Thanatin and vinyl sulfide analogues as narrow spectrum antimicrobial peptides that synergise with polymyxin B

Abstract

Thanatin is a β-hairpin antimicrobial peptide cyclised by a single disulfide bond that has shown potent broad-spectrum activity towards bacterial and fungal pathogens. Towards Gram-negative species, thanatin acts both by forming trans-membranal pores and inhibiting outer membrane biogenesis by binding to LptA and blocking lipopolysaccharide (LPS) transport. Inspired by previous modifications of thanatin, an analogue was prepared which demonstrated potent but selective activity towards E. coli. Furthermore, this compound was shown to act in synergy with the highly potent FDA-approved lipopeptide antibiotic polymyxin B, which engages LPS at the cytoplasmic membrane. Four analogues of thanatin in which the disulfide was substituted for vinyl sulfide bridge mimetics were prepared, all of which retained similar secondary structures. Two of these retained substantial potency and selectivity towards E. coli. Importantly, synergy with polymyxin B was also maintained for the lead analogue. The vinyl sulfide potentially offers a facile replacement strategy for labile disulfide bonds and the selective activity and drug synergy of the reported thanatin analogues is promising for the development of narrow spectrum antimicrobials with reduced likelihood of resistance emerging in clinical settings.

Keywords: AMP; antibiotic synergy; polymyxin B; thanatin; vinyl sulfide; β-hairpin.

FIGURE 1

Example of the two types of combination therapies aimed to target antimicrobials and overcome resistance mechanisms (Shang et al., 2019; van Groesen et al., 2021).

FIGURE 2

AMP thanatin (1), represented by three letter code, disulfide bond between residues Cys¹¹ and Cys¹⁸ shown in orange. Regions essential for antimicrobial activity highlighted as follows; i) C-terminal loop (red), ii) the C-terminal three residue extension (green), iii) a stretch of seven N-terminal mostly hydrophobic residues (blue) and iv) the three final N-terminal residues (pink).

FIGURE 3

Summary of three of the key analogues developed over the years for thanatin (1). Key residue substitutions (red) and disulfides (orange) (Fehlbaum et al., 1996; Wu et al., 2008; Sinha and Bhattacharjya, 2022).

FIGURE 4

Chemical structures of native thanatin (1) and thanatin* (5). Amino acids substitutions (red), disulfide residues (orange), and C-terminal amidation (purple) highlighted.

SCHEME 1

Synthesis of thanatin* (5). Cys residues and disulfides (orange), Cys (Trt) protecting groups (red). (1) 40 s flow (15 mL/min) 30% (v/v) piperidine in DMF; (2) AA coupling following protocol 2A for all non Cys AAs and protocol 2B for Cys residues (see SI); 2A) 30 s flow of AA coupling solution (0.3 M AA, 0.28 M HATU, 20 equiv. DIPEA) at 15 mL/min; 2B) 40 s flow of Cys (Trt) (0.3 M AA 0.28 M PyAOP, 20 equiv. sym-collidine) at 15 mL/min (3) 40 s flow of DMF at 15 mL/min. All flow reactions were performed at 65 °C.

FIGURE 5

Analogue library of thanatin* (5) and vinyl sulfide analogues (8–11). Cys residues and disulfide (orange) and substituted Cys residue and vinyl sulfide bridge (blue).

SCHEME 2

Representative synthetic scheme for cyclisation of vinyl sulfide analogues 8–11. Cys residues and disulfide (orange) and substituted Cys residue and vinyl sulfide bridge (blue).

FIGURE 6

(A) CD spectra overlay of thanatin* (5) and analogues (8–11) at 50 µM in 200 µM sodium phosphate buffer (pH ∼7.4, 20°C). (B) CD spectra overlay of thanatin* (5) and analogues (8–11) at 50 µM in TFE:200 µM sodium phosphate buffer 1:1 (pH ∼7.4, 20°C).