelle. (a) Average structure (stick model) in the presence of DPC micelle over 100 structures sampled during the last 100 ns (thin lines). Hydrophobic side chains are colored in red, and cationic side chains are colored in blue. DPC micelle was omitted for clarity. (b) Last frame of the 250 ns run with DPC micelle. Peptide backbone is in gray cartoon, cationic side chains are colored in blue, hydrophobic side chains are colored in red, and DPC molecules are represented in spheres. (c) Same as (a) for run in water. (d) Comparison of root-mean-square deviation of the peptide backbone relative to the starting coordinates of the α-helix built in PyMol between run with DPC and run in water. (e) Comparison of the number of intramolecular backbone hydrogen bonds between run with DPC and run in water.

Figure 6

Statistical analysis of data set measured on ln65 derivatives. (a) Scatter plot of % helicity in 5 mM DPC against log₂(MIC) for A. baumannii ATCC19606. (b) Same as (a) for log2(MHC). (c) Loading analysis of principal components 1 and 2. α = α-helix, β = β-sheet, t = turn, and u = unordered. Visualization of the (PC1 and PC2) plane. Each point represents one compound and is color coded depending on (d) activity on A. baumannii and (e) hemolytic activity.