Short Antibacterial Peptides with Significantly Reduced Hemolytic Activity can be Identified by a Systematic l-to-d Exchange Scan of their Amino Acid Residues

Abstract

High systemic toxicity of antimicrobial peptides (AMPs) limits their clinical application to the treatment of topical infections; in parenteral systemic application of AMPs the problem of hemolysis is one of the first to be tackled. We now show that the selectivity of lipidated short synthetic AMPs can be optimized substantially by reducing their hemolytic activity without affecting their activity against methicillin resistant Staphylococcus aureus (MRSA). In order to identify the optimized peptides, two sets of 32 diastereomeric H-(D)Arg-WRWRW-(L)Lys(C(O)CnH2n+1)-NH2 (n = 7 or 9) peptides were prepared using a split-split procedure to perform a systematic L-to-D exchange scan on the central WRWRW-fragment. Compared to the all-L C8-lipidated lead sequence, diastereomeric peptides had very similar antibacterial properties, but were over 30 times less hemolytic. We show that the observed hemolysis and antibacterial activity is affected by both differences in lipophilicity of the different peptides and specific combinations of L- and D-amino acid residues. This study identified several peptides that can be used as tools to precisely unravel the origin of hemolysis and thus help to design even further optimized nontoxic very active short antibacterial peptides.

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

“…MP196 therefore is a promising lead structure for derivatization and already has yielded peptides with improved activities and altered pharmacological properties (19). For example, in a recent systematic L-to-D exchange scan peptides with significantly reduced hemolytic activity could be identified (20).…”

Towards Profiles of Resistance Development and Toxicity for the Small Cationic Hexapeptide RWRWRW-NH2

“…Adding a lipidated lysine side chain resulted in peptides with broad-spectrum and/or excellent anti-MRSA activity (Albada et al, 2012b). Lipidation also increased hemolysis, which has been addressed by systematically exchanging L- with D-amino acids resulting in non-hemolytic peptides with excellent activities against multi-resistant bacteria (Albada et al, 2013). The next step will be to investigate some of these advanced derivatives with regard to their toxicity.…”

“…Most importantly, acute toxicity was observed in mice dosed intravenously, likely as a result of toxicity toward erythrocytes. Structural optimization programs exploring peptide lipidation and L- to D-exchange show that peptides with broad-spectrum and/or excellent anti-MRSA activity (Albada et al, 2012b) and very low hemolytic activity (Albada et al, 2013) can be obtained. As the next step toward in vivo efficacy studies, these derivatives should be investigated for their effects on erythrocytes and, where appropriate, tested for acute toxicity.…”

“…Several of the resulting compounds displayed drastically improved activity against both Gram-positive and Gram-negative pathogens but also enhanced hemolytic potential (Albada et al, 2012a,b). Systematic exchange of L- to D-amino acid residues resulted in optimized non-hemolytic linear peptides with excellent anti-MRSA activity (Albada et al, 2013, 2014). Importantly, neither organometallic nor fatty acyl modifications significantly changed the antibacterial mechanism of action compared to the parent peptide MP196, which integrates into the cytoplasmic membrane and by deforming the lipid bilayer delocalizes essential proteins involved in respiration and cell wall biosynthesis as well as cell division (Wenzel et al, 2014, 2016).…”

A Blocking Group Scan Using a Spherical Organometallic Complex Identifies an Unprecedented Binding Mode with Potent Activity In Vitro and In Vivo for the Opioid Peptide Dermorphin

“…Established scan technologies that are used to evaluate the structure–activity relationship (SAR) of peptides, namely, alanine, proline, enantiomeric or aza‐amino acid, and N‐methylation scans, elucidate the influence of individual amino acid residues on the activity of the overall structure . We anticipated that to assess the contribution of a whole peptide domain, imposing steric demand is more appropriate than replacing or omitting multiple amino acid residues.…”