The tridecaptins: non-ribosomal peptides that selectively target Gram-negative bacteria

Abstract

Tridecaptins are a re-emerging class of non-ribosomal antibacterial peptides (NRAPs) with potent activity against highly problematic strains of Gram-negative bacteria. An intricate mode of action has been reported to explain the bactericidal activity of these NRAPs, wherein they bind selectivity to the Gram-negative version of the peptidoglycan precursor lipid II on the outer leaflet of the inner membrane and disrupt the proton-motive force. Tridecaptins are highly amenable to synthetic modification owing to their linear structure, therefore, various synthetic analogues have been reported, several of which have enhanced antimicrobial activity, reduced cost of synthesis and/or improved stability towards d-peptidase mediated hydrolysis. It has also been demonstrated that unacylated tridecaptins can act synergistically with clinically relevant antibiotics by sensitizing the outer membrane. This review will summarize past literature on the development/discovery of novel tridecaptin analogues (up until the end of 2020), some of which may be useful therapeutic agents to treat insidious Gram-negative bacterial infections.

Fig. 1. Chiral lipid tail variants of TriA₁. A methodical approach was employed to assist in the complete stereochemical assignment of TriA₁. Both HPLC and NMR were used to definitively ascertain the lipid tail stereochemistry as 3R,6S.

Fig. 2. A) Biosynthetic gene cluster of TriA₁ (from P. terrae NRRL B-30644) containing a predicted thioesterase (triA), two putative ABC transporter proteins (triB and triC) and NRPSs, triD and triE. B) Predicted domains of the NRPSs, triD and triE. Condensation domains (C) are depicted in gold, adenylation domains in white, thiolation domains (T) in blue, epimerase domains (E) in mint and the thioesterase domain (TE) in red.

Fig. 3. Variants of tridecaptin A₁. The structures of natural tridecaptin A₁ (1) and synthetic lipid tail analogues (14–16) are shown.

Fig. 4. Azidopeptides and covalently linked tridecaptin–antibiotic conjugates. The structures of azidopeptides, H-TriA₁(Glu10-PEG₃N₃) (33) and Oct-TriA₁(Glu10-PEG₃N₃) (34), are shown alongside tridecaptin–antibiotic conjugates, H-TriA₁-Eryc (35), Oct-TriA₁-Eryc (36), H-TriA₁-Van (37), Oct-TriA₁-Van (38), H-TriA₁-Rif (39) and Oct-TriA₁-Rif (40).

Fig. 5. Synthetic Oct-TriA₁ analogue (16) with key residues highlighted. Substitution of these d-amino acid residues, Trp5, Dab8 and aIle12, resulted in the observed reductions in activity (relative to Oct-TriA₁) against E. coli ATCC 25922. The most significant reduction in activity was associated with replacement of the d-Dab8 residue, suggesting it plays an intrinsic role in antimicrobial activity.

Fig. 6. A) Summary of TriA₁ mechanism of action. TriA₁ interacts with LPS on the surface of the outer membrane (1). It then crosses the outer membrane (2) and enters the periplasm. TriA₁ then binds to Gram-negative lipid II on the outer leaflet of the inner membrane (3) and disrupts the proton motive force (4). B) Model of TriA₁-lipid II complex. Figure reproduced from deposited solution NMR structure of Oct-TriA₁ in DPC micelles containing Gram-negative lipid II structure (PDB: 2N5Y) (obtained from Dr Stephen Cochrane). Lipid II is shown as a stick figure, with backbone in purple, and Oct-TriA₁ shown as a surface representation. The interaction between d-Dab8 and DAP is highlighted, with the peptide visible as a stick figure with backbone in grey. C) Structure of synthetic Gram-negative lipid II analogue. In this study a synthetic Gram-negative lipid II analogue containing a shorter prenyl chain was used as this modification improved solubility for NMR studies.

Fig. 7. Cleavage sites on Oct-TriA variants of BogQ and TriF d-peptidase enzymes. BogQ hydrolyses the amide bond at the C-terminus of cationic d-Dab residues whilst TriF voids the peptide of antimicrobial activity by cleaving at the C-terminus of d-Trp5.

Fig. 8. Cyclized Oct-TriA₁ analogues (58–60), in which the characteristic π-stacking interaction has been replaced by regiospecific xylyl linkers. ^a MIC values against A. baumannii NCTC 13304.

Fig. 9. TriA-TAMRA probe (85) used for differential staining of Gram-negative bacteria.

Fig. 10. Structures of tridecaptin B variants: (6S)-TriB₁ (86), (6R)-TriB₁ (87), Oct-TriB₁ (88) and H-TriB₁ (89).

Fig. 11. Elucidation of TriB₁ lipid chain stereochemistry. (6R)-Methyl derivative (90, green) and (6S)-methyl derivative (91, red) were prepared by chemical synthesis. The lipid tail from TriB₁ was obtained by hydrolysis and derivatized with the same anthracenyl unit (blue). ¹H-NMR analysis allowed the chirality of the lipid tail to be determined as S. X-Axis units are parts per million (ppm).