Synthetic Lugdunin Analogues Reveal Essential Structural Motifs for Antimicrobial Action and Proton Translocation Capability

Abstract

Lugdunin, a novel thiazolidine cyclopeptide, exhibits micromolar activity against methicillin-resistant Staphylococcus aureus (MRSA). For structure-activity relationship (SAR) studies, synthetic analogues obtained from alanine and stereo scanning as well as peptides with modified thiazolidine rings were tested for antimicrobial activity. The thiazolidine ring and the alternating d- and l-amino acid backbone are essential. Notably, the non-natural enantiomer displays equal activity, thus indicating the absence of a chiral target. The antibacterial activity strongly correlates with dissipation of the membrane potential in S. aureus. Lugdunin equalizes pH gradients in artificial membrane vesicles, thereby maintaining membrane integrity, which demonstrates that proton translocation is the mode of action (MoA). The incorporation of extra tryptophan or propargyl moieties further expands the diversity of this class of thiazolidine cyclopeptides.

Keywords: aldehyde peptide synthesis; methicillin-resistant Staphylococcus aureus; proton translocation; synthetic membrane vesicles; thiazolidine antibiotics.

Scheme 1

Solid‐phase aldehyde peptide synthesis of 1.

Figure 1

Structure of lugdunin (1) and analogues 9–14.

Figure 2

Exemplified derivatives of 1. 4 is an inactive alanine analogue. The enantiomer 22 shows identical activity as 1. 23 and 24 are specially designed analogues of 1 with twofold and equal activity, respectively.

Figure 3

Effect of 1, 11, 22, and 3 on the S. aureus NCTC8325 membrane potential after 30 (black bars) and 60 minutes (gray bars) of treatment. The protonophore CCCP (5 μm) was used as a positive and DMSO as a negative control. Error bars represent the standard deviation (SD) of two biological replicates including two technical replicates each.

Figure 4

Complementary experiments excluding large pores or lesions. A) Fluorescence microscopy of S. aureus treated with pore‐forming nisin (1–2 × MIC) or 1 (10 × MIC). Scale bars: 1 μm. B) Time course of normalized CF leakage from POPC vesicles, induced by 25 and 1 at concentrations of 5 μm and 1 μm (P/L 1:10 and 1:50).

Figure 5

Time course of normalized pyranine fluorescence after addition of: A,B) 5 μm to 50 nm 1 (P/L 1:10 to 1:1000) with A) proton influx from pH 6.4 to 7.4, B) proton efflux from pH 7.4 to 8.4, C) after addition of 1 μm (P/L 1:50) 11, 25, and 1, proton influx from pH 6.4 to 7.4. The vesicles were composed of POPC, total lipid concentration 50 μm containing 0.5 mm pyranine.