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A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056.

ABSTRACT: The novel antimicrobial peptide with a dimeric dendrimer scaffold, SB056, was empirically optimized by high-throughput screening. This procedure produced an intriguing primary sequence whose structure-function analysis is described here. The alternating pattern of hydrophilic and hydrophobic amino acids suggests the possibility that SB056 is a membrane-active peptide that forms amphiphilic ?-strands in a lipid environment. Circular dichroism confirmed that the cationic SB056 folds as ?-sheets in the presence of anionic vesicles. Lipid monolayer surface pressure experiments revealed unusual kinetics of monolayer penetration, which suggest lipid-induced aggregation as a membranolytic mechanism. NMR analyses of the linear monomer and the dendrimeric SB056 in water and in 30% trifluoroethanol, on the other hand, yielded essentially unstructured conformations, supporting the excellent solubility and storage properties of this compound. However, simulated annealing showed that many residues lie in the ?-region of the Ramachandran plot, and molecular-dynamics simulations confirmed the propensity of this peptide to fold as a ?-type conformation. The excellent solubility in water and the lipid-induced oligomerization characteristics of this peptide thus shed light on its mechanism of antimicrobial action, which may also be relevant for systems that can form toxic ?-amyloid fibrils when in contact with cellular membranes. Functionally, SB056 showed high activity against Gram-negative bacteria and some limited activity against Gram-positive bacteria. Its potency against Gram-negative strains was comparable (on a molar basis) to that of colistin and polymyxin B, with an even broader spectrum of activity than numerous other reference compounds.

SUBMITTER: Scorciapino MA

PROVIDER: S-EPMC3296046 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056.

Scorciapino Mariano A MA Pirri Giovanna G Vargiu Attilio V AV Ruggerone Paolo P Giuliani Andrea A Casu Mariano M Buerck Jochen J Wadhwani Parvesh P Ulrich Anne S AS Rinaldi Andrea C AC

Biophysical journal 20120306 5

The novel antimicrobial peptide with a dimeric dendrimer scaffold, SB056, was empirically optimized by high-throughput screening. This procedure produced an intriguing primary sequence whose structure-function analysis is described here. The alternating pattern of hydrophilic and hydrophobic amino acids suggests the possibility that SB056 is a membrane-active peptide that forms amphiphilic β-strands in a lipid environment. Circular dichroism confirmed that the cationic SB056 folds as β-sheets in ...[more]

PMID: 22404926

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Project description:The biological activity of antimicrobial peptides and proteins is closely related to their structural aspects and is sensitive to certain post-translational modifications such as glycosylation, lipidation and PEGylation. However, PEGylation of protein and peptide drugs has expanded in recent years due to the reduction of their toxicity. Due to their size, the PEGylation process can either preserve or compromise the overall structure of these biopolymers and their biological properties. The antimicrobial peptide LyeTx I-bcys was synthesized by Fmoc strategy and coupled to polyethylene glycol 2.0 kDa. The conjugates were purified by HPLC and characterized by MALDI-ToF-MS analysis. Microbiological assays with LyeTx I-bcys and LyeTx I-bPEG were performed against Staphylococcus aureus (ATCC 33591) and Escherichia coli (ATCC 25922) in liquid medium. MIC values of 2.0 and 1.0 µM for LyeTx I-bcys and 8.0 and 4.0 µM for LyeTx I-bPEG were observed against S. aureus and E. coli, respectively. PEGylation of LyeTx I-bcys (LyeTx I-bPEG) decreased the cytotoxicity determined by MTT method for VERO cells compared to the non-PEGylated peptide. In addition, structural and biophysical studies were performed to evaluate the effects of PEGylation on the nature of peptide-membrane interactions. Surface Plasmon Resonance experiments showed that LyeTx I-b binds to anionic membranes with an association constant twice higher than the PEGylated form. The three-dimensional NMR structures of LyeTx I-bcys and LyeTx I-bPEG were determined and compared with the LyeTx I-b structure, and the hydrodynamic diameter and zeta potential of POPC:POPG vesicles were similar upon the addition of both peptides. The mPEG-MAL conjugation of LyeTx I-bcys gave epimers, and it, together with LyeTx I-bPEG, showed clear α-helical profiles. While LyeTx I-bcys showed no significant change in amphipathicity compared to LyeTx I-b, LyeTx I-bPEG was found to have a slightly less clear separation between hydrophilic and hydrophobic faces. However, the similar conformational freedom of LyeTx I-b and LyeTx I-bPEG suggests that PEGylation does not cause significant structural changes. Overall, our structural and biophysical studies indicate that the PEGylation does not alter the mode of peptide interaction and maintains antimicrobial activity while minimizing tissue toxicity, which confirmed previous results obtained in vivo. Interestingly, significantly improved proteolytic resistance to trypsin and proteinase K was observed after PEGylation.

Project description:Antimicrobial peptides (AMPs) are promising templates for the development of novel antibiofilm drugs. Despite the large number of studies on screening and optimization of AMPs, only a few of these evaluated the antibiofilm activity in physiologically relevant model systems. Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interference of the host microenvironment with peptide stability/availability. Hence, mimicking the complex environment found in biofilm-associated infections is essential to predict the clinical potential of novel AMP-based antimicrobials. In the present study, we examined the antibiofilm activity of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K against Pseudomonas aeruginosa in an in vivo-like three-dimensional (3-D) lung epithelial cell model and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels). Although moderately active when tested alone, lin-SB056-1 was effective in reducing P. aeruginosa biofilm formation in association with 3-D lung epithelial cells in combination with the chelating agent EDTA. The dimeric derivative (lin-SB056-1)2-K demonstrated an enhanced biofilm-inhibitory activity as compared to both lin-SB056-1 and the lin-SB056-1/EDTA combination, reducing the number of biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death. Biofilm inhibition by (lin-SB056-1)2-K was reported both for the reference strain PAO1 and cystic fibrosis lung isolates of P. aeruginosa. In addition, using fluorescence microscopy, a significant decrease in biofilm-like structures associated with 3-D cells was observed after peptide exposure. Interestingly, effectiveness of (lin-SB056-1)2-K was also demonstrated in the wound model with a reduction of up to 1-Log unit in biofilm formation by P. aeruginosa PAO1 and wound isolates. Overall, combination treatment and peptide dendrimerization emerged as promising strategies to improve the efficacy of AMPs, especially under challenging host-mimicking conditions. Furthermore, the results of the present study underlined the importance of evaluating the biological properties of novel AMPs in in vivo-like model systems representative of specific infectious sites in order to make a more realistic prediction of their therapeutic success, and avoid the inclusion of unpromising peptides in animal studies and clinical trials.

Dataset Information

A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056.

Publications

A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056.

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