Aggregation and Its Influence on the Immunomodulatory Activity of Synthetic Innate Defense Regulator Peptides

Abstract

There is increasing interest in developing cationic host defense peptides (HDPs) and their synthetic derivatives as antimicrobial, immunomodulatory, and anti-biofilm agents. These activities are often evaluated without considering biologically relevant concentrations of salts or serum; furthermore certain HDPs have been shown to aggregate in vitro. Here we examined the effect of aggregation on the immunomodulatory activity of a synthetic innate defense regulator peptide, 1018 (VRLIVAVRIWRR-NH). A variety of salts and solutes were screened to determine their influence on 1018 aggregation, revealing that this peptide "salts out" of solution in an anion-specific and concentration-dependent manner. Furthermore, the immunomodulatory activity of 1018 was found to be inhibited under aggregation-promoting conditions. A series of 1018 derivatives were synthesized with the goal of disrupting this self-assembly process. Indeed, some derivatives exhibited reduced aggregation while maintaining certain immunomodulatory functions, demonstrating that it is possible to engineer optimized synthetic HDPs to avoid unwanted peptide aggregation.

Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application

“…Inconsistencies between in vitro and in vivo efficacy might be explained by physiological conditions within the host influencing stability and toxicity of anti-biofilm peptides and thus indirectly interfering with their activity. For instance, 1018 has been shown to precipitate in vitro in the presence of mucin and ions as well as when injected subcutaneously into mice [27,28]. In another study, topical application of the rationally de-DOI: 10.1159/000491497 signed peptide DRGN-1 significantly reduced wound size in a mixed biofilm-infected murine wound model [22].…”

“…In another study, substitutions of multiple amino acid residues in the sequence of LL-37-inspired peptide OP-145 resulted in the production of synthetic peptides with improved stability to plasma proteases [24]. Recently, the synthesis of a series of 1018 derivatives with amino acid substitutions, mainly in the hydrophobic stretch, allowed the investigation of sequence features associated with aggregation [11,27]. The identification of peptide sequences with decreased aggregation tendency but similar immunomodulatory activity suggested that precipitation can be avoided by the rational design of peptides.…”

“…Many parameters such as carbon and nitrogen sources, iron availability, and divalent cation concentrations differ significantly in lab media or buffers from those in an infected host [10,45,46]. HDPs exposed to components of plasma, serum, saliva, mucus, or urine for example, often exhibit very different activity levels from those in lab media for various reasons [24,27,[46][47][48][49]. Furthermore, the environment in which a bacterium grows can substantially affect the biofilm architecture and alter anti-biofilm peptide binding and/or penetration [45].…”

“…Recently used strategies to optimize anti-biofilm peptides include alteration of the amino acid composition, creation of hybrid peptides, and the design of structurally and functionally related compounds referred to as peptidomimetics [22,24,25,27,[29][30][31][32][33].…”

“…Alteration of the amino acid composition is by far the most studied approach and can be applied to modulate activity, stability, solubility/aggregation behavior, and cytotoxicity of anti-biofilm peptides [22,24,25,27,29,33]. Using peptides with known sequences and functions as templates allows for the development of modified peptides with improved properties by testing various amino acid substitutions using systematic or random design principles [12].…”