Indolicidin (IL)是一條有廣效抗生活性的鹼性抗生胜肽，包含對抗許多的病原體，可惜的是其細胞毒性大大的限制了其在臨床之使用。我們曾以置換胺基酸的方式來設計出九條IL的類似胜肽，以期具有低細胞毒性且高抗生活性。本研究探討IL及其類似胜肽之結構與生物活性間的關係，並進一步地提出設計抗生胜肽方面的準則，我們將研究帶正電性、親疏水性、疏水矩與抗菌、抗腫瘤、溶血、正常細胞毒性間的關聯。此外也將胜肽進行螢光標定並以共軛聚焦顯微鏡與螢光定量的方式探討其穿膜特性。結果顯示IL具有抗菌及抗腫瘤活性但卻有高溶血性，而IL類似物(IL-K7F89及IL-RF89)則是除了具有高抗菌及抗腫瘤活性，且同時展現低細胞毒性與溶血性，因此這兩條抗生胜肽在胜肽藥物臨床測試與新藥開發方面具有相當的潛力。另一方面，由結果可推測疏水性(Interface scale)與溶血活性有正相關性，但卻與疏水性(Water-octanol scale)和疏水矩並非直接相關，這可能是由於IL及其類似物與生物活性間的關連十分複雜，因此抗生胜肽之設計原則與胜肽之帶正電性、親疏水性與疏水矩，還需要更進一步的分析與探究才能歸納出彼此間的關聯性。在穿膜特性方面，由實驗結果顯示IL能協助FITC進入細胞內而IL-R57F89則不會，推測兩者的穿膜特性可能有些不同，IL可能擾動膜的程度比IL-R57F89還大，也可能是IL能在膜上形成孔洞或造成膜部分溶解，故能協助小分子物質FITC進入細胞中。另一方面，我們將螢光物質FITC以共價鍵結接枝在IL和具有臨床發展潛力的抗菌胜肽IL-R57F89上，結果顯示IL與IL-R57F89會經由胞吞和直接穿膜之形式進入纖維母細胞內。本研究證實IL-R57F89具有雙功能生物特性:一是具有低細胞毒性的鹼性抗生胜肽，二是可做為讓IL-R57F89攜帶小分子物質進入細胞當中的細胞穿膜胜肽。所以IL-R57F89不僅能作為胜肽抗生藥物，也能當載體運送分子進入細胞內，這在藥物的開發方面也開啟一個新的里程。

Indolicidin (IL) is a cationic antimicrobial peptide with broad-spectrum against many kinds of pathogens; however, the cytotoxicity of IL limits its clinical application. Nine IL analogues with lower hemolysis or cytotoxicity have been designed by engineering the critical amino acids for IL biological action. In this study, the structure-function relationships of IL and its analogues were discussed to provide important implications on antimicrobial IL analogues design. The effects of hydrophobicity, electropositivity, hydrophobic moment of peptides on their biological activities, including antibacterial activity, normal cell toxicity, hemolytic activity, and antitumor cell activity, were examined. Furthermore, transmembranes of peptides were also monitored by the confocal microscopy analysis via image the localization of FITC-conjugated peptides, and quantified through the FITC fluorescence. IL has effective antibacterial and antitumor activities, but it is harmful to human erythrocytes. In conji.;jltrast, IL-K7F89 and IL-R57F89 peptides exhibit not only high antibacterial and antitumor activities but also lower cytotoxicity and hemolytic activity. These two peptides are potential antimicrobial peptides for clinical use. Furthermore, we observed that the hydrophobicity based on interface scale is well correlated to the hemolytic activity rather than water-octanol scale and hydrophobic moment. Apparently, the biological action of IL and its analogues are trickier. Moreover, we found free IL peptide can facilitate FITC into NIH-3T3 cells but IL-R57F89 doesn’t, indicating that IL peptide could make the membrane perturbation, pore forming or membrane local disruption, to transport the FITC into cell. On the other hand, we conjugated the FITC to IL peptide and potential antimicrobial peptide candidate, IL-R57F89 peptide, through the thiourethane linking. We observed that IL and IL-R57F89 peptides could enter the cell via direct transmembrane and endocytosis. Consequently, we suggested that IL-R57F89 peptide has the dual-functional biological actions: one is a lower cytotoxicity cationic antimicrobial peptide; another is a cell penetrating peptide for transporting the small molecule into cell.

[1] Y. Shai, Z. Oren, From "carpet" mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides, Peptides 22 (2001) 1629-1641.
[2] N. Sitaram, R. Nagaraj, Host-defense antimicrobial peptides: importance of structure for activity, Curr Pharm Des 8 (2002) 727-742.
[3] M. Papagianni, Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications, Biotechnol Adv 21 (2003) 465-499.
[4] H.G. Boman, Peptide antibiotics and their role in innate immunity, Annu Rev Immunol 13 (1995) 61-92.
[5] E. Breukink, B. de Kruijff, The lantibiotic nisin, a special case or not?, Biochim Biophys Acta 1462 (1999) 223-234.
[6] T. Ganz, M.E. Selsted, D. Szklarek, S.S. Harwig, K. Daher, D.F. Bainton, R.I. Lehrer, Defensins. Natural peptide antibiotics of human neutrophils, J Clin Invest 76 (1985) 1427-1435.
[7] H.A.A.C. Michl, Isolation and structure of a haemolytic polypeptide from the defensive secretion of European Bombina species., Chemical Mounthly (1970).
[8] H.S. Won, S.J. Kang, W.S. Choi, B.J. Lee, Activity optimization of an undecapeptide analogue derived from a frog-skin antimicrobial peptide, Mol Cells 31 (2011) 49-54.
[9] H.G. Boman, Antibacterial Peptides - Key Components Needed in Immunity, Cell 65 (1991) 205-207.
[10] H. Steiner, D. Hultmark, A. Engstrom, H. Bennich, H.G. Boman, Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292: 246-248. 1981, J Immunol 182 (2009) 6635-6637.
[11] M. Zasloff, Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor, Proc Natl Acad Sci U S A 84 (1987) 5449-5453.
[12] A. Mor, V.H. Nguyen, A. Delfour, D. Migliore-Samour, P. Nicolas, Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin, Biochemistry 30 (1991) 8824-8830.
[13] Z. Oren, Y. Shai, A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole fish Pardachirus marmoratus, European Journal of Biochemistry 237 (1996) 303-310.
[14] J. Johansson, G.H. Gudmundsson, M.E. Rottenberg, K.D. Berndt, B. Agerberth, Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37, J Biol Chem 273 (1998) 3718-3724.
[15] E. Habermann, G. Zeuner, Comparative studies of native and synthetic melittins, Naunyn Schmiedebergs Arch Pharmakol 270 (1971) 1-9.
[16] J.A. Virtanen, K.H. Cheng, P. Somerharju, Phospholipid composition of the mammalian red cell membrane can be rationalized by a superlattice model, Proc Natl Acad Sci U S A 95 (1998) 4964-4969.
[17] K.A. Brogden, Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?, Nat Rev Microbiol 3 (2005) 238-250.
[18] M.E. Selsted, M.J. Novotny, W.L. Morris, Y.Q. Tang, W. Smith, J.S. Cullor, Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils, J Biol Chem 267 (1992) 4292-4295.
[19] A.S. Ladokhin, M.E. Selsted, S.H. White, Bilayer interactions of indolicidin, a small antimicrobial peptide rich in tryptophan, proline, and basic amino acids, Biophys J 72 (1997) 794-805.
[20] W.E. Robinson, Jr., B. McDougall, D. Tran, M.E. Selsted, Anti-HIV-1 activity of indolicidin, an antimicrobial peptide from neutrophils, J Leukoc Biol 63 (1998) 94-100.
[21] I. Ahmad, W.R. Perkins, D.M. Lupan, M.E. Selsted, A.S. Janoff, Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity, Biochim Biophys Acta 1237 (1995) 109-114.
[22] C. Subbalakshmi, V. Krishnakumari, R. Nagaraj, N. Sitaram, Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin, FEBS Lett 395 (1996) 48-52.
[23] A. Giacometti, O. Cirioni, G. Greganti, M. Quarta, G. Scalise, In vitro activities of membrane-active peptides against gram-positive and gram-negative aerobic bacteria, Antimicrob Agents Chemother 42 (1998) 3320-3324.
[24] T.J. Falla, D.N. Karunaratne, R.E. Hancock, Mode of action of the antimicrobial peptide indolicidin, J Biol Chem 271 (1996) 19298-19303.
[25] J.C. Hsu, C.M. Yip, Molecular dynamics simulations of indolicidin association with model lipid bilayers, Biophys J 92 (2007) L100-102.
[26] L. Zhang, A. Rozek, R.E. Hancock, Interaction of cationic antimicrobial peptides with model membranes, J Biol Chem 276 (2001) 35714-35722.
[27] J.E. Shaw, J.R. Alattia, J.E. Verity, G.G. Prive, C.M. Yip, Mechanisms of antimicrobial peptide action: Studies of indolicidin assembly at model membrane interfaces by in situ atomic force microscopy, Journal of Structural Biology 154 (2006) 42-58.
[28] D.J. Schibli, R.F. Epand, H.J. Vogel, R.M. Epand, Tryptophan-rich antimicrobial peptides: comparative properties and membrane interactions, Biochem Cell Biol 80 (2002) 667-677.
[29] W.M. Yau, W.C. Wimley, K. Gawrisch, S.H. White, The preference of tryptophan for membrane interfaces, Biochemistry 37 (1998) 14713-14718.
[30] M. Wu, E. Maier, R. Benz, R.E. Hancock, Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli, Biochemistry 38 (1999) 7235-7242.
[31] C.H. Hsu, C. Chen, M.L. Jou, A.Y. Lee, Y.C. Lin, Y.P. Yu, W.T. Huang, S.H. Wu, Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA, Nucleic Acids Res 33 (2005) 4053-4064.
[32] C. Subbalakshmi, N. Sitaram, Mechanism of antimicrobial action of indolicidin, FEMS Microbiol Lett 160 (1998) 91-96.
[33] C. Subbalakshmi, E. Bikshapathy, N. Sitaram, R. Nagaraj, Antibacterial and hemolytic activities of single tryptophan analogs of indolicidin, Biochem Biophys Res Commun 274 (2000) 714-716.
[34] Y.H. Nan, K.H. Park, Y. Park, Y.J. Jeon, Y. Kim, I.S. Park, K.S. Hahm, S.Y. Shin, Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin, FEMS Microbiol Lett 292 (2009) 134-140.
[35] T.J. Falla, R.E. Hancock, Improved activity of a synthetic indolicidin analog, Antimicrob Agents Chemother 41 (1997) 771-775.
[36] H. Mozsolits, H.J. Wirth, J. Werkmeister, M.I. Aguilar, Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance, Biochim Biophys Acta 1512 (2001) 64-76.
[37] C.L. Friedrich, D. Moyles, T.J. Beveridge, R.E. Hancock, Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria, Antimicrob Agents Chemother 44 (2000) 2086-2092.
[38] C.L. Friedrich, A. Rozek, A. Patrzykat, R.E. Hancock, Structure and mechanism of action of an indolicidin peptide derivative with improved activity against gram-positive bacteria, J Biol Chem 276 (2001) 24015-24022.
[39] W.S. Yew, H.E. Khoo, The role of tryptophan residues in the hemolytic activity of stonustoxin,a lethal factor from stonefish (Synanceja horrida) venom, Biochimie 82 (2000) 251-257.
[40] P. Staubitz, A. Peschel, W.F. Nieuwenhuizen, M. Otto, F. Gotz, G. Jung, R.W. Jack, Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin, J Pept Sci 7 (2001) 552-564.
[41] S.T. Yang, S.Y. Shin, K.S. Hahm, J.I. Kim, Design of perfectly symmetric Trp-rich peptides with potent and broad-spectrum antimicrobial activities, Int J Antimicrob Agents 27 (2006) 325-330.
[42] H.K. Baumgartner, N. Beeman, R.S. Hodges, M.C. Neville, A D-peptide analog of the second extracellular loop of claudin-3 and -4 leads to mislocalized claudin and cellular apoptosis in mammary epithelial cells, Chem Biol Drug Des 77 (2011) 124-136.
[43] I. Nakase, H. Akita, K. Kogure, A. Graslund, U. Langel, H. Harashima, S. Futaki, Efficient Intracellular Delivery of Nucleic Acid Pharmaceuticals Using Cell-Penetrating Peptides, Acc Chem Res (2011).
[44] L. Yang, T.A. Harroun, T.M. Weiss, L. Ding, H.W. Huang, Barrel-stave model or toroidal model? A case study on melittin pores, Biophys J 81 (2001) 1475-1485.
[45] R.M. Srivastava, S. Srivastava, M. Singh, V.K. Bajpai, J.K. Ghosh, Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide, J Biol Chem 287 (2012) 1980-1995.
[46] S. Katayama, H. Hirose, K. Takayama, I. Nakase, S. Futaki, Acylation of octaarginine: Implication to the use of intracellular delivery vectors, J Control Release 149 (2011) 29-35.
[47] G. Drin, S. Cottin, E. Blanc, A.R. Rees, J. Temsamani, Studies on the internalization mechanism of cationic cell-penetrating peptides, J Biol Chem 278 (2003) 31192-31201.
[48] S.W. Jones, R. Christison, K. Bundell, C.J. Voyce, S.M. Brockbank, P. Newham, M.A. Lindsay, Characterisation of cell-penetrating peptide-mediated peptide delivery, Br J Pharmacol 145 (2005) 1093-1102.
[49] M. Tyagi, M. Rusnati, M. Presta, M. Giacca, Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans, J Biol Chem 276 (2001) 3254-3261.
[50] S. Console, C. Marty, C. Garcia-Echeverria, R. Schwendener, K. Ballmer-Hofer, Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans, J Biol Chem 278 (2003) 35109-35114.
[51] C.L. Friedrich, D. Moyles, T.J. Beveridge, R.E.W. Hancock, Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria, Antimicrobial Agents and Chemotherapy 44 (2000) 2086-2092.
[52] C.Y. Jiao, D. Delaroche, F. Burlina, I.D. Alves, G. Chassaing, S. Sagan, Translocation and Endocytosis for Cell-penetrating Peptide Internalization, Journal of Biological Chemistry 284 (2009) 33957-33965.