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To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K4K20-S4 was 2–3-fold more potent against protozoa and RBC, yet K4K20-S4 was more potent by 2 orders of magnitude against bacteria. K4-S4 had similar behavior as K4K20-S4, but K20-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K20-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K4-S4 and K4K20-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity. To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K4K20-S4 was 2–3-fold more potent against protozoa and RBC, yet K4K20-S4 was more potent by 2 orders of magnitude against bacteria. K4-S4 had similar behavior as K4K20-S4, but K20-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K20-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K4-S4 and K4K20-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity. red blood cell(s) N α-9-fluorenylmethyloxycarbonyl high pressure liquid chromatography phosphate-buffered saline hydropathic index Widely distributed in nature, antimicrobial peptides are an essential defense component of invertebrates and vertebrates, destined to control cell proliferation and invading pathogens (1.Nicolas P. Mor A. Annu. Rev. Microbiol. 1995; 4: 277-304Crossref Scopus (536) Google Scholar, 2.Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1526) Google Scholar, 3.Hancock R.E.W. Lancet. 1997; 349: 418-422Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar). Antimicrobial peptides display a large heterogeneity in primary and secondary structures but share common features such as amphipathy and net positive charge. These features seem to form the basis for their cytotoxic function. Although their precise mechanism of action is not fully understood, antimicrobial peptides are believed to kill the target cell by destabilizing the ordered structure of the cell membrane via either a “barrel stave” mechanism or a nonpore carpet-like mechanism (4.Epand M.E. Shai Y. Segrest J.P. Anantharamaiah G.M. Biopolymers. 1995; 37: 319-338Crossref PubMed Scopus (205) Google Scholar, 5.Shai Y. Trends Biochem. Sci. 1995; 20: 460-464Abstract Full Text PDF PubMed Scopus (284) Google Scholar). Antimicrobial peptides are potentially active against a large spectrum of microorganisms, yet they are generally nontoxic for normal mammalian cells. The molecular basis for this selectivity is also ill defined but believed to result from differences inherent to the lipid composition of target versus nontarget cells, such as membrane fluidity and negative charge density. Indeed, a large body of experimental data demonstrates that factors affecting membranolytic properties include peptide amphipathy and positive charge distribution (6.Frohlich D.R Wells A.W. Int. J. Peptide Protein Res. 1991; 37: 2-6Crossref PubMed Scopus (7) Google Scholar, 7.Pouny Y. Rapaport D. Mor A. Nicolas P. Shai Y. Biochemistry. 1992; 31: 12416-12423Crossref PubMed Scopus (577) Google Scholar, 8.Blondelle S.E. Houghten A.H. Biochemistry. 1992; 31: 12688-12694Crossref PubMed Scopus (348) Google Scholar, 9.Bessalle R. Gorea A. Shalit I. Metzger J.W. Dass C. Desidero D.M. Fridkin M. J. Med. Chem. 1993; 36: 1203-1209Crossref PubMed Scopus (78) Google Scholar, 10.Strahilevitz J. Mor A. Nicolas P. Shai Y. Biochemistry. 1994; 33: 10951-10960Crossref PubMed Scopus (122) Google Scholar, 11.Matsuzaki K. Sugishita K. Fujii N. Miyajima K. Biochemistry. 1995; 34: 3423-3429Crossref PubMed Scopus (402) Google Scholar, 12.Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shay Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Moreover, isomers composed of all d-amino acids display identical potency as the all l-amino acid counterparts. This finding represents one of the more fascinating aspects that evolved from the investigation of their mechanism of action, since it implies that if their antimicrobial activity is not mediated by interaction with specific receptors, peptide-based antibiotics could escape some of the mechanisms involved in multidrug resistance. Dermaseptins are a large family of antimicrobial peptides (28–34 amino acids) expressed in the skin of tree frogs belonging to the phyllomedusinae genus (13.Mor A. Nguyen V.H. Delfour A. Migliore S.D. Nicolas P. Biochemistry. 1991; 3: 8824-8830Crossref Scopus (286) Google Scholar, 14.Mor A. Nicolas P. Eur. J. Biochem. 1994; 219: 145-154Crossref PubMed Scopus (158) Google Scholar, 15.Mor A. Amiche M. Nicolas P. Biochemistry. 1994; 33: 6642-6650Crossref PubMed Scopus (106) Google Scholar). They are linear polycationic peptides, structured in amphipathic α-helix in apolar solvents (16.Mor A. Nicolas P. J. Biol. Chem. 1994; 269: 1934-1939Abstract Full Text PDF PubMed Google Scholar) with cytolytic activity in vitro against a broad spectrum of pathogenic microorganisms (bacteria, protozoa, yeast, and filamentous fungi). Using phospholipid liposomes or live cells, the selective antimicrobial action of these peptides was shown to be mediated by selective interaction of the amphipathic α-helix moiety with the plasma membrane phospholipids (7.Pouny Y. Rapaport D. Mor A. Nicolas P. Shai Y. Biochemistry. 1992; 31: 12416-12423Crossref PubMed Scopus (577) Google Scholar, 10.Strahilevitz J. Mor A. Nicolas P. Shai Y. Biochemistry. 1994; 33: 10951-10960Crossref PubMed Scopus (122) Google Scholar, 12.Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shay Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 17.Hernandez C. Mor A. Dagger F. Nicolas P. Hernandez A. Benedetti E.L. Dunia I. Eur. J. Cell Biol. 1992; 59: 414-424PubMed Google Scholar, 18.Mor A. Hani K. Nicolas P. J. Biol. Chem. 1994; 269: 31635-31641Abstract Full Text PDF PubMed Google Scholar, 21.De lucca A.J. Bland J.M. Jacks T.J. Grimm C. Walsh T.J. Med. Mycol. 1998; 36: 291-298Crossref PubMed Google Scholar), leading to cell permeabilization. Dermaseptins were shown also to be potent killers of nongrowing and slowly growing bacteria (19.Jouenne T. Mor A. Bonato H. Junter G.A. J. Antimicrob. Chemother. 1998; 42: 87-90Crossref PubMed Scopus (24) Google Scholar), suggesting a potential interest in the eradication of bacteria placed in a dormant state and/or subject to low oxygen tension, which are major factors affecting susceptibility of bacteria to antimicrobial agents, thus leading to drug resistance. More recently, the potential interest of dermaseptins, alone or in combination with mild heating, in killing spoilage yeast was demonstrated (20.Coot P.J. Holyoak C.D. Bracey D. Ferdinando D.P. Pearce J.A. Antimicrob. Agents Chemother. 1998; 42: 2160-2170Crossref PubMed Google Scholar). Among natural dermaseptins, dermaseptin S4 is highly toxic to erythrocytes, in contrast to other dermaseptin members, such as dermaseptin S3 (12.Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shay Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Moreover, in Plasmodium falciparum-infected red blood cells (RBC),1 dermaseptin S3 selectively killed the intraerythrocytic parasite unlike dermaseptin S4 that was toxic to the parasite and its of the molecular basis for this selective that dermaseptin S3 and S4 were highly as they with similar affinity to and were potent in and indicated that dermaseptin S4 was in a aggregation state in aqueous compared with dermaseptin This to the that the peptide's aggregation state in solution might be an affecting selective (12.Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shay Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In the we experiments to the consequences of dermaseptin S4 aggregation in one of analogs in in or In of the were with These analogs were in of organization in solution and in of cytolytic The results the notion that peptide aggregation in solution cytotoxic properties. and were from other for peptide and cell were were The peptides were by the the active as A. Nicolas P. Eur. J. Biochem. 1994; 219: 145-154Crossref PubMed Scopus (158) Google Scholar). of the from the N-terminal amino the peptide was from the with a of of peptide in a The acid was and the peptide was with by with The peptides were from the with in and to in the of to by with an and were on a a linear of in solvents The peptides were to amino acid and in to their were as to were in and and in the Peptide the N-terminal amino acid with a was by of peptide with of in an in to the of the N-terminal amino acid of the The was for and and the was The peptide was in a solution of in a in the the peptide was with and and and the peptide was from the with and as blood was in by for and RBC in of were to of peptide in alone or were and the hemolytic activity was assessed as a of by the of of data were obtained from experiments in activity was assessed against and in yeast of were The cell were by to a of were to of peptide or various peptide in of proliferation was by the and for Y. and P. activity was assessed against the form of a major by the of of the proliferation was by 2 L. major to of with and in well or various peptide the the of cells was by an from on a cell a of was by the for bacteria and the and in To peptide the of for the were in as (12.Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shay Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) with the were to various peptide in the of the were and of the was by as Peptide was on and The of peptide in the was of for of the peptides was assessed by the of the compared with a The data were with the The properties of and analogs in aqueous solution were peptides as (12.Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shay Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). of or peptide in were to of and the was on a of the of the was obtained by the the of of to a of the peptides were in from in Fluorescence was a of with was on cells, an system The is with a an was were between The peptide hydropathic index was to J. J. Biol. PubMed Scopus Google Scholar), of a hydrophobic hydrophobicity for all amino The represents the of of amino acids from an aqueous to a hydrophobic was from experimental of amino acids in the molecular mechanisms involved in the selective of peptide-based we a in which of dermaseptin S4 analogs and were and tested peptides and their are in The data are for of and properties of dermaseptin S4 and acid the of of RBC selectivity the of of a to that of dermaseptin the J. J. Biol. PubMed Scopus Google Peptide of Peptide of RBC selectivity the of of a to that of dermaseptin S4. in a This involved investigation of dermaseptin S4 and analogs to the of charge on was assessed against 2 of cells on the basis of to their plasma cells that and L. and Gram-negative bacteria Y. and P. plasma membrane is by a of we the peptides organization in the peptides selectively their N-terminal amino acid with of for in the of dermaseptin S4 resulted in to cell a peptide of and RBC or positive charge not affect the peptide's hemolytic on the other had a negative resulted in reduced hemolytic potency was less and positive resulted in increased hemolytic potency was more profiles for of and hemolytic activity of dermaseptin S4 and the analogs a in that to by shown in the antiprotozoan activity of dermaseptin S4 and its analogs that of RBC dermaseptin S4 was potent in major 2 was less and K4K20-S4 was more potent the native positive or negative not dramatically affect the activity. to the hemolytic dermaseptin S4 was to be a weak of of charge or the peptide's antibacterial activity. some positive charge peptides displayed potent antibacterial activity. K4-S4 and K4K20-S4 were and more the native K20-S4 on the other was as active as dermaseptin S4 peptides were not to killing to The results of the experiments are shown in The represent the of of the peptides in the peptides displayed differences in their compared with The of of that the peptides were aggregated to various the peptides were by they similar of In a result of the versus the of The linear of the and to low that these peptides not their state in this the between the and the of or peptides in Peptide are as the of Gram-negative and P. Peptide are as for these results, it can be that the peptides to in aqueous to The state of dermaseptin S4 was not reduced by negative charge In contrast, positive charge in but not in reduced the peptide's aggregation Peptide aggregation was reduced in To affected the peptides cytotoxic the of S4, and K4K20-S4 were in to the was in their antiprotozoan or hemolytic potency as (7.Pouny Y. Rapaport D. Mor A. Nicolas P. Shai Y. Biochemistry. 1992; 31: 12416-12423Crossref PubMed Scopus (577) Google Scholar, 10.Strahilevitz J. Mor A. Nicolas P. Shai Y. Biochemistry. 1994; 33: 10951-10960Crossref PubMed Scopus (122) Google Scholar, 12.Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shay Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). the of aggregation displayed by the peptides correlated with antibacterial potency of dermaseptin S4 and its analogs all aggregated analogs and were active against the aggregated peptide was the K4-S4 displayed an Moreover, the was other Gram-negative bacteria such and P. and These results suggested that K4K20-S4 might high To that and its the of K4K20-S4 was some solution of K4K20-S4 was to be of activity against of activity could be obtained by in the was fully and the low peptide were less potent was increased 2–3-fold compared with of behavior potency high peptide by was with other Gram-negative bacteria P. the tested the peptide to of its potency against either bacteria and with the antibacterial results, the antiprotozoan activity and the hemolytic activity of K4K20-S4 were not reduced high peptide and of these results is of RBC and with dermaseptin S4 that of cells were their of RBC and bacteria the of the are shown in and The suggested but displayed a with in generally to the and/or the with this of the peptides with the for aggregation displayed that to of the by the of of as in The between peptide cell affinity and was by of the the peptide of peptide could be in the of RBC, suggesting that of dermaseptin S4 was of the cells with this that of RBC can be were of RBC with more peptide resulted in the of native S4 to RBC, on Binding on the peptides was similar The that peptide to RBC is linear a of the dermaseptin S4 to S4 between and a of the peptide displayed weak antibacterial activity. a of of dermaseptin S4 was to be of the they displayed or were also to between and version of K4K20-S4 composed with all d-amino acids the results as the l-amino acid version with to and antibacterial suggesting that the properties were not by susceptibility to To the molecular in the structure of dermaseptin S4 that are for we a of analogs in which the native was by a either from the or the or from of these analogs was compared by in of the defined as the peptide that of Gram-negative bacteria the was not high activity was compared in of was assessed by antibacterial activity to hemolytic activity in of and of the had the of antibacterial activity. the was as and the was more active the native peptide Moreover, unlike the native these analogs were to of and had the of reducing the of and the was not to of the N-terminal a peptide that was more active dermaseptin S4 of and were not of hydrophobic the all of the positive also resulted in an peptide shown in the of of to had the of reducing the peptide's hemolytic potency by to of hemolytic and and less active dermaseptin S4. In contrast to antibacterial N-terminal to less hemolytic and and less hemolytic dermaseptin S4. to the of and to these analogs were to RBC to a hemolytic activity. their to with RBC, and between and a of they displayed weak or antibacterial activity. This indicated that not to but the peptide's as The of the of dermaseptin S4 analogs was by the results obtained with the with dermaseptin we for the peptide positive was for potent yet selective antibacterial activity. of these peptides as of the with in of dermaseptin antibacterial since was more potent its and more potent native dermaseptin S4 Moreover, of the negative charge the by of the to a a peptide that displayed a in represents was more potent of the from resulted in the a 15-mer with similar antimicrobial potency a less potent and a less antibacterial activity was for all This all peptides but the native peptide and the analogs and that S4 analogs were not involved in a but they were to kill the bacteria. In the of and composed with all d-amino acids similar results as the acid that the activity of these peptides was not by susceptibility to of the analogs had hemolytic activity compared with dermaseptin S4, their antibacterial activity was or is to that antibacterial activity was with that of dermaseptin S4 displayed hemolytic activity to Fluorescence of is shown in the of peptides to RBC, on The hemolytic less S4, and the the the of the peptides of was to bacteria was with increasing all bacteria are killed The acid of and similar results as the acid with to their to RBC and suggesting that the properties were not by susceptibility to the properties of and were as the the of the The of the obtained suggested that unlike dermaseptin S4, the analogs not their state as that they not the properties of the To peptides from dermaseptin S4 with a reduced hemolytic activity and with an antimicrobial activity and to the such as and that these in and peptides from dermaseptin S4 were in all of the dermaseptin peptides some antibacterial and a reduced hemolytic activity. The peptide of RBC and the peptide of were The selectivity the of a to that of dermaseptin S4, was The selectivity index is thus a of of a high and low antimicrobial of the peptides, from of the peptides, and have an increased selectivity index compared with S4. Dermaseptin S4 and its in aqueous to the state of dermaseptin S4 was not reduced by negative charge substitutions in peptide or positive charge in but not in reduced the peptide's aggregation Peptide aggregation is reduced in other natural dermaseptins, the positive are more or less the peptide dermaseptin S4 positive in the by hydrophobic one of the is involved in hydrophobic with other peptides, of positive might to charge of the peptides in that the peptide was clearly less aggregated native dermaseptin S4. Moreover, the that of the peptides is aggregated but is not that the N-terminal hydrophobic is responsible for experiments on the of dermaseptin S4, the hydrophobic were this In with this the of negative charge a high positive charge is to be or to The factors believed to include peptide and positive charge The positive charge is with their affinity for negative The amphipathy is with their to These are to be by their of the data of antimicrobial hemolytic and aggregation state of the peptides of the that peptide organization in solution represents The aggregation well with their antibacterial aggregated peptides are active against Gram-negative the less aggregated peptides are highly is believed that peptide aggregation in solution the and the selectivity antimicrobial This be of in the and of potent and/or selective peptide-based The that hydrophobic the N-terminal of dermaseptin S4 to a the are responsible for the peptide's aggregation in solution is by the results obtained with the In this dermaseptin S4 a and behavior from the other dermaseptins, since the S4 N-terminal domain by antibacterial activity reducing the hemolytic activity. for this behavior might in a of the N N is hydrophobic had reduced hemolytic such as in N is or all of the as well as all natural N is In contrast, peptides N is highly hydrophobic are include the native dermaseptin S4 and its N is The dermaseptin peptides of the investigation were to be to N-terminal the N-terminal is involved in peptide its aggregation or The N-terminal peptide and other displayed potent antibacterial activity and weak hemolytic activity. In the of peptides indicated that the highly antimicrobial and hemolytic peptides and were less aggregated dermaseptin S4. In contrast, is which this peptide displayed hemolytic activity. was shown that the of various synthetic polycationic peptides their hemolytic peptides display reduced hemolytic by reduced antimicrobial compared with their S.E. Houghten A.H. Biochemistry. 1992; 31: 12688-12694Crossref PubMed Scopus (348) Google Scholar, 9.Bessalle R. Gorea A. Shalit I. Metzger J.W. Dass C. Desidero D.M. Fridkin M. J. Med. Chem. 1993; 36: 1203-1209Crossref PubMed Scopus (78) Google Scholar). In contrast, the dermaseptin S4 display reduced hemolytic activity with antimicrobial activity. The of affecting charge and was to the of selective between bacteria and RBC, data and the not the is the for selectivity. of the hydropathic index and charge with their hemolytic and antimicrobial this These properties of dermaseptin S4 and are in I. of a can be between dermaseptin S4 and the and which have an identical net positive charge. Dermaseptin S4 potent hemolytic activity and weak antibacterial reduced hemolytic activity but antibacterial and less hemolytic activity and antibacterial activity. The selectivity index of the peptides by a of and of a can be between the and These peptides are all the of amino but they increased charge and and reduced and weak hemolytic activity and weak antibacterial activity. its charge from to by with reduced its hydropathic index from to This results in a hemolytic activity and an antibacterial the selectivity index from to increasing the charge of from to by which not the hydropathic results in by Based on these we that hydrophobicity to antimicrobial versus hemolytic charge to The between hydropathic index and positive charge to the activity of dermaseptin S4 be as combination of low hydropathic index with high positive charge is with low hemolytic activity and high antibacterial combination of high hydropathic index and low positive charge is with high hemolytic activity and low antibacterial combination of low hydropathic index and low positive charge is with reduced combination of high hydropathic index and high positive charge is with properties between the that these are highly to peptide organization in K20-S4 a high hydropathic index with a high positive its high aggregation state in solution is responsible for its antibacterial whereas the aggregation state of not its hemolytic and the peptide as In we have demonstrated selectivity reversal in the activity of dermaseptin S4 by the domains that are responsible for hemolytic activity. of these domains in of amphipathy and charge to the of potent and selective antibacterial the the to as a modular synthetic antimicrobial system and that the spectrum of action of other peptide-based antibiotics be to to target cells and by of their The of in is
Feder et al. (Tue,) studied this question.