Conjugation of Aurein 2.2 to HPG Yields an Antimicrobial with Better Properties

Abstract

Aurein 2.2 is an antimicrobial peptide (AMP) whose mechanism of action is quite well-understood and that has good activity against Gram-positive bacteria. It is, however, highly cytotoxic. Poly(ethylene glycol) (PEG) conjugation (PEGylation) of protein and peptide drugs has been used for decades to improve their in vivo efficacy and blood circulation by enhancing the biocompatibility of the protein or peptide in question. However, the relatively large hydrodynamic size, high intrinsic viscosity, the limited number of functional groups available for conjugation, and immunogenicity of high molecular weight PEG limits its use in bioconjugation applications. Recently, hyperbranched polyglycerol (HPG) has been gaining attention as an alternative to PEG due to its excellent biocompatibility. Here, for the first time, we report the synthesis of HPG conjugates of antimicrobial peptides. Aurein 2.2 peptide was conjugated to high molecular weight HPG with a varying number of peptides per polymer, and the biocompatibility and antimicrobial activity of the conjugates were investigated. The antimicrobial activity of the peptide and its conjugates were determined by measuring the minimal inhibitory concentration (MIC) against Staphylococcus aureus and Staphylococcus epidermidis. The interaction of aurein 2.2 peptide and the conjugates with a model bacterial biomembrane was investigated using CD spectroscopy to understand the mode of action of the conjugates. The biocompatibility of the AMP-polymer conjugates was investigated by measuring red cell lysis, platelet activation and aggregation, complement activation, blood coagulation, and cell toxicity. Our results show that the size of the conjugates and the peptide density influence the biocompatibility of the antimicrobial conjugates. These results will help to further define the properties of HPG-AMP conjugates and set the stage for development of better therapeutic agents.

Identification of an IDR peptide formulation candidate that prevents peptide aggregation and retains immunomodulatory activity

“…The presence of the negative charge on the acidic portion of dHPGs provides a potential site to interact with cationic peptides electrostatically and indeed dHPGs have also been explored as potential HDP delivery vectors. For instance, conjugation of a cytotoxic antimicrobial peptide to high molecular weight dHPGs reduced the cytotoxic effects of the peptide while retaining the antibacterial functions in vitro . It is worth noting that these dHPG‐peptide complexes were covalently linked to each other while the dHPG polymers used in the present study were evaluated as excipients to prevent peptide aggregation without chemically modifying the peptide of interest.…”

Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo

“…HPG has been utilized for the development of long circulating drug conjugates [ 178 , 180 ], anticoagulant neutralizing agents [ 177 , 181 ], for cell surface modification [ 182 ], as an osmotic agent in peritoneal dialysis [ 183 ] and cell preservation [ 184 ], making it an excellent candidate to conjugate AMPs. In our initial studies, we conjugated antimicrobial peptide aurein 2.2 to 44 kDa HPG and were able to obtain a conjugate with better therapeutic index [ 185 ]. The HPG-aurein conjugates were not toxic to cell cultures and red blood cells at twice the new minimum inhibitory concentration (MIC) and had excellent blood properties compared to the aurein peptide only.…”

Host defense antimicrobial peptides as antibiotics: design and application strategies

“…Furthermore, the nonantibiotic moiety dithiocarbazate, conjugated to a cell penetrating peptide, is highly active against a wide range of pathogens, especially S. aureus [49]. Hyperbranched polyglycerol conjugated to aurein 2.2, or conjugation of an AMP to polyphosphoester, yields peptides with better biocompatibility and antimicrobial activity [50–51]. …”