De Novo Designed Amphipathic α-Helical Antimicrobial Peptides Incorporating Dab and Dap Residues on the Polar Face To Treat the Gram-Negative Pathogen, Acinetobacter baumannii

Abstract

We have designed de novo and synthesized ten 26-residue D-conformation amphipathic α-helical cationic antimicrobial peptides (AMPs), seven with "specificity determinants", which provide specificity for prokaryotic cells over eukaryotic cells. The ten AMPs contain five or six positively charged residues (d-Arg, d-Lys, d-Orn, l-Dab, or l-Dap) on the polar face to understand their role in hemolytic activity against human red blood cells and antimicrobial activity against seven Acinetobacter baumannii strains, resistant to polymyxin B and colistin, and 20 A. baumannii worldwide isolates from 2016 and 2017 with antibiotic resistance to 18 different antibiotics. AMPs with specificity determinants and with l-Dab and l-Dap residues on the polar face have essentially no hemolytic activity at 1000 μg/mL (380 μM), showing for the first time the importance of these unusual amino acid residues in solving long-standing hemolysis issues of AMPs. Specificity determinants maintained excellent antimicrobial activity in the presence of human sera.

Figure 1.

Helical wheel (upper panel) and helical net (lower panels) representations of our amphipathic helical AMPs. In the helical wheel, the nonpolar face is indicated as a yellow arc (Leu residues are colored yellow and the Lys “specificity determinants” at positions 13 and 16 are colored red). Lys 1 is also on the nonpolar face and colored red. The polar face is indicated as a black arc (positively charged residues at positions 3, 7, 11, 18, 22, and 26 are denoted by X and are colored blue). In the helical net (left side), the residues on the polar face are boxed and shown along the center of the net with the positively charged residues at positions 3, 7, 11, 18, 22, and 26 also colored blue. In the helical net (right side), the residues on the nonpolar face are circled (Leu residues are colored yellow) and shown along the center of the net. The Lys “specificity determinants” are at positions 13 and 16 in the center of the nonpolar face between the hydrophobic clusters of Leu residues.

Figure 2.

Helical wheel (upper panels) and helical net (lower panels) representations of an AMP with and without “specificity determinants”. Lys 13 and Lys 16 (colored red) are in the center of the nonpolar face (left side) between the two clusters of hydrophobic Leu residues (colored yellow). The right side shows the helical net in the absence of specificity determinants, where Lys residues are replaced with Ala 13 and Ala 16, thus maintaining a continuous hydrophobic surface along the center of the helix. The positively charged residues on the polar face are indicated in the helical wheels with an X at positions 3, 7, 11, 18, 22, and 26.

Figure 3.

Percent lysis of human red blood cells vs peptide concentration of AMPs. Panel (A): The sequences of the five peptides (all containing Lys specificity determinants at positions 13 and 16 of the nonpolar face; Figure 1) are shown in Table 1. Note that peptide denotations in this figure have been abbreviated from those shown in Table 1; for example, D87(Lys¹-6 Arg-1) has been shortened to D87(6Arg-1), where 6Arg-1 denotes six Arg residues on the polar face at positions 3, 7, 11, 18, 22, and 26. Panel (B): Comparison of percent lysis of the Dap- and Dab-containing peptides in the presence and absence of Lys specificity determinants at positions 13 and 16. The sequences of the peptides are shown in Table 1. HC₅₀ values (concentration of peptide that results in 50% hemolysis of human red blood cells after 18 h at 37 °C) derived from such data are shown in Table 4.

Figure 4.

Relative hydrophobicity of AMPs as expressed by RP-HPLC elution time. Column: Zorbax 300 SB-C8, 150 × 2.1 mm ID, 5 μm particle size, 300 Å pore size; conditions, linear AB gradient (0.25% acetonitrile/min) at a flow rate of 0.3 mL/min, where eluant A was 20 mM aq TFA and eluant B was 20 mM TFA in acetonitrile and the temperature was 25 °C. The sequences of the ten peptides (with seven peptides containing Lys specificity determinants at positions 13 and 16 of the nonpolar face (Figure 1) are shown in Table 1. Note that peptide denotations in this figure have been abbreviated from those shown in Table 1; for example, D87(Lys¹-6 Arg-1) has been shortened to D87(6 Arg-1). For the peptides without specificity determinants, we have added A13/A16 to the peptide denotations.

Figure 5.

Self-association of AMPs determined by temperature profiling in RP-HPLC. Retention behavior from eight AMPs after normalization to their retention times at 5 °C over the temperature range 5–41 °C in 2 °C—increments or 5–75 °C in 10°C—increments (details shown in Section 2.3). The sequences of the eight peptides are shown in Table 1. D85(A13/A16–6Orn-1), D86(A13/A16–6Dab-1) and D105(A13/A16–6Dap-1) do not contain Lys specificity determinants, whereas the remaining AMPs contain Lys specificity determinants at positions 13 and 16 on the non-polar face (Figure 1). Note that peptide denotations in Figure 5 have been abbreviated from those shown in Table 1; for example, D87(Lys¹-6 Arg-1) has been shortened to D87(6 Arg-1). RC is a random coil control peptide (Ac-ELEKGGLEGEKGGKELEK-amide) used for RP-HPLC temperature profiling. The peptide self-association parameter, P_A, represents the maximum change in peptide retention time relative to the random coil peptide, RC (P_A values shown in Table 5).