A Noncytotoxic Temporin L Analogue with In Vivo Antibacterial and Antiendotoxin Activities and a Nonmembrane-Lytic Mode of Action

Abstract

Cytotoxic frog antimicrobial peptide Temporin L (TempL) is an attractive molecule for the design of lead antimicrobial agents due to its short size and versatile biological activities. However, noncytotoxic TempL variants with desirable biological activities have rarely been reported. TempL analogue Q3K,TempL is water-soluble and possesses a significant antiendotoxin property along with comparable cytotoxicity to TempL. A phenylalanine residue, located at the hydrophobic face of Q3K,TempL and the “d” position of its phenylalanine zipper sequence, was replaced with a cationic lysine residue. This analogue, Q3K,F8K,TempL, showed reduced hydrophobic moment and was noncytotoxic with lower antimicrobial activity. Interestingly, swapping between tryptophan at the fourth and serine at the sixth positions turned Q3K,F8K,TempL totally amphipathic as reflected by its helical wheel projection with clusters of hydrophobic and hydrophilic residues and the highest hydrophobic moment among these peptides. Surprisingly, this analogue, SW,Q3K,F8K,TempL, was as noncytotoxic as Q3K,F8K,TempL but showed augmented antimicrobial and antiendotoxin properties, comparable to that of TempL and Q3K,TempL. SW,Q3K,F8K,TempL exhibited appreciable survival of mice against P. aeruginosa infection and a lipopolysaccharide (LPS) challenge. Unlike TempL and Q3K,TempL, SW,Q3K,F8K,TempL adopted an unordered secondary structure in bacterial membrane mimetic lipid vesicles and did not permeabilize them or depolarize the bacterial membrane. Overall, the results demonstrate the design of a nontoxic TempL analogue that possesses clusters of hydrophobic and hydrophilic residues with impaired secondary structure and shows a nonmembrane-lytic mechanism and in vivo antiendotoxin and antimicrobial activities. This paradigm of design of antimicrobial peptide with clusters of hydrophobic and hydrophilic residues and high hydrophobic moment but low secondary structure could be attempted further.

Switching Bond: Generation of New Antimicrobial Peptides via the Incorporation of an Intramolecular Isopeptide Bond

“…Due to the high potency of AMPs and their various modes of action, they are considered to be a promising alternative to conventional antibiotics. 8 − 13 A particular native AMP can be easily modified to create new peptidic or nonpeptidic analogs, many of which preserve antimicrobial activity. In addition, they gain or lose other activities, such as hemolysis, cell toxicity, immunomodulation, endotoxin neutralization, and others.…”

First-in-Class Cyclic Temporin L Analogue: Design, Synthesis, and Antimicrobial Assessment

“…Considering previous conformational NMR studies performed in both SDS and DPC, 42 − 44 the linear [Pro 3 , d Leu 9 ]TL ( 9 ) peptide displayed an α-helical structure along residues 5–8 in SDS and residues 5–10 in DPC; while in both micelles, it showed a β-turn centered on Pro 3 and Trp 4 . 41 In light of these results, we started replacing Trp 4 with Lys (i) or Pra (i), and Phe 8 with Glu (i+4) or Lys(N 3 ) (i+4), until C-terminal residues, yielding the first series of lactam ( 10–14 ) and 1,4-triazolic ( 15 – 19 ) bridged derivatives. During the systematic incorporation of cyclizing amino acids, positively charged Lys 7 and Arg 11 residues were not taken in consideration because of their essential role for membrane interaction.…”

“…Considering previous conformational NMR studies performed in both SDS and DPC, 42−44 the linear [Pro 3 ,DLeu 9 ]TL (9) peptide displayed an α-helical structure along residues 5−8 in SDS and residues 5−10 in DPC; while in both micelles, it showed a β-turn centered on Pro 3 and Trp 4 . 41 In light of these results, we started replacing 7 and Arg 11 residues were not taken in consideration because of their essential role for membrane interaction. Also, a D-residue in position 9 was still considered for cyclizing amino acids replacing the original D-Leu.…”

Bioevaluation and Targeted Modification of Temporin-FL From the Skin Secretion of Dark-Spotted Frog (Pelophylax nigromaculatus)

“…The helicity of AMPs was shown to have crucial effects on transmembrane pore formation related to lipid–peptide interactions. Cytotoxic temporins assume a helical structure when binding with the zwitterionic membrane, and nontoxic peptides are unable to form a complete helical structure in zwitterionic lipid vesicles ( Kumari et al, 2020 ). The direct correlation between peptide helicity and toxicity toward normal cells was reported previously ( Mangoni et al, 2016 ).…”