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

Abstract

Antimicrobial peptides (AMPs), which can be modified to kill a broad spectrum of microoganisms or a specific microorganism, are considered as promising alternatives to combat the rapidly widespread, resistant bacterial infections. However, there are still several obstacles to overcome. These include toxicity, stability, and the ability to interfere with the immune response and bacterial resistance. To overcome these challenges, we herein replaced the regular peptide bonds with isopeptide bonds to produce new AMPs based on the well-known synthetic peptides Amp1L and MSI-78 (pexiganan). Two new peptides Amp1EP and MSIEP were generated while retaining properties such as size, sequence, charge, and molecular weight. These new peptides have reduced toxicity toward murine macrophage (RAW 264.7) cells, human monocytic (THP-1) cells, and human red blood cells (hRBCs) and enhanced the stability toward proteolytic degradation. Importantly, the new peptides do not repress the pro-inflammatory cytokine and hence should not modulate the immune response. Structurally, the new peptides, Amp1EP and MSIEP, have a structure of random coils in contrast to the helical structures of the parental peptides as revealed by circular dichroism (CD) analysis. Their mode of action, assessed by flow cytometry, includes permeabilization of the bacterial membrane. Overall, we present here a new approach to modulate AMPs to develop antimicrobial peptides for future therapeutic purposes.

Site-Specific Isopeptide Bond Formation: A Powerful Tool for the Generation of Potent and Nontoxic Antimicrobial Peptides

“… 11 − 16 A new approach to modulate AMPs uses an isopeptide bond replacement. 17 The isopeptide bond is an amide bond between the carboxyl group of one amino acid and the epsilon amino group of another amino acid. The isopeptide bond is a natural bond that appears in HK97 bacteriophage capsid formation, Gram-positive bacterial pili, and ubiquitin in humans.…”

“… 21 , 22 Incorporating three isopeptide bonds improves the peptide’s stability and reduces both the toxicity and hemolytic activity but also reduces the antimicrobial activity. 17 In this work, we systematically introduced a single isopeptide bond and examined the effect of the bond switch on the biological and biophysical properties of the peptides. Starting from the parental peptide Amp1L, 14 , 23 we generated six new peptides named Amp1EP2, Amp1EP5, Amp1EP6, Amp1EP9, Amp1EP10, and Amp1EP13.…”

Antimicrobial Peptides against Multidrug-Resistant Pseudomonas aeruginosa Biofilm from Cystic Fibrosis Patients

“… 12 , 13 Generally, AMPs are unstructured, potentially forming amphipathic α-helical or β-sheet structures in the membrane. 14 , 15 AMPs disrupt the bacterial membranes without a specific high-affinity target and are assumed not to evolve resistance in pathogens, although some do. 15 − 19 AMPs bind to the bacterial cell wall by electrostatic interactions with the anionic components.…”

“…In humans, AMPs are stored in phagocytes in large quantities that can be released when invading microorganisms are encountered to stop microbial proliferation . AMPs share biophysical characteristics as they are short and composed of positively charged amino acids and hydrophobic amino acids but are not conserved in their sequences. , Generally, AMPs are unstructured, potentially forming amphipathic α-helical or β-sheet structures in the membrane. , AMPs disrupt the bacterial membranes without a specific high-affinity target and are assumed not to evolve resistance in pathogens, although some do. − AMPs bind to the bacterial cell wall by electrostatic interactions with the anionic components. The hydrophobic interactions between the AMP and the bacterial acyl chains allow cell wall permeation due to the amphipathic structure that enables non-receptor-mediated attraction. , Several AMPs were found to be active against planktonic bacteria and biofilm, e.g., LL-37, histatin, and nisin. − However, these and many other AMPs were found to be toxic to eukaryotic cells at high concentrations; therefore, the discovery of new therapeutics is urgently needed. − De novo -designed AMPs that have high potency against bacterial cells and are not toxic to eukaryotic cells are promising candidates. − …”

Tandem Repeat of a Short Human Chemerin-Derived Peptide and Its Nontoxic d-Lysine-Containing Enantiomer Display Broad-Spectrum Antimicrobial and Antitubercular Activities

“…However, there are some major hurdles in the development of these host defense antimicrobial peptides as alternatives to antibiotics against these pathogens. Thus, despite a huge amount of work being carried out in this area, − it continues to be a challenging task to identify, design, and synthesize novel antimicrobial peptides with broad-spectrum antimicrobial activities, cell selectivity, and stability under physiological conditions.…”