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Identification of antimicrobial peptides by using combinatorial libraries made up of unnatural amino acids. S E Blondelle, E Takahashi, P A Weber and R A Houghten Antimicrob. Agents Chemother. 1994, 38(10):2280. DOI: 10.1128/AAC.38.10.2280. These include: CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Downloaded from http://aac.asm.org/ on April 18, 2014 by PENN STATE UNIV Updated information and services can be found at: http://aac.asm.org/content/38/10/2280 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1994, p. 2280-2286 Vol. 38, No. 10 0066-4804/94/$04.00+0 Copyright C 1994, American Society for Microbiology Identification of Antimicrobial Peptides by Using Combinatorial Libraries Made Up of Unnatural Amino Acids SYLVIE E. BLONDELLE,l* EMA TAKAHASHI,' PATRICIA A. WEBER,2 AND RICHARD A. HOUGHTEN" 2 Torrey Pines Institute for Molecular Studies' and Houghten Pharnaceutical Inc.,2 San Diego, California 92121 The use of water-soluble synthetic peptide combinatorial libraries permits the systematic examination of tens to hundreds of millions of peptides in existing microdilution assays. In the present study, we prepared and determined the antistaphylococcal activities of two new synthetic peptide combinatorial libraries (one N-acetylated, the other not) composed of tetrapeptides having one position defined and the remaining three positions made up of mixtures of L-, D-, and unnatural amino acids (a total of 58 different amino acids). These libraries, when used in conjunction with an iterative selection process, allowed for the development of a series of individually defined tetrapeptides with high levels of activity against Staphylococcus aureus. The activities of the final individual peptides against two additional strains of gram-positive bacteria (methicillin-resistant S. aureus and Streptococcus sanguis), a gram-negative bacterium (Escherichia coli), as well as the yeast Candida albicans were also determined. Cell viability assays showed that the identified peptides are bacteriostatic against both S. aureus and E. coli. MATERIALS AND METHODS Staphylococcus aureus continues to be a problematic pathobecause of the increasing occurrence of strains which are resistant to commonly used antibiotics. Such resistant strains lead to a constant need for new antistaphylococcal agents. In recent years, peptides, either isolated from natural sources or synthetically designed, have emerged as potent antimicrobial agents (for a review, see reference 2). However, the process involved in the development of such new antimicrobial peptides, i.e., their isolation and characterization and the synthesis of large numbers of peptide analogs required for activity optimization, is time-consuming and limited by the large number of individual peptides which can be generated and screened. Highly active antimicrobial peptides have recently been identified through the use of soluble (i.e., non-supportbound), synthetic peptide combinatorial libraries (SPCLs [11, 12, 14, 15]) made up of L-amino acids. This approach permits the systematic screening of hundreds of millions to billions of peptides for the identification of optimal, biologically active peptides (1, 7, 8, 11, 15, 18). Since these newly developed antimicrobial peptides are composed entirely of L-amino acids and are therefore susceptible to proteolytic breakdown and rarely have activity when administered orally, their therapeutic applicability is limited to topical or intravenous use. Antimicrobial peptides with D- and/or unnatural amino acids in their sequences would be expected to enhance the stabilities of these peptides toward proteolysis and, in turn, increase their duration of activity and applicability. In the present study, we extended our search for new antimicrobial agents by determining the activities of two new SPCLs made up of L-, D- and unnatural amino acids against S. aureus. The spectra of activity of the final individual peptides, as well as their toxicities, are described. gen Synthesis of SPCLs and individual peptides. The peptide mixtures making up each of the two SPCLs, as well as those used in the iterative process, were prepared by the process of divide, couple, and recombine (9, 15, 17) in conjunction with simultaneous multiple-peptide synthesis (10). All individual peptides were prepared by simultaneous multiple-peptide synthesis. Peptide mixtures and individual peptides were cleaved from the resulting resins by using low to high concentrations of hydrogen fluoride (13, 20). Twenty-four peptides were individually cleaved simultaneously with HF by using a multiple-vessel cleavage apparatus (Chiron Mimotopes Peptide Systems, San Diego, Calif.) (13). Peptides were exhaustively extracted with TABLE 1. Unnatural amino acids included in the two tetramer SPCLs Abbreviation used in sequences Bala ...... aABA ...... gABA ...... aAIB ...... eAca ...... 7aHa ...... bAsp ...... gGlu ...... Cys[ACM] ...... elys........ oaFmoc-elys ...... MetO2 ...... Nle ...... Nve ...... Orn ...... dOrn ........ NO2F ........ Corresponding author. Mailing address: Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. Phone: (619) 455-3803. Fax: (619) 455-3804. * Hyp ...... Thiopro ...... 2280 Protected amino acid used during synthesis Boc-,3-alanine Boc-L-oa-amino butyric acid Boc-L-'y-amino butyric acid Boc-L-oa-amino isobutyric acid Boc-L-e-amino caproic acid Boc-7-amino heptanoic acid Boc-L-aspartic acid (cs-Bzl) Boc-L-glutamic acid (a-Bzl) Boc-cysteine (acetamidomethyl) N-e-Boc-N-cx-CBZ-L-lysine N-E-Boc-N-a-Fmoc-L-lysine Boc-L-methionine sulfone Boc-L-norleucine Boc-L-norvaline N-a-Boc-N-8-CBZ-L-ornithine N-8-Boc-N-oa-CBZ-L-ornithine Boc-p-nitro-L-phenylalanine Boc-hydroxyproline (Bzl) Boc-L-thioproline Downloaded from http://aac.asm.org/ on April 18, 2014 by PENN STATE UNIV Received 19 April 1994/Returned for modification 6 June 1994/Accepted 22 July 1994 IDENTIFICATION OF NEW ANTIMICROBIAL PEPTIDES VOL. 38, 1994 2281 I 80 60 % inhibition A 20 0 AC DEFGHIKLMNPQRSTVWY a cd e f h i k I m np q r s t vwy 12345678910111213141516171819 I 80 60 % inhibition 40 20 I 0 I DEFGHIKLMNPQRSTVWY a cd e f h i k I m np q r s t vwy 12345678910111213141516171819 FIG. 1. Antistaphylococcal activities of the N-acetylated (A) and non-N-acetylated (B) SPCLs. The D-amino acids are represented by lowercase letters; the unnatural amino acids are numbered as listed in Table 1. Each individual bar is segregated by the first amino acid in the peptide mixtures (U) and represents the percent inhibition of S. aureus growth at a peptide mixture concentration of 2.5 mg/ml. AC water and lyophilized. The identities of the individual peptides determined by laser desorption time-of-flight mass spectroscopy (MALDI-TOF mass spectrometer; Kratos Analytical, Ramsey, N.J.). The peptide mixtures and individual peptides were soluble in H20 at the concentrations tested. Antimicrobial and antifungal assays. The strains S. aureus ATCC 29213, methicillin-resistant S. aureus (MRSA) ATCC 33591, Streptococcus sanguis ATCC 10566, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 were used in were the bioassays (American Type Culture Collection [ATCC], Rockville, Md.). To initiate the exponential phase of bacterial growth prior to the assay, a sample of bacteria grown overnight at 37°C in Mueller-Hinton broth (MH [Becton Dickinson Microbiology Systems] for E. coli and S. aureus) or brain heart infusion broth (Becton Dickinson Microbiology Systems; for S. sanguis) was reinoculated and incubated at 37°C. MRSA was grown at 35°C in cation-adjusted MH broth (BBL, Cockeysville, Md.) in a similar manner. A final concentration of 1 x Downloaded from http://aac.asm.org/ on April 18, 2014 by PENN STATE UNIV 40 2282 BLONDELLE ET AL. RESULTS Preparation of the two SPCLs. The two SPCLs used in the present study, one N-acetylated, the other not, were composed of tetrapeptides in which the first position was defined (U) and three positions (Z) were an equimolar mixture of 56 different L-, D-, and unnatural amino acids (19 L-, 18 D-, and 19 unnatural amino acids; L- and D-cysteine were omitted from the mixture positions but were included in the defined positions). The two SPCLs can be represented by the general formula Ac-UZZZNH2 and UZZZ-NH2. The 19 unnatural amino acids used in the study are listed in Table 1. These were chosen according to their diversities and commercial availabilities. Each tetrapep- TABLE 2. Antistaphylococcal activities of peptide mixtures derived from iterative selection process Position defined Sequence IC50 (Pxg/ml) Second (aFmoc-elys)WZZ-NH2 (aFmoc-Elys)cZZ-NH2 (otFmoc--lys)CZZ-NH2 (aFmoc-elys)wZZ-NH2 (aFmoc-Elys)yZZ-NH2 (aFmoc-elys)FZZ-NH2 (aFmoc-clys)(NO2F)ZZ-NH2 18 23 24 30 33 38 42 Third (otFmoc-elys)WWZ-NH2 (aFmoc-elys)WFZ-NH2 (aFmoc-elys)WfZ-NH2 (otFmoc-Elys)W(Nle)Z-NH2 (aFmoc-slys)WLZ-NH2 (aFmoc-elys)WIZ-NH2 (aFmoc-rlys)WRZ-NH2 (otFmoc-elys)WrZ-NH2 (aFmoc-rlys)WpZ-NH2 (aFmoc-elys)W(Orn)Z-NH2 (oFmoc-elys)W(dOm)Z-NH2 (aFmoc-elys)WKZ-NH2 (aFmoc-Elys)WwZ-NH2 (aFmoc-Elys)WiZ-NH2 (aFmoc-Elys)W(aAIB)Z-NH2 (otFmoc-Elys)WYZ-NH2 (aFmoc-Elys)WsZ-NH2 (aFmoc-elys)W(Nve)Z-NH2 3 4 4 4 5 5 6 6 6 6 6 7 7 7 7 8 8 8 (tFmoc-Elys)cwZ-NH2 (otFmoc-elys)cfZ-NH2 7 (aFmoc-elys)c(NO2F)Z-NH2 (aFmoc-Elys)c(Nve)Z-NH2 (aFmoc-elys)c(Nle)Z-NH2 Fourth (otFmoc-clys)WfR-NH2 (aFmoc-clys)Wfl-NH2 (aFmoc-elys)Wfw-NH2 (aFmoc-elys)Wfr-NH2 (aFmoc-elys)Wf(KCBZ)-NH2 (aFmoc-elys)Wf(Thiopro)-NH2 (aFmoc-Elys)WfL-NH2 (atFmoc-elys)WfP-NH2 (aFmoc-elys)Wfi-NH2 (aFmoc-elys)Wf(Orn)-NH2 (aFmoc-elys)Wf(dOrn)-NH2 (cLFmoc-elys)Wf(aAIB)-NH2 (aFmoc-elys)WKW-NH2 (otFmoc-elys)WKC-NH2 (aFmoc-elys)WYr-NH2 (aFmoc-elys)WY(aABA)-NH2 (aFmoc-rlys)cir-NH2 (aFmoc-elys)ciR-NH2 (otFmoc-elys)ciK-NH2 (etFmoc-elys)ci(aAIB)-NH2 (aFmoc-Elys)ci(Orn)-NH2 (aFmoc-elys)ci(dOm)-NH2 7 8 9 9 2 2 2 2 3 3 4 4 4 4 4 4 4 4 4 4 3 4 4 4 4 4 tide SPCL was therefore composed of 58 peptide mixtures, each containing 175,616 (563) tetrapeptides, for a total of 10,185,728 (58 x 175,616) tetrapeptides per SPCL. Activities of the two SPCLs against S. aureus. Each of the 58 peptide mixtures making up the two SPCLs was examined for its ability to inhibit S. aureus growth in a microdilution assay. The percent inhibition at a concentration of 2.5 mg of each Downloaded from http://aac.asm.org/ on April 18, 2014 by PENN STATE UNIV 105 to 5 x 105 CFU/ml was used in all assays. Prior to the antifungal assay, two colonies of newly grown C. albicans culture were inoculated in 5 ml of phosphate-buffered saline (PBS; 35 mM phosphate buffer, 0.15 M NaCl [pH 7.0]), vortexed, and diluted 10-fold in yeast medium (YM) broth (Difco Laboratories, Detroit, Mich), for an approximate final concentration of 1 X 105 to 5 x 10 CFU/ml. The assays were carried out in 96-well flat-bottom plates (Costar, Pleasanton, Calif.). Bacterial or yeast suspension in 2x concentrated broth was added to the peptide mixtures at concentrations that varied from 2,500 to 1 ,ug/ml in H20 in serial twofold dilutions. The plates were then incubated for 21 h at 37°C for the bacteria or 48 h at 30°C for C. albicans. The relative percent growth of the bacteria or yeast found for each peptide, as determined by measuring the optical density at 620 nm with a Titertek Multiskan Plus apparatus (Flow Laboratories, McLean, Va.), was consistent in three separate determinations. The concentration necessary to inhibit 50% bacterial or yeast growth (IC50) was then calculated by using sigmoidal curve-fitting software (Graphpad; ISI, San Diego, Calif.). The MICs were defined as the lowest concentrations of peptides at which no change in the optical density at 620 nm occurred between time zero and 21 or 48 h. To evaluate whether the peptides were bacteriostatic or bacteriocidal, killing assays were carried out as follows. Aliquots of cells (1 X 105 to 5 x 105 CFU/ml) of 200 ,ul in suspension in MH or 2x concentrated YM broth were allowed to bind to an Eppendorf tube for 15 min at room temperature. An equal volume of peptide solution in H20 was then added, and the tubes were incubated at 37°C for 1 to 24 h. Experiments with control tubes lacking peptide were carried out in an identical manner. Following incubation, 100 ,lI of each solution was diluted in PBS (10-1 to 10-6 dilution) and plated onto MH or YM agar plates. The MICs in the presence of human serum were determined by adding 50 ,ul of heat-inactivated (obtained following a 60-min treatment at 56°C), filtered human serum (Biowhittaker, Walkersville, Md.) or human serum albumin (Sigma, St. Louis, Mo.) to each well and serially diluting the peptides in the serum or albumin solution. The assays were then carried out as described above. Hemolytic assays. The peptides' hemolytic activities were determined by using human erythrocytes (RBCs). The assays were carried out in 96-well flat-bottom plates against a 0.25% RBC suspension as described elsewhere (3). In brief, peptide mixtures were added to the RBC solution at concentrations that varied from 500 to 4 ,ug/ml in serial twofold dilutions. Following a 1-h incubation at 37°C, the plates were centrifuged at 2,800 rpm for 5 min. The supernatant was separated from the pellet, and its optical density at 414 nm was measured. The hemolytic dose to lyse 50% of RBCs (HD50) was calculated by using the sigmoidal curve-fitting software Graphpad. ANTIMICROB. AGENTS CHEMOTHER. IDENTIFICATION OF NEW ANTIMICROBLAL PEPTIDES VOL. 38, 1994 2283 TABLE 3. Spectra of activity of antistaphylococcal tetrapeptides MIC range (,ug/ml) for Tetrapeptide MRSA S. sanguis E. coli C. albicans (oiFmoc-£lys)WfR-NH2 (aFmoc-£lys)Wfl-NH2 (aFmoc-elys)Wfw-NH2 (axFmoc-£lys)Wfr-NH2 (caFmoc-£lys)Wf(KCBZ)-NH2 (aFmoc-£lys)Wf(Thiopro)-NH2 (aFmoc-elys)WfL-NH2 (aFmoc- lys)WfP-NH2 (aFmoc-elys)Wfi-NH2 (aFmoc- 1ys)Wf(Orn)-NH2 (aFmoc-elys)Wf(dOrn)-NH2 4-8 3-4 3-4 (aFmoc--lys)Wf(aAIB)-NH2 48 8-16 4-8 5-8 8-16 8-16 8-16 8-16 8-16 >250 4-8 <2 4-8 8-16 3-5 31-62 125-250 4-8 4-8 8-16 8-16 4-8 3-4 3-4 3-4 5-8 5-8 5-8 5-8 8-16 5-8 5-8 8-16 31-62 62-125 62-125 16-31 31-62 31-62 31-62 31-62 >125 31-62 31-62 31-62 125-250 250-500 250-500 250-500 250-500 250-500 300-500 250-500 >500 250-500 125-250 125-250 (aFmoc-elys)WKW-NH2 (aFmoc-elys)WKC-NH2 5-8 5-8 >250 62-125 4-8 8-16 16-31 31-62 250-500 >500 (aFmoc-elys)WYr-NH2 5-8 5-8 62-125 8-16 5-8 8-16 31-62 62-125 250-500 250-500 4-8 8-16 8-16 8-16 16-32 >250 >250 >250 125-250 8-16 8-16 3-4 5-8 5-8 6-8 4-8 4-8 31-62 31-62 62-125 62-125 31-62 31-62 125-250 250-500 250-500 250-500 125-250 250-500 (aFmoc-elys)WY(aABA)-NH2 (aFmoc- Iys)cir-NH2 (aFmoc-elys)ciR-NH2 (aFmoc-elys)ciK-NH2 (aFmoc-elys)ci(aAIB)-NH2 (aFmoc-elys)ci(Orn)-NH2 8-16 (aFmoc-elys)ci(dOrn)-NH2 peptide mixture per ml is shown in Fig. 1. The IC50s were then determined for those peptide mixtures which exhibited more than 50% inhibition at 2.5 mg/ml. Overall, the non-N-acetylated peptide mixtures were found to be more active than the corresponding N-acetylated ones. The peptide mixture (aFmoc-elys)ZZZ-NH2 showed the greatest activity (IC50 = 44 ,ug/ml; MIC = 78 to 156 ,ug/ml). The next most active peptide mixture, wZZZ-NH2, had an IC50 of 212 ,ug/ml and an MIC ranging from 312 to 625 ,ug/ml [fivefold lower than that of (ctFmoc-slys)ZZZ-NH2]. A single peptide mix- ture, (aFmoc-elys)ZZZ-NH2, was chosen to be followed through the iterative selection process. Identification of individual antistaphylococcal peptides. The identification of individual peptides from an SPCL mixture involves ranking, selecting, and reducing the number of peptide sequences within the mixtures while sequentially defining a single position at a time. By reducing the number of individual peptides within a mixture, the actual concentration of each individual peptide per milligram of each of the new peptide mixtures increases 56-fold with each iteration. Thus, o012 1010 D 108- Elog 0106 ~ 104 0 0.5 1 2 Incubation time (h) by (otFmoc-elys)WfR-NH2. The killing of 105 CFU 24 of S. aureus per ml was evaluated at a peptide FIG. 2. Killing curve of S. aureus concentration of 15.6 jig/ml. The CFU per milliliter of the suspension with (filled squares) and without (open squares) peptide is plotted as a function of the incubation time. Downloaded from http://aac.asm.org/ on April 18, 2014 by PENN STATE UNIV S. aureus 2284 BLONDELLE ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 4. MICs of tetrapeptides for MRSA in presence of seruma MIC (,ug/ml) in presence of serum concn (%) of: Tetrapeptide (otFmoc-elys)Wfl-NH2 (otFmoc-elys)Wfi-NH2 (tFmoc-Elys)ci(Orn)-NH2 0 6.25 12.5 25 50 7.8 7.8 7.8 125 >250 250 125 >250 250 250 >250 250 >250 >250 250 a Heat-inactivated normal human serum pool. 1010 - k 108 0 106 104 0 0.5 1 2 24 Incubation time (h) Killing curve of E. coli by (aFmoc-elys)WfR-NH2. The killing of 105 CFU of E. coli per ml was evaluated at a peptide concentration FIG. 3. of 125 ug/ml. The CFU per milliliter of the suspension with (filled squares) and without (open squares) peptide is plotted as a function of the incubation time. Downloaded from http://aac.asm.org/ on April 18, 2014 by PENN STATE UNIV we synthesized a new series of 58 peptide mixtures in which the second position of (oaFmoc-slys)ZZZ-NH2 was defined. This new series can be represented by the general formula (otFmocElys)UZZ-NH2. Each peptide mixture is then made up of 3,136 (56) individual tetrapeptides. The three most active peptide mixtures were found to be (aFmoc-elys)WZZ-NH2, (aFmocElys)cZZ-NH2, and (otFmoc-elys)CZZ-NH2 (Table 2). The iterative selection process was then repeated in order to define the third positions of (otFmoc-e1ys)WZZ-NH2 and (otFmoc-Clys) cZZ-NH2. The peptide mixtures of (xFmoc-elys)WUZ-NH2 and (otFmoc-elys)cUZ-NH2 then contained 56 individual tetrapeptides. Of the 58 peptide mixtures making up (aFmoc-elys) WUZ-NH2 and of the 58 peptide mixtures making up (oxFmocslys)cUZ-NH2, 18 and 5 peptide mixtures, respectively, had IC50s less than 10 ,ug/ml (Table 2) and MICs ranging from 5 to 18 ,ug/ml. The large number of active sequences indicates the redundancy of the third position (i.e., low relative specificity of each amino acid at this position) on the activities of the two series of peptides being pursued. The follow-up of the iterative process from these 23 active sequences would require the synthesis of 1,334 individual tetrapeptides (23 x 58). In order to decrease the number of peptides required, three main criteria were considered: (i) level of antimicrobial activity, (ii) toxicity as indicated by hemolytic activity, (iii) and differences in the chemical functionalities of the residues making up the sequences. Thus, in our first selection, we synthesized four series of 58 individual tetrapeptides in which the last positions of the active peptide mixtures (oxFmoc-e1ys)WKZ-NH2, (aoFmocelys)WYZ-NH2, (aFmoc-e1ys)WfZ-NH2, and (aFmoc-elys) ciZ-NH2 were defined. The most active tetrapeptides identified from these series had IC50s ranging from 2 to 4 ,ug/ml (Table 2) and MICs ranging from 3 to 16 ,ug/ml (Table 3). It is noteworthy that none of the peptide mixtures and individual peptides with antistaphylococcal activity had aspartic, P-aspartic, glutamic, or y-glutamic acid in their sequences. Viable counts of S. aureus cells in the presence of (aFmocelys)WfR-NH2 were then carried out to determine if this representative peptide was bacteriostatic or bactericidal. As shown in Fig. 2, (ctFmoc-elys)WfR-NH2 had bacteriostatic activity against S. aureus. The tetrapeptides identified above were also tested against an MRSA strain. Twelve of the identified peptides had similar activities against the two strains of S. aureus, while decreases or losses in activity were observed for the remaining nine peptides. Activity in the presence of serum. The activities of the tetrapeptides were also examined in the presence of various concentrations in human serum (Table 4). This furthers the measure of the therapeutic potentials of these peptides. The activities of all of the peptides tested decreased in the presence of 6.5% serum, with (aFmoc-elys)Wfl-NH2 having the greatest activity. Human serum albumin was then used to investigate if this decrease was due to enzyme inactivation or serum protein binding. As shown in Table 5, higher levels of activity were found solely in the presence of human serum albumin. Spectra of activity of the antistaphylococcal peptides. The antimicrobial activities of the peptides found to have antistaphylococcal activities were also assayed against a second gram-positive bacterium (S. sanguis), a gram-negative bacterium (E. coli), and a fungal species (C. albicans). The MICs of each of the tetrapeptides for these three microorganisms were compared with the MICs for S. aureus. Overall, these peptides were found to be more active against gram-positive bacteria. Thus, as seen in Table 3, all of the peptides tested showed VOL. TABLE 5. MICs of (oaFmoc-elys)ci(Orn)-NH2 for S. aureus in presence of serum or albumin TABLE 6. Hemolytic activities of antistaphylococcal peptides ID50 MIC (,ug/ml) in presence of: Concn (%) 0 6.25 12.5 25 50 a 2285 IDENTIFICATION OF NEW ANTIMICROBLAL PEPTIDES 38, 1994 (4/ml) Serum Albumin 15.6 250 250 250 >250 15.6 31.2 31.2 62.5 62.5 Heat-inactivated normal human serum pool. DISCUSSION The widespread use of antibiotics has caused numerous antibiotic-resistant strains to develop. For instance, MRSA is acquiring resistance to antibiotics such as the fluoroquinolones (for a review, see reference 5). With the emergence of vancomycin-resistant Enterococcus strains, there is concern that this resistance will be passed to MRSA. Such new resistant strains underscore the need for new antibiotics that are effective against them. The rational design of new antimicrobial agents requires years of effort to screen a large number of new compounds. The introduction of soluble combinatorial peptide and organic chemical libraries now enables the identification of antimicrobial agents in a matter of weeks. A series of successful uses of such SPCLs has been reported for the identification of biologically active peptide sequences in various assay systems such as enzyme-linked immunosorbent assays (15, 18), radioreceptor assays (7), enzyme inhibitor assays (8), and microdilution assays (11, 12, 14, 15). In particular, investigators have identified a number of hexapeptides composed of Lamino acids which exhibit antimicrobial activities against S. aureus (11, 12, 15) and E. coli (14). These new hexapeptides have MICs that vary from 3 to 20 jig/ml. These activities are greater or equivalent to those found for naturally occurring peptides, which are generally greater in length (i.e., 10 residues (aFmoc-elys)Wfl-NH2 (aFmoc-elys)Wfw-NH2 (aFmoc-elys)Wfr-NH2 (aFmoc-clys)Wf(KCBZ)-NH2 (aFmoc-elys)Wf(Thiopro)-NH2 (oaFmoc-elys)WfL-NH2 (aFmoc-EIys)WfP-NH2 (aFmoc-EIys)Wfi-NH2 (aFmoc-elys)Wf(Orn)-NH2 (aFmoc-elys)Wf(dOrn)-NH2 (aFmoc-elys)Wf(aAIB)-NH2 (aFmoc-elys)WKW-NH2 (aFmoc-elys)WKC-NH2 (aFmoc-clys)WYr-NH2 (aFmoc-elys)WY(aABA)-NH2 (aFmoc-elys)cir-NH2 (aFmoc-elys)ciR-NH2 (aFmoc-elys)ciK-NH2 (aFmoc-elys)ci(aAIB)-NH2 (aFmoc-elys)ci(Orn)-NH2 (aFmoc-clys)ci(dOrn)-NH2 16 >gml 17-46 18-26 100 100 100 100 10-22 100 0 49-76 69-98 13-20 3-4 0 0-1 3-4 0 6-7 0 0-1 0 0 5-7 0 32-37 40-43 80-82 56-77 10-12 8-9 31-66 114-143 100 27-51 7-8 48-66 50-77 89-94 100 4-5 79-173 70-71 28-53 100 77-114 77-119 54-79 122-130 197-208 36-63 42-54 52-55 45-52 294-320 40-58 >500 64-98 42-60 383-410 62-70 1-5 2-3 1-2 11-12 0 0-1 for the gramicidins, 23 for the magainins, 35 to 37 for the cecropins, and 30 to 43 for the defensins; for a review, see reference 2). To expand the chemical diversity of the original SPCLs and to decrease the risk of enzymatic degradation in in vivo systems, D- and unnatural amino acids have been incorporated, thus permitting the screening of a larger variety of peptides. The generation of such libraries can be easily performed by using the divide, couple, and recombine synthetic approach (9, 15, 17), which ensures the equimolarity of each peptide within a mixture. The tetrapeptide format was chosen so that the number of individual peptides in each peptide mixture was in the same range as that in the initial hexapeptide library used for the identification of new antimicrobial peptides (11, 12, 14, 15). The individual tetrapeptides found in the current study to exhibit antimicrobial activities against S. aureus were similar to those described earlier, which were made up of L-amino acids only. The greater antistaphylococcal activities observed for the non-N-acetylated peptide mixtures compared with the activities of the equivalent N-acetylated peptide mixtures suggests that hydrogen bonding or electrostatic interactions through the protonated N terminus are involved in the antimicrobial activities of these series of tetrapeptides. The lack of activity of those tetrapeptides with either a glutamic acid or an aspartic acid supports this hypothesis. The presence of negative charges in such peptides may prevent the initial electrostatic interactions between the peptides and the negatively charged phospholipids that make up the bacterial cells. The higher level of activity found for the (oFmoc-elys)ZZZ-NH2 mixture compared with that of the Ac-KZZZ-NH2 mixture, which differ only by their protecting N-terminal groups, may be due to the hydrophobicity of the Fmoc group relative to that of an acetyl group. Longer Fmoc-protected peptides have been reported to have potent antimicrobial activities (6). The tetrapeptides studied here appear to exhibit their Downloaded from http://aac.asm.org/ on April 18, 2014 by PENN STATE UNIV similar activities against the two gram-positive bacteria. Lower levels of activity, although similar within the series, were found for all of the tetrapeptides against E. coli and C. albicans, with the exception of (aFmoc-e1ys)Wfi-NH2 and (otFmoc-slys) WKC-NH2. (otFmoc-e1ys)Wfi-NH2 was highly selective for gram-positive bacteria, while (ctFmoc-e1ys)WKC-NH2 was active against bacteria but was inactive against C. albicans. Kinetic studies of the activity of (aFmoc-e1ys)WfR-NH2 against E. coli (Fig. 3) were carried out in a manner similar to that described above for S. aureus. Those studies indicated that (aoFmoc-s1ys)WfR-NH2 exhibits bacteriostatic activity against E. coli. Hemolytic activity of the antistaphylococcal peptides. The ability of the antistaphylococcal peptides to lyse eukaryotic cells was determined by using human RBCs. The HD50 of each peptide was calculated (Table 6). The HD5Os were generally found to be greater than the MICs for the gram-positive bacteria. At 16 j,g/ml, the concentration corresponding to the MICs for gram-positive bacteria, the majority of the peptides showed less than 5% hemolysis (Table 6). At a higher concentration (125 ,ug/ml), only (aFmoc-elys)Wfi-NH2 had no hemolytic activity, with (aFmoc-e1ys)WfL-NH2 and (aFmoc-elys) Wf(aAIB)-NH2 being 10 to 20% hemolytic (Table 5). Interestingly, (otFmoc-s1ys)Wfi-NH2 was found to be the only peptide of the series highly selective for gram-positive bacteria. (oFmoc-elys)WfR-NH2 % Hemolysis at: 12 jml 2286 BLONDELLE ET AL. 8. 9. 10. 11. 12. 13. ACKNOWLEDGMENTS We thank Mary Buck and Adam Lucka for technical assistance and Eileen Silva for editing the manuscript. These studies were supported by Houghten Pharmaceuticals Inc. 1. 2. 3. 4. 5. 6. 7. REFERENCES Appel, J. R., C. Pinilla, and R. A. Houghten. 1992. Identification of related peptides recognized by a monoclonal antibody using a synthetic peptide combinatorial library. Immunomethods 1:1723. Blondelle, S. E., and R A. Houghten. 1992. Progress in antimicrobial peptides. Annu. Rep. Med. Chem. 27:159-168. Blondelle, S. E., L. R Simpkins, E. Perez-Paya, and R A. Houghten. 1993. Influence of tryptophan residues on melittin's hemolytic activity. Biochim. Biophys. Acta 1202:331-336. Boman, H. G., I. Faye, G. H. Gudmundsson, J. Lee, and D. Lidholm. 1991. Cell-free immunity in cecropia: a model system for antibacterial proteins. Eur. J. Biochem. 201:23-31. Brighty, K. E., W. Kohlbrenner, and P. R McGuirk 1993. Recent developments in antibacterial resistance mechanisms. Annu. Rep. Med. Chem. 28:141-150. DeGrado, W. F. 1983. Solution phase synthesis of cecropin A 1-22 and potent analogues thereof using segments prepared on an oxime solid phase support, p. 195-198. In J. H. Hruby and D. H. Rich (ed.), Peptides: structure and function. Proceedings of the Eighth American Peptide Symposium. Pierce Chemical Co., Rockford, Ill. Dooley, C. T., N. N. Chung, P. W. Schiller, and R. A. Houghten. 1993. Acetalins: new opioid receptor antagonists determined 14. 15. 16. 17. 18. 19. 20. through the use of synthetic peptide combinatorial libraries. Proc. Natl. Acad. Sci. USA 90:10811-10815. Eichler, J., and R A. Houghten. 1993. Identification of substrateanalog trypsin inhibitors through the screening of synthetic peptide combinatorial libraries. Biochemistry 32:11035-11041. Furka, A., F. Sebestyen, M. Asgedom, and G. Dibo. 1991. General method for rapid synthesis of multicomponent peptide mixtures. Int. J. Pept. Protein Res. 37:487-493. Houghten, R A. 1985. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigenantibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. Houghten, R. A., J. R Appel, S. E. Blondelle, J. H. Cuervo, C. T. Dooley, and C. Pinilla. 1992. The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides. BioTechniques 13:412-421. Houghten, R A., S. E. Blondelle, and J. H. Cuervo. 1992. Development of new antimicrobial agents using a synthetic peptide combinatorial library involving more than 34 million hexamers, p. 237-239. In R. Epton (ed.), Innovation and perspectives in solid phase synthesis-peptides, polypeptides and oligonucleotides. Solid Phase Conference Coordination, Oxford. Houghten, R. A., M. K. Bray, S. T. De Graw, and C. J. Kirby. 1986. Simplified procedure for carrying out simultaneous multiple hydrogen fluoride cleavages of protected peptide resins. Int. J. Pept. Protein Res. 27:673-678. Houghten, R A., K. T. Dhin, D. E. Burcin, and S. E. Blondelle. 1993. The systematic development of peptides having potent antimicrobial activity against E. coli through the use of synthetic peptide combinatorial libraries, p. 249-256. In R. H. Angeletti (ed.), Techniques in protein chemistry IV. Academic Press, Inc., Orlando. Houghten, R. A., C. Pinilla, S. E. Blondelle, J. R Appel, C. T. Dooley, and J. H. Cuervo. 1991. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature (London) 354:84-86. Jackson, M., H. H. Mantsch, and J. H. Spencer. 1992. Conformation of magainin-2 and related peptides in aqueous solution and membrane environments probed by Fourier transform infrared spectroscopy. Biochemistry 31:7289-7293. Lam, K. S., S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R J. Knapp. 1991. A new type of synthetic peptide library for identifying ligand-binding activity. Nature (London) 354:82-84. Pinilla, C., J. R. Appel, P. Blanc, and R A. Houghten. 1992. Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. BioTechniques 13:901-905. Rana, F. R, E. A. Macias, C. M. Sultany, M. C. Modzrakowski, and J. Blazyk. 1991. Interactions between magainin 2 and Salmonella typhimurium outer membranes: effect of lipopolysaccharide structure. Biochemistry 30:5858-5866. Tam, J. P., W. F. Heath, and R. B. Merrifield. 1983. SN2 deprotection of synthetic peptides with a low concentration of HF in dimethyl sulfide: evidence and application in peptide synthesis. J. Am. Chem. Soc. 105:6442-6455. Downloaded from http://aac.asm.org/ on April 18, 2014 by PENN STATE UNIV activities by inhibiting cell growth and development rather than lysing the cells. This effect may be due to the shortness of these peptides, which would prevent them from spanning the lipid bilayer of a membrane and/or forming a channel through the membranes, as has been proposed for longer antimicrobial peptides such as magainins (16) and cecropins (4). Because of their cationic character, these short peptides may bind to the lipopolysaccharides of the bacterial cell membranes in a manner similar to that of the magainins (19), thereby increasing cell permeability. This is supported by their higher levels of activity against gram-positive bacteria, which have a less complex cell wall and lack an outer membrane, compared with their activities against gram-negative bacteria. The results of the experiments with serum suggest that the activities of the tetrapeptides decrease because of degradation by heat-stable proteases rather than binding to serum proteins. Further studies are necessary to determine the cause of the decrease in activity that was observed. The studies described above indicate that new potent antimicrobial compounds can be readily and rapidly identified through the examination of extendeddiversity peptide libraries. ANTIMICROB. AGENTS CHEMOTHER.