Linear Analogues of Human β-Defensin 3: Concepts for Design of Antimicrobial Peptides with Reduced Cytotoxicity to Mammalian Cells

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<ul><li><p>DOI: 10.1002/cbic.200700560</p><p>Linear Analogues of Human b-Defensin 3: Concepts forDesign of Antimicrobial Peptides with ReducedCytotoxicity to Mammalian CellsShouping Liu,[a] Lei Zhou,[a, b] Jing Li,[a, b] Anita Suresh,[c] Chandra Verma,[c] Yong Hwee Foo,[a]</p><p>Eric P. H. Yap,[d] Donald T. H. Tan,[a, b, e] and Roger W. Beuerman*[a, b]</p><p>Introduction</p><p>The defensins are small cationic and cysteine-rich moleculeswith molecular weights between 36 kDa, and are about 45amino acids in length. Based on the spatial distribution of thethree cysteine intramolecular bonds, mammalian defensins canbe divided into two major groups termed a and b-defensins.q-Defensins make up a third group, but to-date are found onlyin nonhuman primates, and have only 18 amino acids.[13] De-fensins are an important component of the innate immunesystem and provide an initial antimicrobial barrier for mucosalsurfaces such as the surface of the eye, oral tissues, the air-ways and lungs, and also the skin.[48] As antimicrobial agentsthey show broad-spectrum activity against Gram-positive andGram-negative bacteria, fungi and enveloped viruses, and alsoagainst bacteria that have demonstrated resistance to thecurrently used antibiotics.[9] Because of this ability, they havecome to be known as natural antibiotics. Interest in the de-fensins has also been aided by an increase in bacterial strainswith resistance to the standard antibiotics, an increase in thepopulation with reduced immunity, and the water solubilityof defensins, which makes them potentially rapidly deploy-able in response to a pathogen release into the environ-ment.[10] Current efforts are focused toward designing ana-logues of defensins that have properties that are appropriatefor therapeutic application. However, as improvements to thestructureactivity relationship are attempted, issues such ascharge density and hydrophobicity emerge because analoguescan interact with both the membrane of a pathogen as wellas the membrane of human cells. Toxicity to human cells, ofboth native forms and of analogues, has been a concern.[1112]</p><p>Peptide analogues have been developed with diverse cap-abilities, and it appears that the disulfide bonds can be rear-</p><p>ranged or removed with preservation of antimicrobial activi-ty.[1315]</p><p>The b-defensins have been discovered more recently thana-defensins, and have been given special attention becausethey are found in skin and epithelial cells that line mucosal sur-faces.[7, 16] In b-defensins, the disulfide bridges that likely stabi-lize these molecules are Cys1Cys5, Cys2Cys4 and Cys3Cys6while the a-defensins, which are largely produced by thePaneth cells of the intestine and neutrophils, have bridges at</p><p>[a] Dr. S. Liu,+ Dr. L. Zhou,+ Dr. J. Li,+ Y. H. Foo, Prof. D. T. H. Tan,Prof. Dr. R. W. BeuermanSingapore Eye Research Institute11 Third Hospital Avenue, #06-00, Singapore 16875 (Singapore)Fax: (+65)6322-4599E-mail :</p><p>[b] Dr. L. Zhou,+ Dr. J. Li,+ Prof. D. T. H. Tan, Prof. Dr. R. W. BeuermanDepartment of Ophthalmology, Yong Loo Lin School of MedicineNational University of SingaporeSingapore 16875 (Singapore)</p><p>[c] A. Suresh, Dr. C. VermaBioinformatics InstituteSingapore 16875 (Singapore)</p><p>[d] Dr. E. P. H. YapDefence Medical &amp; Environmental Research InstituteSingapore 16875 (Singapore)</p><p>[e] Prof. D. T. H. TanSingapore National Eye CenterSingapore 16875 (Singapore)</p><p>[+] These authors contributed equally to this work.</p><p>Supporting information for this article is available on the WWW under or from the author: RP-HPLC-UV chromato-grams and MS spectra of the six linear analogues of hBD3. (NMRC/CPG/007/2004)</p><p>A series of engineered linear analogues [coded as F6, W6, Y6, A6,S6 and CACHTUNGTRENNUNG(Acm)6] were modeled, designed, synthesized and struc-turally characterized by mass spectra, circular dichroism, hydro-phobicity analysis and molecular modeling. We have screenedantimicrobial activity, hemolysis to rabbit erythrocytes, and cyto-toxicity to human conjunctival epithelial cells. No significantACHTUNGTRENNUNGhemolytic effect was observed for hBD3 or from five of the sixACHTUNGTRENNUNGanalogues [F6, Y6, A6, S6 and C ACHTUNGTRENNUNG(Acm)6] over the range of 3100 mgmL1. The six linear analogues have reduced cytotoxicityto human conjunctival epithelial cells over the range of 6</p><p>100 mgmL1 compared to hBD3. By tuning the overall hydropho-bicity of linear hBD3 analogues, reduced cytotoxicity and hemoly-sis were obtained while preserving the antimicrobial properties.The decreased cytotoxicity of the linear analogues is suggested tobe structurally related to the removal of disulfide bridges, andthe flexible structure of the linear forms, which seem to be associ-ated with loss of secondary structure. These results suggest anew approach for guiding the design of new linear analogues ofdefensin peptides with strong antibiotic properties and reducedcytotoxicity to mammalian cells.</p><p>964 @ 2008 Wiley-VCH Verlag GmbH&amp;Co. KGaA, Weinheim ChemBioChem 2008, 9, 964 973</p></li><li><p>Cys1Cys6, Cys2Cys4 and Cys3Cys5. However, in the eye,both of these are active in response to injury, as has beenfound in tears.[6, 17] Within the family of b-defensins, hBD1,hBD2 and hBD3 have been the most extensively studied; addi-tionally hBD46 have been recently reported. Further, genomebioinformatics studies have pointed to the existence of an ad-ditional 28 b-defensins that have yet to be found by proteomicstudies.[1820] Studies of the tissue distribution of hBD3 by usingRNA levels found only low levels in most tissues, except in oralmucosa and more recently in epithelial cells from the ocularsurface.[7,21] Of the b-defensins, hBD3 has been of particularACHTUNGTRENNUNGinterest as it appears to possess better broad-spectrum anti-ACHTUNGTRENNUNGmicrobial activity than either hBD1 or hBD2.[22]</p><p>The goal of this study was to design analogues of hBD3 thatexhibit good antimicrobial properties and display reduced tox-icity to human cells. We designed and synthesized linear ana-logues of hBD3 by retaining the same net positive charge(+11), and by replacing the six bridging cysteine residues withresidues of varying hydrophobicities. Factors including linearity,hydrophobicity, antimicrobial activity, hemolysis and epithelialcell cytotoxicity were determined. Within this group of linearanalogues, the overall hydrophobicity was generally but notspecifically related to decreased cytotoxicity to human ocularsurface cells ; however, antimicrobial capability remained at thesame level as native hBD3.</p><p>Results and Discussion</p><p>Synthesis of analogues of hBD3</p><p>The solid-phase synthesis of six linear analogues of hBD3 with45 residues was performed by using Fmoc chemistry. The pep-tide sequences are illustrated in Scheme 1. In the analogues,all cysteine residues were uniformly replaced by one of the fol-lowing amino acids: alanine (A), serine (S), cysteine that wasprotected by Acm [CACHTUNGTRENNUNG(Acm)], tryptophan (W), tyrosine (Y), orphenylalanine (F). The six analogues were coded as follows:A6, S6, C ACHTUNGTRENNUNG(Acm)6, W6, Y6 and F6, respectively. Physicochemicalproperties of the peptides are shown in Table 1 (sequencelength, numbers of hydrophobic and aromatic residues, netcharge, relative hydrophobicity) and Table 2 (retention time inHPLC). The UV spectroscopy and mass spectrometry (MS) ofthese peptides are shown in Figure S1 in the Supporting Infor-mation.</p><p>Compared with native hBD3, the six linear analogues ofhBD3 are of the same length and net positive charge (+11) be-cause they are designed and synthesized by uniform replace-ment of the six bridging cysteine residues in hBD3 with sixACHTUNGTRENNUNGresidues of varying hydrophobicities (viz. , F, W, Y, A, S and C-ACHTUNGTRENNUNG(Acm)). Therefore, these linear analogues provide a well-de-fined model to study the effect of overall hydrophobicity, sec-ondary structure conformation, removal of the native disulfidebridges on antimicrobial, hemolytic and cytotoxic activities.</p><p>Molecular hydrophobicity</p><p>We have measured the relative molecular hydrophobicity byRP-HPLCMS in terms of retention time at 500 mgmL1 and100 mgmL1 (Table 2). RP-HPLC is an approach that is common-</p><p>ly employed for comparisons of peptides or aminoacid side chains on antibacterial peptides.[3135] Be-cause the stationary phase of C18-modified silica ishydrophobic and the mobile phase (water/acetoni-trile) is hydrophilic, a longer retention time is a mea-sure of greater hydrophobicity. The measured orderfor the relative molecular hydrophobicity of the pep-tides was as follows: W6&gt;F6&gt;Y6&gt;native hBD3&gt;A6&gt;S6&gt;CACHTUNGTRENNUNG(Acm)6. The relative hydrophobicities ofthe peptides were also calculated based on theHoppWoods hydrophilicity scale[23] (Table 1). Thescale is a hydrophilic index in which apolar residueshave been assigned negative values, and is typicallyScheme 1. Sequence of wild-type hBD3 and its linear analogues.</p><p>Table 1. Physicochemical properties of the peptides.</p><p>Variant Number of residues Net positive Relativetotal hydrophobic charge hydrophobicity[a]</p><p>ACHTUNGTRENNUNG(aromatic)</p><p>CACHTUNGTRENNUNG(Acm)6 45 14 (2) 11 n.c.S6 45 14 (2) 11 28.3A6 45 14 (2) 11 23.5wt-hBD3 45 14 (2) 11 20.5Y6 45 14 (8) 11 12.7F6 45 14 (8) 11 11.5W6 45 14 (8) 11 6.1</p><p>[a] The overall hydrophobicity was calculated based on the hydrophobici-ty scale of HoppWoods hydrophilicity scale.[23] The lower value corre-sponds to lower hydrophilicity or higher hydrophobicity; n.c. , not calcu-lated.</p><p>Table 2. The overall hydrophobicity of the peptides in terms of retentiontime (tR) in RP-HPLCMS.</p><p>Variant tR [min]500 mgmL1 100 mgmL1</p><p>CACHTUNGTRENNUNG(Acm)6 20.150.19 20.940.21S6 20.330.25 20.440.15A6 22.360.07 22.360.28wt-hBD3 23.240.03 24.280.13Y6 23.850.16 24.400.05F6 27.650.13 28.250.12W6 29.290.03 29.660.06</p><p>ChemBioChem 2008, 9, 964 973 @ 2008 Wiley-VCH Verlag GmbH&amp;Co. KGaA, Weinheim 965</p><p>Linear Analogues of Human b-Defensin 3</p></li><li><p>used to identify antigenic regions based on hydrophilic patch-es. For each peptide, the values that correspond to each resi-due were summed to give an overall measure. The generaltrend in computed hydrophobicity of the peptides by usingthis scale matched that of the experimental HPLC retentiontime data. CACHTUNGTRENNUNG(Acm)6, was excluded because Acm has not beenparameterized in the HoppWoods scale.[23]</p><p>CD spectroscopy</p><p>Previous X-ray and NMR spectroscopic studies of human b-de-fensins (hBD13)[27,3637] have shown that the tertiary structuresare similar, and have a short a-helical segment before a triple-stranded antiparallel b-sheet, all rigidly held together by threedisulfide bonds. It follows that in the absence of the con-straints that are imposed by these disulfide bonds, the linearanalogues of hBD3 are likely to be flexible and random inaqueous solution, and they could adopt conformations thatare dictated by the environment.[13,15] Therefore, it was of inter-est to examine the solution conformations of the linear ana-logues in different media. CD spectroscopy of the hBD3 deriva-tives was carried out in aqueous solution in the presence of50% organic modifier trifluoroethanol (TFE), and in the pres-ence of 20 mm SDS micelles, which is a membrane-mimetic(Figures 1, 2, and 3). TFE has been widely used in protein struc-</p><p>ture studies because it can induce stable, structured conforma-tions from otherwise unstructured/random peptides in aque-ous solution.[3841] The membrane-mimicking SDS was chosenbecause it is most similar to the prokaryotic membrane, and istherefore a good model for the natural target of an antibiot-ic.[42] While all of the tested peptides appeared unstructured inaqueous solution, they displayed an observable level of struc-turing in the presence of 50% TFE or 20 mm SDS micelles, aswas determined by CD spectroscopy.</p><p>The CD spectrum of wild-type hBD3 agrees very well withthat of native hBD3, which was reported elsewhere.[13,42] Thespectra of the six linear analogues and wild-type hBD3 in aque-ous solution were similar, and suggested a mix of random and</p><p>b-hairpin conformations, although to different extents becausethe intensities at the negative extrema varied (Figure 1). Theabsence of prominent crossover at wavelengths of 195 nmwas indicative of an absence of significant amounts of structur-ally distinct conformers, while the minima at ~200 nm wassuggestive of transient b-hairpin or turn-like conformer. Thissuggests that the existence of disulfide bonds and the type ofCys-replacing residues impose no significant effect on theACHTUNGTRENNUNGsecondary structure of hBD3 derivatives. The minima at ~195200 nm for native defensin has been observed previously inthe case of native HNP-1[43] and BNBD-12,[44] and is characteris-tic of peptides that contain b-sheet or b-turn secondary struc-ture with some amount of random coil.[42] Thus, the secondarystructure seems to be preformed in the peptides independentof the presence of multiple disulfide bridges or the Cys-replac-ing residues. This observation is in accordance with the resultthat was reported by Kluver et al. ,[13] and is further supportedby molecular dynamics simulations (Figure 4) that show thatthe linear variants adopt a variety of conformations in aqueousconditions.</p><p>Figure 1. CD spectra of hBD3 and its linear analogues in water.</p><p>Figure 2. CD spectra of hBD3 and its linear analogues in 50% TFE solution.</p><p>Figure 3. CD spectra of hBD3 and its linear analogues in 20 mm SDS mi-celles.</p><p>966 @ 2008 Wiley-VCH Verlag GmbH&amp;Co. KGaA, Weinheim ChemBioChem 2008, 9, 964 973</p><p>R. Beuerman et al.</p></li><li><p>In the presence of 50% TFE in water, all linear analogues aswell as native hBD3 underwent a marked conformational tran-sition (Figure 2). Their CD spectra became red-shifted withclearly observable double minima at wavelengths of 209 and222 nm, and strong positive peaks at 190193 nm. Such aspectrum is characteristic of a-helical structures.[41] Because theN terminus of native hBD3 can assume a helical conformation,it might be reasonable to conclude that the presence of TFEincreased the proportion of helical conformation in these pep-tides.[42] The a-helical content of the six analogues and wild-type hBD3 perceptibly increased in the presence of TFE, whichis known to stabilize this type of conformation;[43] this is alsoseen in our molecular dynamics simulations (Figure 5). Thenegative ellipticity near 205 nm and crossover at 200 nm forY6, F6, CACHTUNGTRENNUNG(Acm)6 and A6 spectra in TFE suggest populationswith b-hairpin conformations. A pronounced positive peak at~195 nm for F6 indicates the presence of a b-sheet conforma-tion. The double minima at ~205 nm and ~223 nm, with acrossover at ~200 nm indicates the presence of populationswith both b-hairpin and helical conformations.</p><p>Examination of the populations of hydrogen bonds from ourmolecular dynamics (MD) simulations show that in the wild-type, the induction of structure in the presence of TFE (in con-trast to water) is brought about by an increase in the num...</p></li></ul>


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