| The widespread use of traditional antibiotics in clinic has resulted in the emergence of many antibiotic-resistant strains. Therefore, the development of a new class of antibiotics is critical. Antimicrobial peptides (AMPs), with broad-spectrum activity, rapid action and difficult development of resistance, have become promising molecules as new potential anti-infection drugs. Moreover, it is generally accepted that the bacterial cytoplasmic membrane is the primary target of most amphiphilicα-helical antimicrobial peptides, whereby peptide accumulation in the membrane causes increased permeability and loss of barrier function resulting in leakage of cytoplasmic components and cell death. Although antimicrobial peptides are extremely active to kill a broad spectrum range of Gram-negative and Gram-positive bacteria, the specificity for prokaryotic and eukaryotic cells could be a serious challenge of peptides using in clinical practices.The 26-residue amphiphilicα-helical antimicrobial peptide V13K adopts a stationaryα-helical conformation in a hydrophobic environment and contains a hydrophilic lysine residue on the center of the non-polar face. V13K possesses high antimicrobial activity and, more importantly, low hemolytic activity. In order to improve antimicrobial activity and specificity of V13K, we utilized de novo approach to design amphiphilicα-helical antimicrobial peptide V13K analoges, based on the secondary structure of peptides and the knowledge of mechanism of action. We used V13K as a framework to systematically introduce single/double hydrophilic amino acid lysine on the non-polar face of the helix to modify the peptide hydrophobicity. We selected positions further away from the center of peptide sequence as the lysine substituting positions. We used single or double lysine substitution to investigate the effect of hydrophilic and charged residue on the non-polar face on peptide biological activities. Moreover, we utilized leucine with higher hydrophobicity to replace original alanine in order to compensate the loss of overall hydrophobicity by introducing lysine on the non-polar face of V13K. The criteria of designingα-helical antimicrobial peptides are as follows: (a) We selected positions 5, 9, 17 and 21 of peptide V13K as single lysine substitution positions. Positions 9 and 17 are located in the center of sequence, in contrast, positions 5 and 21 are on N- and C-terminals, respectively. The single Lys-substituting peptides are named as F5K, F9K, L17K and L21K, respectively. We compensated the decrease of hydrophobicity on the non-polar face by utilizing leucine to replace alanine on the same side of peptide sequence with substituting lysine. We selected alanine 12(N-terminal)and alanine 20(C-terminal), hence, peptides F5K/A12L, F9K/A12L, L17K/A20L and L21K/A20L were designed as hydrophobicity compensating analogs; (b) Double lysine-substituting analogs were F9K/L17K, F5K/L21K, the two substitution positions of former were closer to the center of sequence; in contrast, the two substitution positions of F5K/L21K were on N- and C-terminals. The purpose of such replacements was exploring the location effect of lysine substitution on the peptide non-polar face. Similarly, hydrophobicity-compensating analogs were made as F9K/L17K/A12L/A20L and F5K/L21K/A12L/A20L, respectively. The research methods used in the experiments: (1) Circular dichroism (CD) spectra of the peptide analogs were measured in KP buffer and in KP buffer containing 50%TFE in order to measure the effect of amino acid substitutions on peptides secondary structure and the relationship between secondary structure and biological activities of peptides; (2) RP-HPLC was utilized to measure hydrophobic and self-association ability of peptides analoges; (3) In order to determine activities against eukaryocyte and prokaryocyte, we measured peptide MHC and MIC, respectively; (4) By calculating therapeutic index of peptide analoges, we further verified the"membrane discrimination"mechanism.By comparing CD spectra and RP-HPLC retention behavior of peptides F9K and F5K, L17K and L21K, F9K/L17K/A12L/A20L and F5K/L21K/A12L/A20L, we concluded that the central lysine substitution on non-polar face causing the greater effect on peptide analoges hydrophobicity and helicity. Moreover, lysine substitution on the non-polar face of peptide V13K results in the decrease of hydrophobicity, thus improves the peptide hemolytic activity. However, the greater decrease of hydrophobicity also prevents peptides entering into the bacterial membrane to kill the cells, such as F9K/L17K and F5K/L21K. Therefore, using leucine to substitute alanine to compensate the decrease of hydrophobicity on the non-polar face, we made peptides F9K/L17K/A12L/A20L,F5K/L21K/A12L/A20L with stronger antimicrobial activity and better therapeutic index than peptide V13K, their therapeutic index for gram-negtive and gram-positive are 217.3 and 52.9 versus 176.8 and 52.6, respectively. Peptide hydrophobicity is very important to the balance between antimicrobial activity and hemolytic activity. Decreasing hydrophobicity can improve peptide hemolytic activity, but if reducing peptide hydrophobicity too much, the peptide will lose its antimicrobial activity. These results in this study are consistent with the"membrane discrimination mechanism", peptide analoges take a carpet-like mechanism to kill the prokaryocyte, in contrast, for hemolytic activity of eukaryotic cells, peptides adopt barrel-stave mechanism. In conclusion, utilizing de novo design approach to introduce hydrophilic amino acid into the non-polar face of the peptide then to compensate the loss of overall hydrophobicity peptide by other amino acid substitutions can be a practical way to obtain antimicrobial peptides with great specificity. |
