Abstract
To become clinically effective, antimicrobial peptides (AMPs) should be non-cytotoxic to host cells. Piscidins are a group of fish-derived AMPs with potent antimicrobial and antiendotoxin activities but limited by extreme cytotoxicity. We conjectured that introduction of cationic residue(s) at the interface of polar and non-polar faces of piscidins may control their insertion into hydrophobic mammalian cell membrane and thereby reducing cytotoxicity. We have designed several novel analogs of piscidin-1 by substituting threonine residue(s) with L and D-lysine residue(s). L/D-lysine-substituted analogs showed significantly reduced cytotoxicity but exhibited either higher or comparable antibacterial activity akin to piscidin-1. Piscidin-1-analogs demonstrated higher efficacy than piscidin-1 in inhibiting lipopolysaccharide (LPS)-induced pro-inflammatory responses in THP-1 cells. T15,21K-piscidin-1 (0.5 mg/Kg) and T15,21dK-piscidin-1 (1.0 mg/Kg) demonstrated 100% survival of LPS (12.0 mg/Kg)-administered mice. High resolution NMR studies revealed that both piscidin-1 and T15,21K-piscidin-1 adopted helical structures, with latter showing a shorter helix, higher amphipathicity and cationic residues placed at optimal distances to form ionic/hydrogen bond with lipid A of LPS. Remarkably, T15,21dK-piscidin-1 showed a helix-loop-helix structure in LPS and its interactions with LPS could be sustained by the distance of separation of side chains of R7 and D-Lys-15 which is close to the inter-phosphate distance of lipid A.
Highlights
Introduction of cationic residues within anantimicrobial peptides (AMPs) could strongly influence its biological properties
Piscidin-1-analogs containing D-amino acids have been omitted in the helical wheel diagrams since these amino acids are known to impair the helical structure of a peptide[23,24]
HPLC retention times for purification of piscidin-1 and its analogs are shown in Table 1 and their HPLC profiles are shown in Supplementary Figure S2
Summary
Introduction of cationic residues within anAMP could strongly influence its biological properties. We envisioned that placement of cationic residues just outside its non-polar faces or at the interfaces of hydrophobic and hydrophilic faces of piscidin-1 could weaken the interaction between its hydrophobic face and hydrophobic outer membrane of mammalian cell, resulting in its weaker penetration in the mammalian cell membrane and reducing its cytotoxicity. The impact of substitution of single threonine residue by a lysine residue in the interface of non-polar and polar faces was investigated. Since many times incorporation of D-amino acids in AMPs results in the reduction of their cytotoxic properties[23,24], by perturbing their helical structures, the effect of substitution with D-lysine residue(s) instead of L-lysine residue(s) in the interface of polar and non-polar faces of piscidin-1 was investigated. High resolution NMR structures were determined for piscidin-1, its double L-lysine and D-lysine substituted analogs in LPS to understand their interactions with LPS, the basis of their anti-endotoxin properties as well as the influence of double L and D- lysine substitutions in the atomic structure of piscidin-1 in LPS
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