Rational Design of Antimicrobial C3a Analogues with Enhanced Effects against Staphylococci Using an Integrated Structure and Function-Based Approach

Abstract

The anaphylatoxin C3a and its inactivated derivative C3adesArg, generated during complement activation, exert direct antimicrobial effects, mediated via its C-terminal region [Nordahl et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 16879-16884]. During evolution, this region of C3a displays subtle changes in net charge, while preserving a moderate but variable amphipathicity [Pasupuleti et al. (2007) J. Biol. Chem. 282, 2520-2528]. In this study, we mimic these evolutionary changes, employing a design approach utilizing selected amino acid substitutions at strategic and structurally relevant positions in the original human C3a peptide CNYITELRRQHARASHLGLA, followed by structure-activity studies incorporating sequence-dependent QSAR models as tools for generation of C3a peptide variants with enhanced effects. While the native peptide and related amphipathic analogues of moderate positive net charge were active against the Gram-negative Escherichia coli, activity against the Gram-positive Staphylococcus aureus was primarily observed for peptides characterized by a combination of a relatively high net charge (+6-7) and a propensity to adopt an alpha-helical conformation with amphipathic character. Such increased helicity and charge also conferred activity against the fungus Candida albicans. A central histidine residue (H11), evolutionarily conserved among vertebrates, conferred high selectivity toward microbes, while substitutions with leucine rendered the peptides hemolytic. Selected C3a analogues retained their specificity against staphylococci in the presence of human plasma, while showing low cytotoxicity. The work illustrates structure-activity relationships underlying the function and specificity of antimicrobial C3a and related analogues and provides insights into the forces that drive evolution of antimicrobial peptides.

Structure-Activity Relations of Myxinidin, an Antibacterial Peptide Derived from the Epidermal Mucus of Hagfish

“…Other studies previously identified charge as a critical parameter for activity of helical AMPs (48); decreasing the net charge below ϩ3 reduces potency, while increasing the net charge gradually increases activity (49,50). In this context, it is interesting that positive charge selection appears to characterize the evolution of ␤-defensins and that hBD3, the human defensin with the highest positive net charge (ϩ10), also has the highest activity against S. aureus (51).…”

Structure-Activity Studies and Therapeutic Potential of Host Defense Peptides of Human Thrombin

“…In most cases, trials are aimed at topical indications where direct microbicidal effects are utilized. Considering this property of AMPs, various strategies have been employed in order to optimize the therapeutic index, including the use of combinational library approaches (1), stereoisomers composed of Damino acids (40) or cyclic D,L-␣-peptides (8), and quantitative structure-activity relationship (QSAR) and high-throughputbased screening assays (9,15,18,35,44). Furthermore, a novel approach for boosting AMPs through end-tagging with hydrophobic oligopeptide stretches has recently been demonstrated (23,32,33,41).…”

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

“…Minimal Inhibitory Concentration Assay-MIC assay was carried out by a microtiter broth dilution method as described previously (11,29) with slight modifications. For preparation of refined media, 100 ml of Luria Bertani broth was prepared in 10 mM Tris, pH 7.4, and applied to a column packed with 40 ml of DEAE Sephacel (9013-34-7; Sigma-Aldrich).…”