Articles A Different Molecular Mechanism Underlying Antimicrobial and Hemolytic Actions of Temporins A and L Alfonso Carotenuto, § Stefania Malfi, § Maria Rosaria Saviello, § Pietro Campiglia, † Isabel Gomez-Monterrey, § Maria Luisa Mangoni, # Ludovica Marcellini Hercolani Gaddi, # Ettore Novellino, § and Paolo Grieco* ,§ Department of Pharmaceutical and Toxicological Chemistry, UniVersity of Naples “Federico II”, I-80131 Naples, Italy, Department of Pharmaceutical Science, UniVersity of Salerno, I-84084 Fisciano, Salerno, Italy, and Department of Biochemical Science, Cenci Bolognetti FoundationsPasteur Institute, “A. Rossi Fanelli”, UniVersity of Rome “La Sapienza”, I-00185 Rome, Italy ReceiVed December 20, 2007 In this work, the naturally occurring antimicrobial peptides temporin A (TA) and L (TL) are studied by spectroscopic (CD and NMR) techniques and molecular dynamics simulation. We analyzed the interactions of TA and TL with sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelles, which mimic bacterial and mammalian membranes, respectively. In SDS, the peptides prefer a location at the micelle-water interface; in DPC, they prefer a location perpendicular to the micelle surface, with the N-terminus imbedded in the hydrophobic core. TL shows higher propensity, with respect to TA, in forming R-helical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles. Hence, we have proposed a different molecular mechanism underlying the antimicrobial and hemolytic activities of the two peptides. We also designed new analogues of TA and TL and found interesting differences in their efficacy against microbial species and human erythrocytes. Introduction Gene-encoded antimicrobial peptides (AMPs) are considered key components of the innate immunity, which is found throughout the living world. 1–3 An ever increasing number of these molecules have been isolated from a vast array of biological sources, either prokaryotic or eukaryotic organisms, including humans, which they protect from the invasion of bacteria, protozoa, fungi, and viruses. 2,4,5 AMPs display an extreme diversity in their primary and secondary structures and usually have a rather large spectrum of antibiotic activity. As highlighted by Hans Boman, 6 the low selectivity and fast killing of microbes are crucial features of this peptide-based defense mechanism and make it as an “instant” immune system against microbial invaders. This immediate host response to infections plays an important role not only in invertebrates, which exclusively depend on it, but also in higher vertebrates, where it comes into action before the adaptive immunity is activated. 7,8 To face the challenge posed by the spreading resistance of pathogenic microorganisms to conventional drugs, the produc- tion of alternative antibiotics with new modes of action and resistance-avoiding properties has become an emergency. Among the possible candidates, AMPs are actually attractive molecules to be potentially developed as therapeutic anti- infective agents 2,9,10 and even as food preservatives. 11 Amphib- ian skin represents an incredibly rich source of AMPs, which are stored in granules of dermal glands and secreted upon stress or contact with microorganisms. 3,12 Among AMPs from am- phibia, temporins are a family of related molecules, first isolated from the skin of the European red frog Rana temporaria. 13 Many other members of this group, counting now over 50 peptides, have later been found in several other Rana species and also in the venom of wasps. 14–17 Structurally, temporins are character- ized by being short peptides (10–14 residues), with a net positive charge at neutral pH and a potential to adopt an amphipathic R-helix structure upon contact with membranes or in hydro- phobic environments. Their consensus sequence is FLPLI- ASLLSKLL-NH 2 . 16 Previously, temporins were found to be active particularly against Gram-positive bacteria, Candida species, fungi and with the ability to bind and permeate both artificial and biological membranes. 12,18–21 In this work, we focused on temporin A (TA) and temporin L (TL, Table 1) because of their different spectra of antimicrobial activity and toxicity. In particular, temporin A is preferentially active against Gram-positive bacteria, 14,19 including some clinically important antibiotic-resistant isolates, 14 displays a low lytic activity against human erythrocytes, 19 and kills efficiently the human parasitic protozoan Leishmania. 22 Differently, among all temporins studied to date, temporin L has the strongest activity against fungi, yeasts, Gram-positive and Gram-negative bacteria, eryth- rocytes, and cancer cells. 18,23 It is widely accepted that the main target of AMPs is the lipid bilayer of the bacterial membrane instead of specific * To whom correspondence should be addressed. Phone: +39-081- 678620. Fax: +39-081-678644. E-mail: pagrieco@unina.it. § University of Naples “Federico II”. † University of Salerno. # University of Rome “La Sapienza”. Table 1. Sequences of Analyzed Peptides peptide sequence TA H-Phe 1 -Leu 2 -Pro 3 -Leu 4 -Ile 5 -Gly 6 -Arg 7 -Val 8 - Leu 9 -Ser 10 -Gly 11 -Ile 12 -Leu 13 -NH 2 TL H-Phe 1 -Val 2 -Gln 3 -Trp 4 -Phe 5 -Ser 6 -Lys 7 -Phe 8 - Leu 9 -Gly 10 -Arg 11 -Ile 12 -Leu 13 -NH 2 Gln 3 -TA H-Phe 1 -Leu 2 -Gln 3 -Leu 4 -Ile 5 -Gly 6 -Arg 7 -Val 8 - Leu 9 -Ser 10 -Gly 11 -Ile 12 -Leu 13 -NH 2 Pro 3 -TL H-Phe 1 -Val 2 -Pro 3 -Trp 4 -Phe 5 -Ser 6 -Lys 7 -Phe 8 - Leu 9 -Gly 10 -Arg 11 -Ile 12 -Leu 13 -NH 2 J. Med. Chem. 2008, 51, 2354–2362 2354 10.1021/jm701604t CCC: $40.75  2008 American Chemical Society Published on Web 03/28/2008