In Vivo Behavior of the Antibacterial Peptide Cyclo[RRRWFW], Explored Using a 3-Hydroxychromone-Derived Fluorescent Amino Acid.

Sergii Afonin,Serhii Koniev,Anne S Ulrich,Oleg Babii,Vasyl G Pivovarenko,Margitta Dathe,Sepand Rastegar,Ferdinand Le Noble,Masanari Takamiya,Andrii Hrebonkin,Igor V Komarov,Laetitia Préau,Alexander V Strizhak,Uwe Strähle

doi:10.3389/fchem.2021.688446

Abstract

Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly. FHC thus can be considered an adequate label for studies of the parent peptide. Several analytical and imaging techniques were used to establish the activity of the obtained labeled cWFW analogues toward animal cells and to study the behavior of the peptides in a multicellular organism. The 3-hydroxychromone fluorophore can undergo excited-state intramolecular proton transfer (ESIPT), resulting in double-band emission from its two tautomeric forms. This feature allowed us to get insights into conformational equilibria of the labeled peptides, localize the cWFW analogues in human cells (HeLa and HEK293) and zebrafish embryos, and assess the polarity of the local environment around the label by confocal fluorescence microscopy. We found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the utility of fluorescent labeling by double-emission labels to evaluate biologically active peptides as potential drug candidates in vivo.

Highlights

Antimicrobial peptides (AMPs) have received growing attention from the medicinal chemistry community in recent decades, as AMPs offer solutions to problems caused by acquired microbial resistance to conventional antibiotics, and they tend to demonstrate a broad spectrum antibacterial activities (Bahar and Ren, 2013; Mantravadi et al, 2019)
Culture media and supplements for animal cells were from Thermo Fisher, those for bacterial culturing were from Becton, and those for zebrafish embryo handling were freshly prepared
It should be confirmed that the label, incorporated instead of an appropriate peptide fragment, disturbs neither the parent peptide structure nor functions significantly

Summary

Introduction

Antimicrobial peptides (AMPs) have received growing attention from the medicinal chemistry community in recent decades, as AMPs offer solutions to problems caused by acquired microbial resistance to conventional antibiotics, and they tend to demonstrate a broad spectrum (i.e., species-nonselective) antibacterial activities (Bahar and Ren, 2013; Mantravadi et al, 2019). Among the peptides considered promising candidates for further development into antibacterial drugs, the cyclic arginine-/tryptophan-rich peptide cyclo[RRRWFW] (cWFW, Figure 1A and Table 1) stands out due to its prominent features. This peptide is one of the smallest known AMPs. This peptide is one of the smallest known AMPs It possesses enhanced biostability and remarkable selectivity against prokaryotic cells, and it effectively kills Gram-positive and Gram-negative bacteria through a unique mechanism of action that is unlikely to induce bacterial resistance (Peschel and Sahl, 2006). The unique mode of action, high bactericidal potency, low hemolytic activity, and synthetic availability make cWFW a viable antibacterial drug candidate, justifying further studies of its behavior in vivo

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Conclusion