Semisynthetic Macrocyclic Lipo-lanthipeptides Display Antimicrobial Activity Against Bacterial Pathogens

Abstract

A large number of antimicrobial peptides depend on intramolecular disulfide bonds for their biological activity. However, the relative instability of disulfide bonds has limited the potential of some of these peptides to be developed into therapeutics. Conversely, peptides containing intramolecular (methyl)lanthionine-based bonds, lanthipeptides, are highly stable under a broader range of biological and physical conditions. Here, the class-II lanthipeptide synthetase CinM, from the cinnamycin gene cluster, was employed to create methyllanthionine stabilized analogues of disulfide-bond-containing antimicrobial peptides. The resulting analogues were subsequently modified in vitro by adding lipid tails of variable lengths through chemical addition. Finally, the created compounds were characterized by MIC tests against several relevant pathogens, killing assays, membrane permeability assays, and hemolysis assays. It was found that CinM could successfully install methyllanthionine bonds at the intended positions of the analogues and that the lipidated macrocyclic core peptides have bactericidal activity against tested Gram-positive and Gram-negative pathogenic bacteria. Additionally, fluorescence microscopy assays revealed that the lipidated compounds disrupt the bacterial membrane and lyse bacterial cells, hinting toward a potential mode of action. Notably, the semisynthesized macrocyclic lipo-lanthipeptides show low hemolytic activity. These results show that the methods developed here extend the toolbox for novel antimicrobial development and might enable the further development of novel compounds with killing activity against relevant pathogenic bacteria.

Ribosomally synthesized peptides, foreground players in microbial interactions: recent developments and unanswered questions

“…(iii) The deep knowledge of the RSP biosynthetic rules to engineer and produce antimicrobials with improved efficiency, such as recently shown with semi-synthesized lipo-lanthipeptides. 267…”

Exploring the Heterogeneous Structural Dynamics of Class II Lanthipeptide Synthetases with Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS)

“…CinA binding (Figure and Figure S20) triggers a further organization in the capping domain (peptides D42-L56, A109-L118, and E196-E204), motif 1 (D225-L230), and in several dehydratase active site peptides containing catalytic residues involved in phosphoryl transfer (D402, H404, and N407) . Alanine mutational scanning of CinA localized the CinM binding site to an element near the N terminus of the CinA leader peptide (CinA residues V11-A17) . This segment of the CinA leader peptide is predicted by AlphaFold to interact directly with the D42-L56 helical element of the CinM capping domain (Figure and Figure S33).…”

“…29 Alanine mutational scanning of CinA localized the CinM binding site to an element near the N terminus of the CinA leader peptide (CinA residues V11-A17). 56 This segment of the CinA leader peptide is predicted by AlphaFold to interact directly with the D42-L56 helical element of the CinM capping domain (Figure 5 and Figure S33). Similar to all the other LanMs investigated here, additional interactions are predicted by AlphaFold between the helical region at the C terminus of the CinA leader peptide and the CinM kinase activation domain, which also becomes organized in the [CinM:CinA] complex (motif 4, peptide A569-E586, Figures 5 and 6).…”

Modular Use of the Uniquely Small Ring A of Mersacidin Generates the Smallest Ribosomally Produced Lanthipeptide

“…(C) After chemical handles have been installed, functional groups such as fatty acid chains can be added to the ring A construct to obtain the intended functionality. 20 (D) Linear peptides can be added to the minimal ring a construct in a ribosomal way to obtain lanthionine-stabilized linear peptides.…”

“…It has recently been shown that by adding functional groups, for example, fatty acid chains, to otherwise nonactive lanthipeptides, good antimicrobial activity can be obtained. 20 Lanthipeptides or fragments thereof, can in this sense, be used as a stable module to which all kinds of modifications can be added in vitro. The proposed ring A module could be obtained through the incorporation of noncanonical amino acids that have convenient chemical handle side chains.…”