Aurein-2.2

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.

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an alpha-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The (31)P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC(3)5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.

The naturally occurring host defense peptide (HDP), aurein 2.2, secreted by the amphibian Litoria aurea, acts as a moderate antibacterial, affecting Gram positive bacteria such as Staphylococcus aureus by forming selective ion pores. In a quest to find more active analogues of aurein 2.2, peptides 73 and 77 were discovered. These peptides were rich in arginine and tryptophan and found to have MICs of 4 μg/mL. Here we examined what impact the increased charge from +2 to +3 and a slight increase in hydrophobic moment relative to aurein 2.2 had on the mechanism of action of these two analogues. Using a time-kill assay, both peptides 73 and 77 were found to kill bacteria more effectively than the parent peptide. Using solution CD and NMR, the peptides were found to not adopt a continuous α-helical structure, i.e. the analogues were not helical from residue 1-13 like the parent peptide. Results obtained from oriented CD (OCD), DiSC35 and pyranine assays and a gel retardation experiment showed that the peptides did not function by membrane perturbation and further showed that peptide 73 and 77 did not interact with DNA. Overall, the data were consistent with these peptides acting as cell penetrating peptides with intracellular targets, which did not appear to be DNA.