Augmentation of Antimicrobial Activity of Spiniferin by Introducing an Arginine Residue Toward Its Amino Terminus: A Possible Role of Cation-π Interaction.

Spiniferin is a 13-mer scorpion-origin antimicrobial peptide having poor antimicrobial activity. To augment Spiniferin's antimicrobial activity, we enhanced its net positive charge by replacing a glutamic acid residue with an arginine residue toward its amino terminus. We envisaged that a cation-π interaction could be introduced between this arginine residue and the tryptophan residue located near the middle of Spiniferin. This cation-π interaction could promote stronger interaction of the peptide with a negatively charged bacterial membranes, resulting in its increased antimicrobial activity. Though glutamic acid-to-arginine substitution [Spiniferin-(E4R)] enhanced both the antimicrobial and toxic properties of Spiniferin, the same replacement with a d-arginine residue [Spiniferin-(E4dR)] significantly enhanced its antimicrobial activity against selected Gram-negative/positive bacteria and a MRSA strain while maintaining low hemolytic/cytotoxic properties. Interestingly, Spiniferin-(E4dR) analogs, with its aromatic-tryptophan residue substituted with an aromatic phenylalanine or an aliphatic valine residue, and its d-arginine residue replaced with a d-lysine residue, showed much lesser antibacterial activity than Spiniferin-(E4dR) or Spiniferin-(E4R). The results indicated a crucial role of the tryptophan and l-/d-arginine combination in augmenting the antimicrobial activity of Spiniferin analogs, Spiniferin-(E4R) and Spiniferin-(E4dR). Spiniferin-(E4dR) showed bactericidal properties against selected Gram-positive/negative bacteria. It permeabilized bacterial membranes and induced damages in bacterial membrane organization, suggesting that the bacterial plasma membrane is its target for exhibiting antimicrobial activity. Further, Spiniferin-(E4dR) in the intravenous route demonstrated the survival of E. coli ATCC 25922-infected mice and the clearance of bacteria from the visceral organs of these mice. Computational studies showed the requisite distance between the arginine's cationic side chain and the π-electron site of the tryptophan residue for a possible intramolecular cation-π interaction in Spiniferin-(E4dR)/Spiniferin-(E4R).