Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index

Abstract

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.

Fig. 1. Representation of the “host” peptide V₆₈₁ as helical nets showing the polar/hydrophilic face (boxed residues) and nonpolar/hydrophobic face (circled residues) and helical wheel and the sequences of the synthetic peptide analogs used in this work.

The hydrophobic face is indicated as an open arc, and the hydrophilic face is shown as a solid arc in the helical wheel. The substitution sites at positions 11 and 13 are in triangles on the polar face and the nonpolar face. S11X _l and S11X _d denote polar face substitutions of Ser-11. V13X _l and V13X _d denote nonpolar face substitutions of Val-13. Ac denotes N ^α-acetyl, and amide denotes C ^α-amide. One-letter codes are used for the amino acid residues.

Fig. 2. CD spectra of peptides V13L_d and V13L_l (panel A) and peptides V13K_d and V13K_l (panel B) at pH 7 and 25 °C in 50 mm aqueous PO₄ containing 100 mm KCl.

Panels A and B, solid symbols represent the CD spectra of peptide analogs in KP buffer without TFE, and open symbols represent CD spectra obtained in the presence of 50% TFE. The symbols used are as follows: circle for V13L_d and square for V13L_l in panel A; diamond for V13K_dand triangle for V13K_l in panel B.

Fig. 3. CD temperature denaturation of peptide V₆₈₁.

Panel A, the experiment was carried out in 0.05% aqueous trifluoroacetic acid, pH 2, in the presence of 50% TFE. CD spectra at different temperatures are shown as different lines in the figure. Panel B, stability plot of peptide V₆₈₁ during CD temperature denaturation.

Fig. 4. Effect of temperature on RP-HPLC profiles of peptide C, peptide V13K_l, and V13L_l.

Conditions are as follows: RP-HPLC, narrow-bore SB-C8 column (150 × 2.1-mm inner diameter; 5-μm particle size, 300-Å pore size), linear A-B gradient (1% acetonitrile/min) at a flow rate of 0.25 ml/min, where eluent A is 0.05% aqueous trifluoroacetic acid and eluent B is 0.05% trifluoroacetic acid in acetonitrile. Only RP-HPLC profiles of peptide C, peptide V13K_l, and peptide V13L_l at 5, 35, and 80 °C were selected as examples to show the temperature effect.

Fig. 5. RP-HPLC temperature profiles of peptide V₆₈₁ and its analogs.

Column and conditions are as in Fig. 4. Retention data have been collected in 3 °C increments within the temperature range of 5–80 °C. Open symbols represent the temperature profiles of l-amino acid substituted peptides on either the nonpolar or polar face of V₆₈₁ (panels A and C); solid symbols represent the temperature profiles of d-amino acid substituted peptides on either the nonpolar or polar face of V₆₈₁ (panels B and D). In all panels, the substituting amino acids used in either the nonpolar or polar face of V₆₈₁ are Val (circle), Leu (square), Ala (diamond), Ser (triangle), Lys (inverted triangle), and Gly (×). The temperature profile of the random coil control peptide (C) is also shown in the figure (+).

Fig. 6. Normalized RP-HPLC temperature profiles of peptide V₆₈₁ and its analogs.

Temperature profiles normalized to retention behavior of random coil peptide C. Column and conditions are as in Fig. 4. The retention behavior of the peptides was normalized to that of the random coil peptide C through the expression (t _R ^t − t _R ⁵ for peptides) minus (t _R ^t − t _R ⁵ for C), where t _R ^tis the retention time at a specific temperature of an antimicrobial peptide or the random coil peptide, and t _R ⁵ is the retention time at 5 °C. Open symbols represent the temperature profiling of l-amino acid substituted peptides on either the nonpolar or polar face of V₆₈₁ (panels A and C, respectively); solid symbols represent d-amino acid substituted peptides on either the nonpolar or polar face of V₆₈₁ (panels B and D, respectively). In all panels, amino acids used for substitution in either the nonpolar or polar face of V₆₈₁ are Val (circle), Leu (square), Ala (diamond), Ser (triangle), Lys (inverted triangle), and Gly (×).