β-Lactamase cleavable antimicrobial peptide-drug conjugates

Abstract

Antimicrobial resistance attracts a considerable amount of attention as it threatens the efficiency of current antibacterial treatments. Besides a more considerate use of current antibiotics to slow down the spread of antimicrobial resistance, there is ample need for new therapeutic avenues to treat already resistant strains. Here, we describe the use of a cleavable peptide-drug conjugate to target bacteria with diverse resistance strategies. The conjugate consists of three main components: a β-lactamase cleavable linker, a positively charged stapled antimicrobial peptide, and an antibiotic. The linker ensures selective cleavage and provides the prospect of lowering systemic toxicity of the conjugate. The positively charged peptide targets the negatively charged bacterial membrane, and stapling pre-organises it in a helical structure. Finally, the drug provides another, distinct mode of action to the peptide, which should overall reduce the development of resistance. A series of peptides was prepared and the most promising one was then developed into a stapled conjugate. The factors affecting the activity of this conjugate were investigated, proving cleavage by β-lactamase and superior potency compared to the non-cleavable control, as shown by its minimal inhibitory concentrations.

Fig. 1. Previous research that this work (d) builds on. It combines the concepts of stapled antimicrobial peptides (e.g. work by Pham et al., (a)) with β-lactamase cleavable ciprofloxacin prodrugs (Evans et al., (c)). In the context of this work, the prodrug will be attached to the peptide via a functionalised staple and the divinylpyrimidine methodology previously reported by the Spring group (b) will be used for the stapling.

Fig. 2. Further analysis of peptide P2. (a) Minimal inhibitory concentration (MIC) for the peptide P2a, its analogue with cysteine residues (P2) and the stapled peptide (P2-1). Measured for P. aeruginosa strains PAO1 and PA14. (b) The circular dichroism (CD) spectra of P2-1 and P2a in MeCN/H₂O (1 : 1). The percent helicity is given in parentheses in the legend.

Fig. 3. A study to establish the influence of the staple core on the activity of the stapled AMPs. (a) Staples based on triazine or pyrimidine, with or without N–Me were prepared. (b) Two additional peptides with staple on the hydrophobic face of the helix were also prepared. (c) Heat map showing the MIC of the different peptide-staple combinations.

Scheme 1. (a) The initial functionalised staple design and the updated design. The changes between the two designs are highlighted. The linker between cephalosporin and divinylpyrimidine is shown in pink and connection between cephalosporin and ciprofloxacin is shown in blue. (b) Synthesis of the second generation staple 10a. Compound 7 was prepared using a previously reported procedure (Scheme S1c). (c) Stapling of peptide with staple and removal of the t-Bu protecting group. (d) Preparation of the non-cleavable stapled peptide–ciprofloxacin conjugate. (e) Preparation of a peptide stapled with cephalosporin only, acting as a control.

Fig. 4. Curve showing growth of bacteria measured by OD₆₀₀ after incubation at different concentrations of P2-10 (a) or P2a (b). Each point was done in triplicate; error bars represent standard deviation. NC stands for negative control (incubated without added bacteria, serves as sterility control); GC stands for growth control (incubated without any drugs). Tested on strain PAO1 with and without plasmid pUCP20.

Fig. 5. (a) In vitro cleavage of P2-10 by 1 nm recombinant β-lactamase compared to the background stability in PBS buffer, pH 7.4. (b) Serum stability of peptides P2a, P2-8 and P2-10.