Enhancement of the anti-inflammatory activity of temporin-1Tl-derived antimicrobial peptides by tryptophan, arginine and lysine substitutions Ganesan Rajasekaran, a Radhakrishnan Kamalakannan b and Song Yub Shin a,c * Temporin-1Tl (TL) is a 13-residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti-inflammatory activity. To develop novel AMP with improved anti-inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin-resistant Staphylococcus aureus strains compared with TL. TL-1 and TL-4 showed a little increase in antimicrobial selectivity, while TL-2 and TL-3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti-inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor-α (TNF-α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti-inflammatory activity is as follows: TL-2 ≈ TL-3 ≈ TL-4 > TL-1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti-inflammatory activity. These results apparently suggest that the anti-inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti-inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram-negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: temporin-1T1; temporin-1T1 analogs; antimicrobial activity; anti-inflammatory activity; gram-negative infection Introduction Amphibian skin temporins represent one of the largest families (over 100 members) of antimicrobial peptides (AMPs) and are among the smallest (8–14 amino acids in length) identified in na- ture to date [1–4]. The primary structures of temporins are rich in non-polar residues with fewer cationic and polar amino acids, yielding a net charge of 0 to +3 at neutral pH. Most of these peptides adopt an amphipathic α-helical conformation in a membrane-mimetic environment [5]. Temporins are particu- larly active against gram-positive bacteria and fungi. Interest- ingly, temporin-1Tl (TL: FVQWFSKFLGRIL-NH 2 ) exhibits higher antimicrobial activity against gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, and yeast strains than other isoforms [6,7]. Lipopolysaccharide (LPS), also termed endotoxin, is the major com- ponent of the outer membrane of gram-negative bacteria [8]. LPS is a well-known activator of the humoral and cellular immune systems in the host [9,10]. Uncontrolled stimulation of the host immune system by LPS can result in excessive release of inflammatory cytokines, leading to lethal septic shock, which often causes death [11–13]. Recently, some AMPs have emerged as attractive therapeutic candi- dates for treating endotoxin shock and sepsis caused by gram- negative bacterial infections because in addition to a broad-spectrum activity against bacteria, they also possess the potential ability to bind LPS and block LPS-stimulated cytokine release [14,15]. LL-37, in particular, has been shown to be effective in neutralizing LPS and consequently has considerable potential in the treatment of sepsis associated with gram-negative bacterial infections [16–19]. TL is also known to have significant anti-inflammatory activity against LPS-stimulated macrophage cells [20]. In order to develop novel TL analogs with improved anti-inflammatory activity, we designed and synthesized a series of TL analogs by amino acid substitution. TL forms an amphipathic α-helical structure in membrane-mimicking environment [6]. The amphipathicity of AMPs promotes better membrane interaction by enabling optimized electrostatic and hydrophobic interactions; highly positive charges on the hydrophilic region lead to * Correspondence to: Song Yub Shin, Department of Cellular and Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-759, Korea. E-mail: syshin@chosun.ac.kr a Department of Medical Science, Graduate School, Chosun University, Gwangju 501-759, Korea b Department of Pharmacology, School of Medicine, Chosun University, Gwangju 501-759, Korea c Department of Cellular and Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-759, Korea J. Pept. Sci. 2015; 21: 779–785 Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Research article Received: 3 June 2015 Revised: 10 July 2015 Accepted: 10 July 2015 Published online in Wiley Online Library: 27 August 2015 (wileyonlinelibrary.com) DOI 10.1002/psc.2807 779