Releasable and traceless PEGylation of arginine-rich antimicrobial peptides

Abstract

Arginine-rich antimicrobial peptides (AMPs) are emerging therapeutics of interest. However, their applicability is limited by their short circulation half-life, caused in part by their small size and digestion by blood proteases. This study reports a strategy to temporarily mask arginine residues within AMPs with methoxy poly(ethylene glycol). Based on the reagent used, release of AMPs occurred in hours to days in a completely traceless fashion. In vitro, conjugates were insensitive to serum proteases, and released native AMP with full in vitro bioactivity. This strategy is thus highly relevant and should be adaptable to the entire family of arginine-rich AMPs. It may potentially be used to improve AMP-therapies by providing a more steady concentration of AMP in the blood after a single injection, avoiding toxic effects at high AMP doses, and reducing the number of doses required over the treatment duration.

Fig. 1. rPEGylation of arginine-rich AMPs. (a) Synthesis of arginine-reactive polymers ((i): tosyl chloride, triethylamine; (ii): o-substituted p-hydroxyacetophenone, K₂CO₃). (b) Sequence and 3D structure (inset from ref. 21 with permission) of model AMP (3) and controls with one (4) or no arginine residues (5). Structure of three forms (6–8) of the conjugates obtained. Letters represent single letter codes for amino acids (underlined are d-amino acids).

Fig. 2. PEGylation of AMPs. (a) Kinetics of reaction of 3–5 with 2a–d in phosphate buffer pH 7.4. Mean + SD (n = 3). (Conjugate formation = 100 × (1 – fraction residual AMP)); (b) representative chromatograms; (c) mass spectrum of [4 + 2a] ([M + H]⁺ calcd 2790.43 (found: 2790.43); [M – H₂O + H]⁺ calcd 2772.42 (found: 2772.42)).

Fig. 3. Influence of AMP sequence on PEGylation kinetics. Half-life of reaction of peptides with 1.5 eq. 2a–d at pH 7.4 to assess the influence of steric hindrance and charge on reactivity. Mean + SD (n = 3). Full ANOVA in Table S1.

Fig. 4. Release of AMPs from AMP–mPEG conjugates. (a and b) Influence of substituents on the release of peptide from conjugates of 4 or 3 with 2a–d. Mean + SD (n = 3). (c) Effect of pH on the release of 4 from [4 + 2c] conjugates (n = 1).