Title: Rational Design of α-Helical Antimicrobial Peptides with Enhanced Activities and Specificity/Therapeutic Index
Abstract: In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 °C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the d-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with l-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected d-/l-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities. In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 °C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the d-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with l-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected d-/l-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities. The extensive clinical use of classical antibiotics has led to the growing emergence of many medically relevant resistant strains of bacteria (1.Neu H.C. Science. 1992; 257: 1064-1073Crossref PubMed Scopus (2303) Google Scholar, 2.Travis J. Science. 1994; 264: 360-362Crossref PubMed Scopus (172) Google Scholar). Moreover, only three new structural classes of antibiotics (the oxazolidinone, linezolid, the strepto-gramins, and the lipopeptide, daptomycin (3.Calza L. Manfredi R. Chiodo F. Expert Opin. Pharmacother. 2004; 5: 1899-1916Crossref PubMed Scopus (7) Google Scholar, 4.Jacqueline C. Asseray N. Batard E. Mabecque V.L. Kergueris M.F. Dube L. Bugnon D. Potel G. Caillon J. Int. J. Antimicrob. Agents. 2004; 24: 393-396Crossref PubMed Scopus (35) Google Scholar, 5.Wagenlehner F.M. Naber K.G. Int. J. Antimicrob. Agents. 2004; 24: 39-43Crossref PubMed Scopus (28) Google Scholar)) have been introduced into medical practice in the past 40 years. Therefore, the development of a new class of antibiotics has become critical. The cationic antimicrobial peptides could represent such a new class of antibiotics (6.Hancock R.E. Lancet. 1997; 349: 418-422Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 7.Andreu D. Rivas L. Biopolymers. 1998; 47: 415-433Crossref PubMed Scopus (503) Google Scholar, 8.Sitaram N. Nagaraj R. Curr. Pharm. Des. 2002; 8: 727-742Crossref PubMed Scopus (99) Google Scholar). Although the exact mode of action of antimicrobial peptides has not been established, all cationic amphipathic peptides interact with membranes, and it has been proposed that the cytoplasmic membrane is the main target of some peptides, whereby peptide accumulation in the membrane causes increased permeability and loss of barrier function (9.Hancock R.E. Lehrer R. Trends Biotechnol. 1998; 16: 82-88Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar, 10.Duclohier H. Molle G. Spach G. Biophys. J. 1989; 56: 1017-1021Abstract Full Text PDF PubMed Scopus (197) Google Scholar). The development of resistance to membrane active peptides whose sole target is the cytoplasmic membrane is not expected because this would require substantial changes in the lipid composition of cell membranes of microorganisms. Two major classes of the cationic antimicrobial peptides are the α-helical and the β-sheet peptides (6.Hancock R.E. Lancet. 1997; 349: 418-422Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 7.Andreu D. Rivas L. Biopolymers. 1998; 47: 415-433Crossref PubMed Scopus (503) Google Scholar, 11.van't Hof W. Veerman E.C. Helmerhorst E.J. Amerongen A.V. Biol. Chem. 2001; 382: 597-619PubMed Google Scholar, 12.Devine D.A. Hancock R.E. Curr. Pharm. Des. 2002; 8: 703-714Crossref PubMed Scopus (144) Google Scholar). The β-sheet class consists of cyclic peptides constrained in this conformation either by intramolecular disulfide bonds, e.g. defensins (13.Ganz T. Lehrer R.I. Curr. Opin. Immunol. 1994; 6: 584-589Crossref PubMed Scopus (374) Google Scholar) and protegrins (14.Steinberg D.A. Hurst M.A. Fujii C.A. Kung A.H. Ho J.F. Cheng F.C. Loury D.J. Fiddes J.C. Antimicrob. Agents Chemother. 1997; 41: 1738-1742Crossref PubMed Google Scholar), or by an N-terminal to C-terminal covalent bond, e.g. gramicidin S (15.Khaled M.A. Urry D.W. Sugano H. Miyoshi M. Izumiya N. Biochemistry. 1978; 17: 2490-2494Crossref PubMed Scopus (26) Google Scholar) and tyrocidines (16.Mootz H.D. Marahiel M.A. J. Bacteriol. 1997; 179: 6843-6850Crossref PubMed Google Scholar). Unlike the β-sheet peptides, α-helical peptides are linear molecules that mainly exist as disordered structures in aqueous media and become amphipathic helices upon interaction with the hydrophobic membranes, e.g. cecropins (17.Christensen B. Fink J. Merrifield R.B. Mauzerall D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5072-5076Crossref PubMed Scopus (446) Google Scholar), magainins (18.Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Crossref PubMed Scopus (1985) Google Scholar), and melittins (19.Andreu D. Ubach J. Boman A. Wahlin B. Wade D. Merrifield R.B. Boman H.G. FEBS Lett. 1992; 296: 190-194Crossref PubMed Scopus (234) Google Scholar). From numerous structure/activity studies on both natural and synthetic antimicrobial peptides, a number of factors believed to be important for antimicrobial activity have been identified, including the presence of both hydrophobic and basic residues, an amphipathic nature that segregates basic and hydrophobic residues, and an inducible or preformed secondary structure (α-helical or β-sheet). The major barrier to the use of antimicrobial peptides as antibiotics is their toxicity or ability to lyse eukaryotic cells. This is perhaps not a surprising result if the target is indeed the cell membrane (6.Hancock R.E. Lancet. 1997; 349: 418-422Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 7.Andreu D. Rivas L. Biopolymers. 1998; 47: 415-433Crossref PubMed Scopus (503) Google Scholar, 8.Sitaram N. Nagaraj R. Curr. Pharm. Des. 2002; 8: 727-742Crossref PubMed Scopus (99) Google Scholar, 9.Hancock R.E. Lehrer R. Trends Biotechnol. 1998; 16: 82-88Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar). To be useful as a broad spectrum antibiotic, it would be necessary to dissociate anti-eukaryotic activity from antimicrobial activity, i.e. increasing the antimicrobial activity and reducing toxicity to normal cells. Recent studies on a number of α-helical and β-sheet peptides have attempted to delineate features responsible for these activities and found that high amphipathicity (20.Dathe M. Wieprecht T. Nikolenko H. Handel L. Maloy W.L. MacDonald D.L. Beyermann M. Bienert M. FEBS Lett. 1997; 403: 208-212Crossref PubMed Scopus (327) Google Scholar, 21.Blondelle S.E. Houghten R.A. Biochemistry. 1992; 31: 12688-12694Crossref PubMed Scopus (343) Google Scholar, 22.Lee D.L. Hodges R.S. Biopolymers. 2003; 71: 28-48Crossref PubMed Scopus (98) Google Scholar, 23.Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), high hydrophobicity (20.Dathe M. Wieprecht T. Nikolenko H. Handel L. Maloy W.L. MacDonald D.L. Beyermann M. Bienert M. FEBS Lett. 1997; 403: 208-212Crossref PubMed Scopus (327) Google Scholar, 23.Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 24.Oren Z. Hong J. Shai Y. J. Biol. Chem. 1997; 272: 14643-14649Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 25.Kondejewski L.H. Lee D.L. Jelokhani-Niaraki M. Farmer S.W. Hancock R.E. Hodges R.S. J. Biol. Chem. 2002; 277: 67-74Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), as well as high helicity or β-sheet structure (23.Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 26.Shai Y. Oren Z. J. Biol. Chem. 1996; 271: 7305-7308Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 27.Oren Z. Shai Y. Biochemistry. 1997; 36: 1826-1835Crossref PubMed Scopus (371) Google Scholar) were correlated with increased toxicity as measured by hemolytic activity. In contrast, antimicrobial activity was found to be less dependent on these factors, compared with hemolytic activity (20.Dathe M. Wieprecht T. Nikolenko H. Handel L. Maloy W.L. MacDonald D.L. Beyermann M. Bienert M. FEBS Lett. 1997; 403: 208-212Crossref PubMed Scopus (327) Google Scholar, 21.Blondelle S.E. Houghten R.A. Biochemistry. 1992; 31: 12688-12694Crossref PubMed Scopus (343) Google Scholar, 22.Lee D.L. Hodges R.S. Biopolymers. 2003; 71: 28-48Crossref PubMed Scopus (98) Google Scholar, 23.Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 24.Oren Z. Hong J. Shai Y. J. Biol. Chem. 1997; 272: 14643-14649Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 26.Shai Y. Oren Z. J. Biol. Chem. 1996; 271: 7305-7308Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 27.Oren Z. Shai Y. Biochemistry. 1997; 36: 1826-1835Crossref PubMed Scopus (371) Google Scholar, 28.Lee D.L. Powers J.P. Pflegerl K. Vasil M.L. Hancock R.E. Hodges R.S. J. Pept. Res. 2004; 63: 69-84Crossref PubMed Scopus (77) Google Scholar). Therefore, specificity (or therapeutic index, which is defined as the ratio of MHC 1The abbreviations used are: MHC, minimal hemolytic concentration; RP-HPLC, reversed-phase high performance liquid chromatography; MIC, minimal inhibitory concentration; TFE, trifluoroethanol; PE, phosphatidylethanolamine; PC, phosphatidylcholine.1The abbreviations used are: MHC, minimal hemolytic concentration; RP-HPLC, reversed-phase high performance liquid chromatography; MIC, minimal inhibitory concentration; TFE, trifluoroethanol; PE, phosphatidylethanolamine; PC, phosphatidylcholine. (hemolytic activity) and MIC (antimicrobial activity), MHC/MIC for bacteria over erythrocytes) could be increased in one of the following three ways: increasing antimicrobial activity, decreasing hemolytic activity while maintaining antimicrobial activity, or a combination of both increasing antimicrobial activity and decreasing hemolytic activity. We believe that a synthetic peptide approach to examining the effect of small incremental changes in hydrophobicity/hydrophilicity, amphipathicity, and helicity of cationic antimicrobial peptides will enable rapid progress in the rational design of peptide antibiotics. Our previous studies (22.Lee D.L. Hodges R.S. Biopolymers. 2003; 71: 28-48Crossref PubMed Scopus (98) Google Scholar, 23.Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 28.Lee D.L. Powers J.P. Pflegerl K. Vasil M.L. Hancock R.E. Hodges R.S. J. Pept. Res. 2004; 63: 69-84Crossref PubMed Scopus (77) Google Scholar) have successfully utilized such an approach to dissociate antimicrobial and hemolytic activities of de novo designed cyclic β-sheet gramicidin S analogs, by systematic alterations in amphipathicity/hydrophobicity through d-amino acid substitutions. In recent work, we demonstrated that the helix-destabilizing properties of d-amino acids offer a systematic approach to the controlled alteration of the hydrophobicity, amphipathicity, and helicity of amphipathic α-helical model peptides (29.Chen Y. Mant C.T. Hodges R.S. J. Pept. Res. 2002; 59: 18-33Crossref PubMed Scopus (80) Google Scholar). By single substitutions of different d-amino acids into the center of the hydrophobic face of an amphipathic α-helical model peptide, we demonstrated that different d-amino acids disrupted α-helical structure to different degrees, whereas the destabilized structure could still be induced to fold into an α-helix in hydrophobic medium. The advantage of this method of single d- or l-amino acid substitutions at a specific site is that it enables a greater understanding of the mechanism of action of these peptides. In this study, we have utilized the structural framework of an amphipathic α-helical antimicrobial peptide V681 (30.Zhang L. Benz R. Hancock R.E. Biochemistry. 1999; 38: 8102-8111Crossref PubMed Scopus (131) Google Scholar, 31.Zhang L. Falla T. Wu M. Fidai S. Burian J. Kay W. Hancock R.E. Biochem. Biophys. Res. Commun. 1998; 247: 674-680Crossref PubMed Scopus (77) Google Scholar) to systematically change peptide amphipathicity, hydrophobicity, and helicity by single d- or l-amino acid substitutions in the center of either the polar or nonpolar faces of the amphipathic helix. Peptide V681, with excellent antimicrobial activity and strong hemolytic activity (30.Zhang L. Benz R. Hancock R.E. Biochemistry. 1999; 38: 8102-8111Crossref PubMed Scopus (131) Google Scholar, 31.Zhang L. Falla T. Wu M. Fidai S. Burian J. Kay W. Hancock R.E. Biochem. Biophys. Res. Commun. 1998; 247: 674-680Crossref PubMed Scopus (77) Google Scholar), was selected as an ideal candidate for our study. By introducing different d- or l-amino acid substitutions, we report here that hydrophobicity/amphipathicity and helicity have dramatic effects on the biophysical and biological activities, and by utilizing this method, a significant improvement in antimicrobial activity and specificity can be achieved. In addition, it is plausible that high peptide hydrophobicity and amphipathicity also result in greater peptide self-association in solution. Because we have developed a novel method to measure self-association of small amphipathic molecules, namely temperature profiling in reversed-phase chromatography (32.Mant C.T. Chen Y. Hodges R.S. J. Chromatogr. A. 2003; 1009: 29-43Crossref PubMed Scopus (49) Google Scholar, 33.Lee D.L. Mant C.T. Hodges R.S. J. Biol. Chem. 2003; 278: 22918-22927Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), we have applied this technique for the first time to investigate the influence of peptide dimerization ability on biological activities of α-helical antimicrobial peptides. Thus, our objectives in this study were 3-fold. First was to demonstrate the importance of the peptide self-association parameter in the de novo design of amphipathic α-helical antimicrobial peptides. Second was to test the hypothesis that disruption of α-helical structure in benign conditions by d-amino acid substitutions or substitutions of hydrophilic/charged l-amino acids on the nonpolar face can dramatically alter specificity in a similar manner to our previous work on cyclic β-sheet antimicrobial peptides (23.Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 28.Lee D.L. Powers J.P. Pflegerl K. Vasil M.L. Hancock R.E. Hodges R.S. J. Pept. Res. 2004; 63: 69-84Crossref PubMed Scopus (77) Google Scholar). Third was to observe whether these substitutions will enhance antimicrobial activity, decrease toxicity, and improve antimicrobial specificity while maintaining broad spectrum activity for Gram-negative and Gram-positive bacteria. Peptide Synthesis and Purification—Syntheses of the peptides Ac-KWKSFLKTFKXD/LAVKTVLHTALKAISS-amide and Ac-KWKSFLKTFKSAXD/LKTVLHTALKAISS-amide, with substitution sites at positions 11 and 13, respectively, were carried out by solid phase peptide synthesis using t-butyloxycarbonyl chemistry and 4-methylbenzhydrylamine resin (0.97 mmol/g), as described previously (29.Chen Y. Mant C.T. Hodges R.S. J. Pept. Res. 2002; 59: 18-33Crossref PubMed Scopus (80) Google Scholar). The crude peptides were purified by preparative RP-HPLC using a Zorbax 300 SB-C8 column (250 × 9.4-mm inner diameter; 6.5-μm particle size, 300-Å pore size; Agilent Technologies) with a linear AB gradient (0.2% acetonitrile/min) at a flow rate of 2 ml/min, where mobile phase A was 0.1% aqueous trifluoroacetic acid in water, and B was 0.1% trifluoroacetic acid in acetonitrile. The purity of peptides was verified by analytical RP-HPLC as described below. The peptides were further characterized by electrospray mass spectrometry and amino acid analysis. Analytical RP-HPLC of Peptides—Peptides were analyzed on an Agilent 1100 series liquid chromatograph (Little Falls, DE). Runs were performed on a Zorbax 300 SB-C8 column (150 × 2.1-mm inner diameter; 5-μm particle size, 300-Å pore size) from Agilent Technologies using a linear AB gradient (1% acetonitrile/min) and a flow rate of 0.25 ml/min, where solvent A was 0.05% aqueous trifluoroacetic acid, pH 2, and solvent B was 0.05% trifluoroacetic acid in acetonitrile. Temperature profiling analyses were performed in 3 °C increments, from 5 to 80 °C. Characterization of Helical Structure—The mean residue molar ellipticities of peptides were determined by CD spectroscopy, using a Jasco J-720 spectropolarimeter (Jasco, Easton, MD), at 25 °C under benign (nondenaturing) conditions (50 mm KH2PO4/K2HPO4, 100 mm KCl, pH 7), hereafter referred to as KP buffer, as well as in the presence of an α-helix inducing solvent, 2,2,2-trifluoroethanol (TFE) (50 mm KH2PO4/K2HPO4, 100 mm KCl, pH 7 buffer, 50% TFE). A 10-fold dilution of an ∼500 μm stock solution of the peptide analogs was loaded into a 0.02-cm fused silica cell and its ellipticity scanned from 190 to 250 nm. The values of molar ellipticities of the peptide analogs at a wavelength of 222 nm were used to estimate the relative α-helicity of the peptides. CD Temperature Denaturation Study of Peptide V681—The native peptide V681 was dissolved in 0.05% aqueous trifluoroacetic acid containing 50% TFE, pH 2, loaded into a 0.02-cm fused silica cell, and peptide ellipticity scanned from 190 to 250 nm at temperatures of 5, 15, 25, 35, 45, 55, 65, and 80 °C. The spectra at different temperatures were used to mimic the alteration of peptide conformation during temperature profiling analysis in RP-HPLC. The ratio of the molar ellipticity (222 nm) at a particular temperature (t) relative to that at 5 °C ([θ]t – [θ]u)/([θ]5 – [θ]u) was calculated and plotted against temperature in order to obtain the thermal melting profiles, where [θ]5 and [θ]u represent the molar ellipticity values for the fully folded and fully unfolded species, respectively. [θ] u was determined in the presence of 8 m urea with a value of 1500 degree·cm2·dmol–1 to represent a totally random coil state (34.Monera O.D. Sereda T.J. Zhou N.E. Kay C.M. Hodges R.S. J. Pept. Sci. 1995; 1: 319-329Crossref PubMed Scopus (258) Google Scholar). The melting temperature (Tm) was calculated as the temperature at which the α-helix was 50% denatured (([θ]t – [θ]u)/([θ]5 – [θ]u) = 0.5), and the values were taken as a measure of α-helix stability. Determination of Peptide Amphipathicity—Amphipathicity of peptide analogs was determined by the calculation of hydrophobic moment (35.Eisenberg D. Weiss R.M. Terwilliger T.C. Nature. 1982; 299: 371-374Crossref PubMed Scopus (819) Google Scholar) using the software package Jemboss version 1.2.1 (36.Carver T. Bleasby A. Bioinformatics. 2003; 19: 1837-1843Crossref PubMed Scopus (100) Google Scholar), modified to include a hydrophobicity scale determined in our laboratory. The hydrophobicity scale used in this study is listed as follows: Trp, 33.1; Phe, 30.1; Leu, 24.7; Ile, 22.8; Met, 17.3; Tyr, 16.0; Val, 15.1; Pro, 10.4; Cys, 9.2; His, 4.7; Ala, 4.1; Arg, 4.1; Thr, 4.1; Gln, 1.7; Ser, 1.3; Asn, 1.0; Gly, 0.0; Glu, –0.3; Asp, –0.8; and Lys, –2.0. 2J. Kovacs, C. T. Mant, and R. S. Hodges, unpublished data. These hydrophobicity coefficients were determined from reversed-phase chromatography at pH 7 (10 mm Na2HPO4 buffer containing 50 mm NaCl) of a model random coil peptide with single substitution of all 20 naturally occurring amino acids. We propose that this HPLC-derived scale reflects the relative differences in hydrophilicity/hydrophobicity of the 20 amino acid side chains more accurately than previously determined scales. Measurement of Antibacterial Activity—Minimal inhibitory concentrations (MICs) were determined using a standard microtiter dilution method in a modified Luria-Bertani medium with no added salt (LB, composed exclusively 10 g of tryptone and 5 g of yeast extract/liter). Briefly, cells were grown overnight at 37 °C in LB and diluted in the same medium. Serial dilutions of the peptides were added to the microtiter plates in a volume of 100 μl followed by 10 μl of bacteria to give a final inoculum of 5 × 105 colony-forming units/ml. Plates were incubated at 37 °C for 24 h, and MICs were determined as the lowest peptide concentration that inhibited growth. Measurement of Hemolytic Activity (MHC)—Peptide samples were added to 1% human erythrocytes in phosphate-buffered saline (0.08 m NaCl, 0.043 m Na2HPO4, 0.011 m KH2PO4), and reactions were incubated at 37 °C for 12 h in microtiter plates. Peptide samples were diluted 2-fold in order to determine the concentration that produced no hemolysis. This determination was made by withdrawing aliquots from the hemolysis assays, removing unlysed erythrocytes by centrifugation (800 × g), and determining which concentration of peptide failed to cause the release of hemoglobin. Hemoglobin release was determined spectrophotometrically at 562 nm. The hemolytic titer was the highest 2-fold dilution of the peptide that still caused release of hemoglobin from erythrocytes. The control for no release of hemoglobin was a sample of 1% erythrocytes without any peptide added. Because erythrocytes were in an isotonic buffer, no detectable release (<1% of that released upon complete hemolysis) of hemoglobin was observed from this control during the course of the assay. Calculation of Therapeutic Index (MHC/MIC Ratio)—It should be noted that both the MHC and MIC values are carried out by serial 2-fold dilutions; thus, for individual bacteria and individual peptides, the therapeutic index (MHC/MIC) could vary as much as 4-fold if the peptide is very active in both hemolytic and antimicrobial activities. However, if there is no detectable hemolytic activity, then the maximum possible error in the therapeutic index would be only 2-fold from variations in the antimicrobial activity. When there was no detectable hemolytic activity at 250 μg/ml, a minimal hemolytic concentration of 500 μg/ml was used to calculate the therapeutic index. Peptide Design—Peptide V681, a 26-residue amphipathic antimicrobial peptide with a polar and nonpolar face (30.Zhang L. Benz R. Hancock R.E. Biochemistry. 1999; 38: 8102-8111Crossref PubMed Scopus (131) Google Scholar), was selected as the parent peptide in this study (Fig. 1). Its polar face consisted of 14 residues: six lysine residues, one histidine, four serines, and three threonines. In contrast, the nonpolar face consisted of 12 residues: three alanines, two valines, three leucines, two phenylalanines, one isoleucine, and one tryptophan residue. In this study, we chose d-/l-amino acid substitution sites at the center of the hydrophobic face (position 13) and at the center of the hydrophilic face (position 11) of the helix, such that these substitution sites were also located in the center of the overall peptide sequence. This was based on our previous model peptide studies (29.Chen Y. Mant C.T. Hodges R.S. J. Pept. Res. 2002; 59: 18-33Crossref PubMed Scopus (80) Google Scholar, 34.Monera O.D. Sereda T.J. Zhou N.E. Kay C.M. Hodges R.S. J. Pept. Sci. 1995; 1: 319-329Crossref PubMed Scopus (258) Google Scholar, 37.Zhou N.E. Monera O.D. Kay C.M. Hodges R.S. Protein Pept. Lett. 1994; 1: 114-119Google Scholar) that demonstrated that these central location substitutions had the greatest effect on peptide secondary structure. To study the effects of varying hydrophobicity/hydrophilicity on peptide biological activities, in the design of V681 analogs, five l-amino acids (Leu, Val, Ala, Ser, and Lys) and Gly were selected out of the 20 natural amino acids as the substituting residues, representing a wide range of hydrophobicity. The hydrophobicity of these six amino acid residues decreased in the order Leu > Val > Ala > Gly > Ser > Lys (29.Chen Y. Mant C.T. Hodges R.S. J. Pept. Res. 2002; 59: 18-33Crossref PubMed Scopus (80) Google Scholar). Based on the relative hydrophobicity of amino acid side chains (29.Chen Y. Mant C.T. Hodges R.S. J. Pept. Res. 2002; 59: 18-33Crossref PubMed Scopus (80) Google Scholar), leucine was used to replace the native valine on the nonpolar face to increase peptide hydrophobicity and amphipathicity. Alanine was selected to reduce peptide hydrophobicity/amphipathicity while maintaining high helicity. A hydrophilic amino acid, serine, was selected to decrease the hydrophobicity/amphipathicity of V681 in the nonpolar face. Positively charged lysine was used to decrease further peptide hydrophobicity and amphipathicity. In contrast, the same amino acid substitutions on the polar face would have different effects on the alteration of hydrophobicity/hydrophilicity and amphipathicity, because the native amino acid residue is serine on the polar face of V681. As a result, on the polar face leucine, valine, and alanine were used to increase peptide hydrophobicity as well as to decrease the amphipathicity of V681, whereas lysine was selected to increase peptide hydrophilicity and amphipathicity. In previous studies, Kondejewski and co-workers (23.Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 38.McInnes C. Kondejewski L.H. Hodges R.S. Sykes B.D. J. Biol. Chem. 2000; 275: 14287-14294Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and Lee et al. (28.Lee D.L. Powers J.P. Pflegerl K. Vasil M.L. Hancock R.E. Hodges R.S. J. Pept. Res. 2004; 63: 69-84Crossref PubMed Scopus (77) Google Scholar) successfully utilized d-amino acid substitutions to dissociate the antimicrobial activity and hemolytic activity of the cyclic β-sheet gramicidin S analogs. In the present study, d-enantiomers of the five l-amino acid residues were also incorporated at the same positions on the nonpolar/polar face of V681 to change not only peptide hydrophobicity/hydrophilicity and amphipathicity but, more importantly, to disrupt peptide helical structure. Because glycine does not exhibit optical activity and has no side chain, the Gly-substituted analog was used as a reference for diastereomeric peptide pairs. Because all peptide analogs were made based on a single amino acid substitution in either the polar or nonpolar faces of V681, peptides were divided into two categories, V13X peptides (nonpolar face substitutions) and S11X peptides (polar face substitutions). Each peptide was named after the substituting amino acid residue, e.g. the peptide analog with l-leucine substitution on the nonpolar face of V681 is called V13LL. It is important to note that because the l-valine of the nonpolar face and l-serine of the polar face are the original amino acid residues in the V681 sequence (Fig. 1), peptide analogs V13VL and S11SL are the same peptide as V681. A control peptide (peptide C) designed to exhibit negligible secondary structure, i.e. a random coil, was employed as a standard peptide for temperature profiling during RP-HPLC to monitor peptide dimerization. As shown in the prev