Antibiotic activity and structural analysis of the scorpion-derived antimicrobial peptide IsCT and its analogs Kyungik Lee a , Song Yub Shin b, * , Kyoungho Kim a , Shin Saeng Lim b , Kyung-Soo Hahm b , Yangmee Kim a, * a Department of Chemistry and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea b Department of Bio-Materials, Graduate School and Research Center for Proteineous Materials, Chosun University, Gwangju 501-759, Republic of Korea Received 4 August 2004 Abstract IsCT is a non-cell-selective antimicrobial peptide isolated from the scorpion Opisthacanthus madagascariensis that has potent cytolytic activity against both mammalian and bacterial cells. To investigate the structure–activity relationships of IsCT and to design novel peptide antibiotics with bacterial cell selectivity, we synthesized several analogs of IsCT and determined their three-di- mensional structures in solution by 2D-NMR spectroscopy. IsCT has a linear a-helical structure from Gly 3 to Phe 13 , and [K 7 ]-IsCT has a linear a-helical structure from Leu 2 to Phe 13 . [K 7 ,P 8 ,K 11 ]-IsCT, which has a bend in its middle region, exhibited the highest antibacterial activity without hemolytic activity, suggesting that its proline-induced bend is an important determinant of this selec- tivity. Tryptophan fluorescence showed that the high selectivity of [K 7 ,P 8 ,K 11 ]-IsCT toward bacterial cells is closely correlated with its highly selective interaction with negatively charged phospholipids. Its potent activity against antibiotic-resistant bacteria suggests that [K 7 ,P 8 ,K 11 ]-IsCT may serve as a promising lead candidate in the development of new peptide antibiotics. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Antimicrobial peptide; IsCT; NMR; Bacterial cell selectivity; Phospholipid selectivity Antimicrobial peptides are important components of the innate immune system in all living organisms [1–3]. Many of these peptides rapidly kill invading pathogens by causing membrane permeabilization, although this may not be their sole mode of action [4–7]. Because these peptides have a mechanism of action that differs from that of currently available therapeutic antibiotics, the former may constitute a class of attractive new drugs against microorganisms resistant to currently available antibiotics. Of the antimicrobial peptides, one group, which in- cludes the cecropins, magainins, and seminal plasmin, has been shown to possess activity only against micro- bial cells, with little or no activity against mammalian cells [8–10], whereas the second group, which includes melittin, LL-37, indolicidin, the protegrins, and the tachyplesins, is toxic to both bacterial and mammalian cells [11–15]. For therapeutic use, antimicrobial peptides must have high bacterial cell selectivity, with high anti- bacterial activity and no hemolytic activity. IsCT is a natural a-helical antimicrobial peptide iso- lated from the scorpion Opisthacanthus madagascarien- sis [16,17]. It is one of the shortest of these peptides, consisting of only 13 amino acid residues, without any cysteines. IsCT shows potent cytolytic activity against both mammalian and bacterial cells. Since a short linear antimicrobial peptide with bacterial cell selectivity would be an attractive candidate as a therapeutic agent, we designed several analogs of IsCT with point 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.08.144 * Corresponding authors. Fax: +82 2 447 5987 (Y. Kim), +82 62 227 8345 (S.Y. Shin). E-mail addresses: syshin@chosun.ac.kr (S.Y. Shin), ymkim@ konkuk.ac.kr (Y. Kim). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 323 (2004) 712–719 BBRC