From a marine neuropeptide to antimicrobial pseudopeptides containing aza-β(3)-amino acids: structure and activity

Abstract

Incorporation of aza-β(3)-amino acids into an endogenous neuropeptide from mollusks (ALSGDAFLRF-NH(2)) with weak antimicrobial activity allows the design of new AMPs sequences. Depending on the nature of the substitution, this can render the pseudopeptides inactive or lead to a drastic enhancement of the antimicrobial activity without high cytotoxicity. Structural studies of the pseudopeptides carried out by NMR and circular dichroism show the impact of aza-β(3)-amino acids on peptide structure. The first three-dimensional structures of pseudopeptides containing aza-β(3)-amino acids in aqueous micellar SDS were determined and demonstrate that the hydrazino turn can be formed in aqueous solution. Thus, AMP activity can be modulated through structural modifications induced by the nature and the position of such amino acid analogues in the peptide sequences.

Figure 1

Hemolytic activity of analogs on rabbit erythrocytes.

Figure 2

Cytotoxic activity of analogs on Chinese Hamster Ovarian cells (CHO-K1 cell line).

Figure 3

CD spectra of peptides AD and AK (A), and pseudopeptides Aβ³K, K2-Nal7 (B), K-1Nal (C), temperature dependence of K-2Nal (D). Buffer: 10 mM Phosphate buffer, SDS: 30 mM SDS in buffer

Figure 4

The 20 lowest energy structures with no violations > 0.3 Å from the 60 structures calculated in the final iteration of ARIA for neuropeptide analogs in SDS micelles. (A) AD, (B) AK, the two set of structures of (C) Aβ³K, (D) K-2nal7 and (E) K-2Nal for the two possible configurations of the aza-β³-lysine and the aza-β³-naphtylalanine, respectively. The superposition was performed using the backbone atoms of residues 5–10. For clarity, the side chains of residues 1–4 are not drawn. The backbone of all structures is shown in black while the side chains of the Phe residues are displayed in red, the Asp or Lys residues in green, the Ala residues in orange, the Leu residues in purple, and the Arg residues in blue.

Figure 4

The 20 lowest energy structures with no violations > 0.3 Å from the 60 structures calculated in the final iteration of ARIA for neuropeptide analogs in SDS micelles. (A) AD, (B) AK, the two set of structures of (C) Aβ³K, (D) K-2nal7 and (E) K-2Nal for the two possible configurations of the aza-β³-lysine and the aza-β³-naphtylalanine, respectively. The superposition was performed using the backbone atoms of residues 5–10. For clarity, the side chains of residues 1–4 are not drawn. The backbone of all structures is shown in black while the side chains of the Phe residues are displayed in red, the Asp or Lys residues in green, the Ala residues in orange, the Leu residues in purple, and the Arg residues in blue.

Figure 4

The 20 lowest energy structures with no violations > 0.3 Å from the 60 structures calculated in the final iteration of ARIA for neuropeptide analogs in SDS micelles. (A) AD, (B) AK, the two set of structures of (C) Aβ³K, (D) K-2nal7 and (E) K-2Nal for the two possible configurations of the aza-β³-lysine and the aza-β³-naphtylalanine, respectively. The superposition was performed using the backbone atoms of residues 5–10. For clarity, the side chains of residues 1–4 are not drawn. The backbone of all structures is shown in black while the side chains of the Phe residues are displayed in red, the Asp or Lys residues in green, the Ala residues in orange, the Leu residues in purple, and the Arg residues in blue.

Figure 5

The best structures (with the lowest energies) of AD (green), AK (cyan), Aβ³K (yellow), K-2Nal7 (pink) and K-2Nal (grey). The side chains of the polar and hydrophobic residues are respectively in blue and orange. Aza-β³ residues snapshots are available for the Aβ³K and K-2Nal7 peptides (see also Table S2).

Figure 6

(A) The structural features of the Aβ³K. The carbon atoms of the R-hydrazino turn are displayed in green and the two β-turn carbon atoms involving the residue 2-5 and 5-8, surrounding this N-N turn, are respectively shown in cyan and in magenta, side chains have been removed for clarity. Hydrogen bonds are represented with dashed lines. (B) Distances and torsion angles in the hydrazino turn.