New indolicidin analogues with potent antibacterial activity*
T.S. Ryge
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorX. Doisy
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorD. Ifrah
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorJ.E. Olsen
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorP.R. Hansen
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorT.S. Ryge
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorX. Doisy
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorD. Ifrah
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorJ.E. Olsen
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorP.R. Hansen
T.S. Ryge , X. Doisy , D. Ifrah , J.E. Olsen and P.R. Hansen , Department of Chemistry, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark J.E. Olsen , Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen 1871, Denmark
Search for more papers by this authorA preliminary account of this work was presented at the 18th American Peptide Symposium, Boston, MA, 2003 and will be published in the proceedings.
Abstract
Abstract: Indolicidin is a 13-residue antimicrobial peptide amide, ILPWKWPWWPWRR-NH2, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two-fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR-NH2 in which Trp-residues 4,6,8,9,11 were replaced in all positions with X = a single non-natural building block; N-substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non-natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N-terminus with a hydrophobic non-natural building block. We prepared 22 analogues of indolicidin and [Phe4,6,8,9,11] indolicidin, 11 of each, carrying a hydrophobic non-natural building block attached to the N-terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non-natural building blocks, are promising lead structures for developing future therapeutics.
References
- 1 Boman, H.G. (2000) Innate immunity and the normal microflora. Immunol. Rev. 173, 5–16.
- 2 Lehrer, R. & Ganz, T. (1999) Antimicrobial peptides in mammalian and insect host defence. Curr. Opin. Immunol. 11, 23–27.
- 3 Hancock, R.E.W. & Scott, M.G. (2000) The role of antimicrobial peptides in animal defenses. PNAS 97, 8856–8861.
- 4 Hancock, R.E.W. & Diamond, G. (2000) The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 8, 402–410.
- 5 Huttner, K.M. & Bevins, C.L. (1999) Antimicrobial peptides as mediators of epithelial host defense. Pediatr. Res. 45, 785–794.
- 6 Nicolas, P. & Mor, A. (1995) Peptides as weapons against microorganisms in the chemical defense system of vertebrates. Annu. Rev. Microbiol. 49, 277–304.
- 7 These sequences are available at: http://www.bbcm.univ.trieste.it/tossi/antimic.html. (Last accessed 7th July 2004).
- 8 Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms. Nature 415, 389–395.
- 9 K. Lohner (ed.) (2001) Development of Novel Antimicrobial Agents: Emerging Strategies. Horizon Scientific Press, Norwich, UK.
- 10
Andreu, D. &
Rivas, L. (1998) Animal antimicrobial peptides: an overview.
Biopolymers
47, 415–433.
10.1002/(SICI)1097-0282(1998)47:6<415::AID-BIP2>3.0.CO;2-D CAS PubMed Web of Science® Google Scholar
- 11 Hancock, R.E.W. & Lehrer, R. (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16, 82–88.
- 12 van't Hof, W., Veerman, E.C.I., Helmerhorst, E.J. & Amerongen, A.V.N. (2001) Antimicrobial peptides: properties and applicability. Biol. Chem. 382, 597–619.
- 13 C.J. Dutton, M.A. Haxell, H.A.I. McArthur & R.G. Wax (eds) (2002) Peptide Antibiotics: Discovery, Modes of Action and Application. Marcel-Dekker, New York.
- 14 Wu, M., Maier, E., Benz, R. & Hancock, R.E. (1999) Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 38, 7235–7242.
- 15
Simmaco, M.,
Mignogna, G. &
Barra, D. (1998) Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers
47, 435–450.
10.1002/(SICI)1097-0282(1998)47:6<435::AID-BIP3>3.0.CO;2-8 CAS PubMed Web of Science® Google Scholar
- 16 K. Lohner (ed.) (2001) Development of Novel Antimicrobial Agents: Emerging strategies. Horizon Scientific Press, Norwich, UK, pp. 149–165.
- 17 Shai, Y. (2002) From innate immunity to de novo design of antimicrobial peptides. Curr. Pharm. Des. 8, 715–725.
- 18 Hong, J., Oren, Z. & Shai, Y. (1999) Structure and organization of hemolytic and nonhemolytic diastereomers of antimicrobial peptides in membranes. Biochemistry 38, 16963–16973.
- 19 Shai, Y. (1999) Mechanism of the binding, insertion, and destabilisation of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1462, 55–70.
- 20 Matsuzaki, K. (1999) Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta 1462, 1–10.
- 21 Yang, L., Weiss, T.M., Lehrer, T.I. & Huang, H.W. (2000) Crystallization of antimicrobial pores in membranes: magainin and protegrin. Biophys. J. 79, 2002–2009.
- 22 Huang, H.W. (2000) Action of antimicrobial peptides: two-state model. Biochemistry 39, 8347–8352.
- 23 Tossi, A., Sandri, L. & Giangaspero, A. (2000) Amphipathic, α-helical antimicrobial peptides. Biopolymers 55, 4–30.
- 24 Selsted, M.E., Novotny, M.J., Morris, W.L., Tang, Y.Q., Smith, W. & Cullor, J.S. (1992) Indolicidin a novel bactericidal tridecapeptide amide from neutrophils. J. Biol. Chem. 267, 4292–4295.
- 25 Zanetti, M., Gennaro, R., Scocchi, M. & Skerlavaj, B. (2000) Structure and biology of cathelicidins. Adv. Exp. Med. Biol. 479, 203–218.
- 26 Falla, T.J., Karunaratne, D.N. & Hancock, R.E. (1996) Mode of action of the antimicrobial peptide indolicidin. J. Biol. Chem. 271, 19298–19303.
- 27 Aley, S.B., Zimmerman, M., Hetsko, M., Selsted, M.E. & Gillin, F.D. (1994) Killing of Giardia-lamblia by cryptdins and cationic neutrophil peptides. Infect. Immun. 62, 5397–5403.
- 28 Lee, D.G., Kim, H.K., Kim, S.A., Park, Y., Park, S.C., Jang, S.H. & Hahm, K.S. (2003) Fungicidal effect of indolicidin and its interaction with phospholipid membranes. Biochem. Biophys. Res. Commun. 305, 305–310.
- 29 Robinson, W.E. Jr, McDougall, B., Tran, D. & Selsted, M.E. (1998) Anti-HIV-1 activity of indolicidin, an antimicrobial peptide from neutrophils. J. Leukoc. Biol. 63, 94–100.
- 30 Schluesener, H.J., Radermacher, S., Melms, A. & Jung, S. (1993) Leukocytic antimicrobial peptides kill autoimmune T-cells. J. Neuroimmunol. 47, 199–202.
- 31 Rozek, A., Friedrich, C.L. & Hancock, R.E. (2000) Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles. Biochemistry 39, 15765–15774.
- 32 Uchida, Y. & Shindo, M. (1999) Antibacterial activity of indolicidin and its analogues. In: Peptides 1998 ( S. Bajusz & F. Hudescz, eds), pp. 802–803. Akademiai Kiado, Hungary.
- 33 Staubitz, P., Peschel, A., Nieuwenhuizen, W.F., Otto, M., Götz, F., Jung, G. & Jack, R.W. (2001) Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin. J. Pept. Sci. 7, 552–564.
- 34 Lim, Y.B., Pyun, J.-C. & Park, J.-S. (1997) Synthesis and biological characterization of indolicidin analogues. J. Biochem. Mol. Biol. 30, 229–233.
- 35 Falla, T.J. & Hancock, R.E.W. (1997) Improved activity of a synthetic indolicidin analogue. Antimicrob. Agents Chemother. 41, 771–775.
- 36 Ösapay, K., Tran, D., Ladokhin, A.S., White, S.H., Henschen, A.H. & Selsted, M.E. (2000) Formation and characterization of a single trp-trp cross-link in indolicidin that confers protease stability without altering antimicrobial activity. J. Biol. Chem. 275, 12017–12022.
- 37 Subbalakshmi, C., Krishnakumari, V., Nagaraj, R. & Sitaram, N. (1996) Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin. FEBS Lett. 395, 48–52.
- 38 Subbalakshmi, C., Bikshapathy, E., Sitaram, N. & Nagaraj, R. (2000) Antibacterial and hemolytic activities of single tryptophan analogues of indolicidin. Biochem. Biophys. Res. Commun. 274, 714–716.
- 39 Kolodkin, N.I., Smirnova, M.P., Afonin, V.G., Shpen, V.M. & Tyagotin, J.V. (2002) Design and synthesis of indolicidin analogues with enhanced positive net charge and amphipathicity. In: Proceedings of the Twenty-seventh European Peptide Symposium ( E. Benedetti & C. Pedone, eds), p. 538, Edizioni Ziino, Napoli, Italy.
- 40 Hruby, V.J. & Matsunaga, T.O. (2002) Applications of synthetic peptides. In: Synthetic Peptides: a user's guide ( G.A. Grant ed.), pp. 272–376, Oxford University Press, Oxford, UK.
- 41 Zuckermann, R.N., Kerr, J.M., Kent, S.B.H. & Moos, W.H. (1992) Efficient method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase synthesis. J. Am. Chem. Soc. 114, 10646–10648.
- 42 Goodson, B., Ehrhardt, A., Ng, S., Nuss, J., Johnson, K., Giedlin, M., Yamamoto, R., Moos, W.H., Krebber, A., Ladner, M., Giacona, M.B., Vitt, C. & Winter, J. (1999) Characterization of novel antimicrobial peptoids. Antimicrob. Agents Chemother. 43, 1429–1434.
- 43 Humet, M., Carbonell, T., Masip, I., Sanchez-Baeza, F., Mora, P., Canton, E., Gobernado, M., Abad, C., Perez-Paya, E. & Messeguer, A. (2003) A positional scanning combinatorial library of peptoids as a source of biological active molecules: identification of antimicrobials. J. Comb. Chem. 5, 597–605.
- 44 Oh, J.E. & Lee, K.H. (1999) Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide. Bioorg. Med. Chem. 7, 2985–2990.
- 45 Cohen, S.A. & Strydom, D.J. (1988) Review: Amino acid analysis utilizing phenylisothiocyanate derivates. Anal. Biochem. 174, 1–16.
- 46 W.C. Chan & P.D. White (eds) (2000) Fmoc Solid-phase Peptide Synthesis. Oxford University Press, Oxford.
- 47 Available at: http://www.cmdr.ubc.ca/bobh/lab_methods.php. (Last accessed 7th July 2004).
- 48 Mshana, R., Tadesse, G., Abate, G. & Miorner, H. (1998) Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis. J. Clin. Microbiol. 36, 1214–1219.
- 49 Park, Y., Lee, D.G., Jang, S.H., Woo, E.R., Jeong, H.G., Choi, C.H. & Hahm, K.S. (2003) A Leu-Lys-rich antimicrobial peptide: activity and mechanism. Biochim. Biophys. Acta 1645, 172–182.
- 50 Blondelle, S.E. & Lohner, K. (2000) Combinatorial libraries: a tool to design antimicrobial and antifungal peptide analogues having lytic specificities for structure-activity relationship studies. Biopolymers 55, 74–87.
- 51 Haug, B.E. & Svendsen, J.S. (2001) The role of tryptophan in the antibacterial activity of a 15-residue bovine lactoferricin peptide. J. Pept. Sci. 7, 190–196.
- 52 Blondelle, S.E., Takahashi, E., Weber, P.A. & Houghten, R.A. (1994) Identification of antimicrobial peptides by using combinatorial libraries made up of unnatural amino acids. Antimicrob. Agents Chemother. 38, 2280–2286.
- 53 Vigano, C., Manciu, L., Buyse, F., Goormaghtigh, E. & Ruysschaert, J.M. (2000) Attenuated total reflection IR spectroscopy as a tool to investigate the structure, orientation and tertiary structure changes in peptides and membrane proteins. Biopolymers 55, 373–380.
- 54 Avrahami, D. & Shai, Y. (2002) Conjugation of a magainin analogue with lipophilic acids controls hydrophobicity, solution assembly, and cell selectivity. Biochemistry 41, 2254–2263.
- 55 Ding, L., Yang, L., Weiss, T.M., Waring, A.J., Lehrer, R.I. & Huang, H.W. (2003) Interaction of antimicrobial peptides with lipopolysaccharides. Biochemistry 42, 12251–12259.
- 56 Ahmad, I., Perkins, W.R., Lupan, D.M., Selsted, M.E. & Janoff, A.S. (1995) Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity. Biochim. Biophys. Acta 1237, 109–114.
- 57 Helmerhorst, E.J., Reijnders, I.M., van't Hof, W., Veerman, E.C.I. & Amerongen, A.V.N. (1999) A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides. FEBS Lett. 449, 105–110.
- 58 Ladokhin, A.S., Selsted, M.E. & White, S.H. (1999) CD spectra of indolicidin antimicrobial peptides suggest turns, not polyproline helix. Biochemistry 38, 12313–12319.
- 59 Ronish, E.W. & Krimm, S. (1974) The calculated circular dichroism of polyproline II in the polarizability approximation. Biopolymers 13, 1635–1651.
