Patent Publication Number: US-2022211796-A1

Title: Peptide antibiotics and methods of use thereof

Description:
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under grant GM065500 awarded by the National Institutes of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 15, 2021, is named 51415-003002_Sequence_Listing_11_15_21_ST25 and is 102,989 bytes in size. 
     BACKGROUND OF THE INVENTION 
     Bacterial resistance to antibiotics in clinical use is a clear and present danger to public health. Because development of resistance is inevitable, new strategies for discovery of compounds with antimicrobial activity are urgently needed.  Staphylococcus aureus  is a leading cause of community-associated and nosocomial infections, and its ability to become resistant to existing therapeutics poses a significant concern. Although vancomycin, often referred to as ‘the antibiotic of last resort’, has been effective in the treatment of infections caused by  S. aureus  and methicillin-resistant  S. aureus  (MRSA) for more than three decades, emergence of vancomycin-resistant strains underscore the critical need for expansion of the pipeline of lead compounds. Several approaches are currently being pursued in an attempt to develop compounds that are active against resistant bacteria, such as modification of existing antibiotics, finding new leads from nature, and expansion of new synthetic classes through rational design. The mode of action of antibiotics have typically consisted of the selective inhibition of bacterial enzymes, exploiting differential structures of the bacterial versus the human ribosomes, selective membrane disruption, and the binding of DNA or RNA sequences that are unique to bacteria. Among these approaches, targeting bacterial cell wall biosynthesis has been a fruitful line of investigation for the discovery of several classes of antibiotics. 
     Peptide based therapeutics afford a high level of specificity. However, the use of these compounds is limited because of hydrolytic cleavage by proteases, possible induction of a vigorous humoral immune response and administration mostly as injectables. 
     SUMMARY OF THE INVENTION 
     As described below, the present invention features peptide antibiotic compositions and methods of using such compositions for the treatment of bacterial infections (e.g., vancomycin resistant infections). In particular embodiments, the invention features peptides composed of D-amino acids, which are metabolically stable, are either less or non-immunogenic, and short sequences (&lt;12 residues) have been known to be absorbed through the gut making them viable oral drugs. These favorable features make them a particularly attractive class of new therapeutics. 
     Other features and advantages of the invention will be apparent from the detailed description, and from the claims. 
     Definitions 
     Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale &amp; Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. 
     By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. 
     By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. 
     By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.” 
     By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a peptide analog retains the biological activity of a corresponding naturally-occurring peptide, while having certain biochemical modifications that enhance the analog&#39;s function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog&#39;s protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid. 
     In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. 
     “Detect” refers to identifying the presence, absence or amount of the analyte to be detected. 
     By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens. 
     By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include bacterial infections (e.g., antibiotic resistant infections). 
     By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. 
     The invention provides a number of targets that are useful for the development of highly specific drugs to treat a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity. 
     By “fragment” is meant a portion of a peptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. 
     “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. 
     The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. 
     By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. 
     By an “isolated polypeptide” is meant a polypeptide or fragment thereof (e.g., peptide) of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. 
     By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. 
     As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent. 
     By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%. 
     By “reference” is meant a standard or control condition. 
     A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. 
     Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). 
     For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. 
     For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. 
     By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. 
     Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e −3  and e −100  indicating a closely related sequence. 
     By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. 
     Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. 
     As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. 
     Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural. 
     Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. 
     The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. 
     Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . a Schematic representation of the mirror image phage display concept: the phage display peptide library is screened for identification of L-peptides that bind the antipode of the target molecule. Due to symmetry relations, L-peptides identified through phage display can be synthesized in the D-form that would now bind the original L-target. b Chemical structures of cephalosporin and N-acyl-D-Ala-D-Ala terminus of cell wall peptidoglycan precursor showing structural similarity as well as N-acyl-D-Ala-D-Lac terminus of cell wall peptidoglycan precursor of vancomycin-resistant bacteria. c Structures of target molecules used in phage display screenings, Cep-1, Ala-2, and Lac-4. 
         FIG. 2 . Peptides identified from phage display screening against Cep-1 and Ala-2 were identical. a Sequences of peptides are shown with phage display libraries isolated from libraries depicted in the table. b Phage-ELISA showing the binding differences of identical peptide sequences from each target molecule. Data are shown as mean±standard deviation of three independent experiments. 
         FIG. 3 . Phage display of bicyclic peptides. a Structure and diversity of two bicyclic peptide libraries used in the phage display screening. b Schematic representation of bicyclization reaction on phage protein pIII with 1,3,5-tris(bromomethyl)benzene (TBMB) through three cysteine residues in peptides expressed on the surface. 
         FIG. 4 . Properties of antibiotic D-peptides. a The binding capabilities of potent peptides (MIC value of &lt;256 μg/ml) from phage display screening with linear 12-mer, linear 7-mer and cyclic 7-mer libraries against target molecules, Cep-1 and Ala-2, by phage-ELISA. A control peptide containing “HPQ” consensus sequence that specifically binds to streptavidin was also included. The experiments were performed in triplicate and data are presented as mean±standard deviation. b Time-dependent killing kinetics of  S. aureus  in presence of P14 and P15 compared to vancomycin at 10×MIC; both peptides show bactericidal activity similar to vancomycin. Data are shown as mean±SD (n=3). c Accumulation of the soluble cell wall precursor UDP-MurNAc-pentapeptide of  S. aureus  after incubation with P14 (red), P15 (green) and vancomycin (blue). d MALDI-TOF mass spectrum confirms accumulation of UDP-MurNAc-pentapeptide of  S. aureus  (MW=1149.52). 
         FIG. 5 . Peptides identified from phage display screening against target molecules Lac-4 and Ala-2 with identical sequences. a Peptides with their sequences and which commercially available phage display libraries they were identified. b Peptides showed different binding affinities for target molecules, Lac-4 and Ala-2, in a phage-ELISA assay. Data are shown as mean±standard deviation (n=3). 
         FIG. 6 . Toxicity of D-peptides. a Percentage hemolysis of hRBCs by P14 and P15 with melittin (100%) and vancomycin (0%) as controls. b Percentage hemolysis of hRBCs by P18. c Cytotoxicity of potent D-peptides, P14, P15 and P18, on HeLa cells compared to non-toxic vancomycin and toxic melittin. Data are depicted as mean±S.D. (n=3). 
         FIG. 7 . a Drug-like properties of potent D-peptide antibiotics. P14, P15 and P18, identified from phage display screening against vancomycin-sensitive, P14 and P15, and vancomycin-resistance bacteria target molecules, P18. b Chemical structures of bicyclic D-peptides, P14, P15 and P18. 
         FIG. 8  provides a schematic for the experimental scheme. 
         FIG. 9  shows the synthesis of construct Cep-1 that contains the enantiomeric cephalosporin scaffold. Reagents and conditions. a PhtCO 2 Et, Na 2 CO 3 , H 2 O, rt, 2 h, 45%; b BnBr, Et 3 N, DMF, rt, 6 h, 60%; c DBU (cat.) in CH 2 Cl 2 , rt, 90 min, 64%; d SO 2 Cl 2  (1 equiv.), CCl 4 , 0° C., 30 min; e SnCl 2 , THF, rt, 2 h reflux, 30% over 2 steps; f 03, Me 2 CO, −78° C., quantitative yield; g pTSA (cat.), DMF, 100° C., 90 min, 52%; h 1M N 2 H 4  in DMF, −78° C. to rt, 30 min, 40%; i NHS-PEG 12 -Biotin, DIPEA, DMF, 0° C., overnight, 65%; j AlCl 3 , PhOMe, CH 2 Cl 2 , CH 3 NO 2 ; 0° C. to rt, 8 h, 28% 
         FIG. 10  is a  1 H NMR spectrum of compound 10, Cep-1. 
         FIG. 11  is a  13 C NMR spectrum of compound 10, Cep-1. 
         FIG. 12  is a RP-HPLC chromatogram of Cep-1. Linear gradient was applied from 20% to 40% solvent B over 40 min. 
         FIG. 13  is a MALDI TOF-MS spectrum of pure Cep-1. Calculated mass [M+Na+H] + , 1062.62, observed mass 1062.42. 
         FIG. 14  is a RP-HPLC chromatogram of Ala-2. Linear gradient was applied from 5% to 20% solvent B over 30 min. 
         FIG. 15  is a MALDI TOF-MS spectrum of pure Ala-2. Calculated mass [M+H] + , 962.46, observed mass 962.121. 
         FIG. 16  is a RP-HPLC chromatogram of Ala-3. Linear gradient was applied from 5% to 20% solvent B over 30 min. 
         FIG. 17  is a MALDI TOF-MS spectrum of pure Ala-3. Calculated mass [M+H] + , 962.46, observed mass 962.121. 
         FIG. 18  is a RP-HPLC chromatogram of Lac-4. Linear gradient was applied from 5% to 20% solvent B over 30 min. 
         FIG. 19  is a MALDI TOF-MS spectrum of pure Lac-4. Calculated mass [M+H] + , 963.26 [M], observed mass 963.115. 
         FIG. 20  is a RP-HPLC chromatogram of P14. Linear gradient was applied from 5% to 30% solvent B over 30 min. 
         FIG. 21  is a MALDI TOF-MS spectrum of pure P14. Calculated mass [M+H] + , 1711.17 [M], observed mass 1711.716. 
         FIG. 22  is a RP-HPLC chromatogram of P15 Linear gradient was applied from 5% to 30% solvent B over 30 min. 
         FIG. 23  is a MALDI TOF-MS spectrum of pure P15. Calculated mass [M+H] + , 1582.69 [M], observed mass 1582.92. 
         FIG. 24  is a RP-HPLC chromatogram of P18 Linear gradient was applied from 20% to 40% solvent B over 30 min. 
         FIG. 25  is a MALDI TOF-MS spectrum of pure P18. Calculated mass [M+H] + , 1562.61 [M], observed mass 1562.29. 
         FIG. 26  shows the Structural similarities between peptidoglycan cell wall termini L-Lys-D-Ala-D-Ala and penicillin. Calculated structural overlays of the best conformations of N-Ac-D-Ala-D-Ala and Ampicillin (a β-lactam). Longest RMS distance=1.70 Å; shortest 0.29 Å. (Figure is based on the calculations done by Boyd et al J. Med. Chem. 1975, 18, 408-417). 
         FIG. 27  shows identical peptides isolated from phage display experiments of target molecules Ala-2 and Ala-3. a Sequences of peptides that were selected as well as phage display libraries are shown in the table. b Binding affinities of each peptide for target molecules Ala-2 and Ala-3 were evaluated by phage-ELISA experiments. Data are shown as mean±standard deviation. (n=3). 
         FIG. 28  shows Hemolytic activity of potent D-peptides showing minimum inhibitory activity less than 256 μg ml −1  against both vancomycin-sensitive and resistant bacteria compared to vancomycin (blue) and melittin (red). All peptides showed no toxicity on red human blood cells. Data are shown as mean±standard deviation (n=3). 
         FIG. 29  shows Proteolytic and serum stability of potent D-peptides. RP-HPLC analysis of 1 mg/ml solutions of three potent bicyclic D-peptides, P14, P15 and P18, a After incubation with 50 μg/ml of pancreatin, and digestion of samples analyzed after 0, 15, 30, 60, 120, 180, 240, 360 min and overnight. b After incubation with human serum and digestion of samples analyzed after 0, 5, 30, 60, 120, 240 min and overnight. 
         FIG. 30  is a table showing phage display peptide libraries used during affinity selections. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention features peptide antibiotic compositions and methods of using such compositions for the treatment of bacterial infections (e.g., vancomycin resistant infections). 
     A paucity of viable programs and pipelines for the discovery of new antibiotics poses a significant public health threat. The emergence of resistant strains against vancomycin is particularly dangerous in hospital settings. Here we report the design of enantiomeric targets based on bacterial cell wall biosynthesis precursors that allow for selection and identification of short linear, cyclic and bicyclic peptides that are composed of D-amino acids. These compounds are active against  S. aureus , Methicillin-resistant  S. aureus  (MRSA), and vancomycin-resistant Enterococci (VRE) that possess moderately high antibacterial activity, and furthermore display no toxicity to both human red blood cells (hRBCs) and mammalian cells (HeLa) at these concentrations. This ‘mirror image phage display’ approach yielded templates that can serve as scaffolds for further improvements in activity based structural modifications. This strategy has potential to provide a new class of antimicrobials that are metabolically stable and have the promise for oral delivery. The use of this platform combined with traditional medicinal chemistry approaches could rapidly yield large numbers of new therapeutic peptides. Peptides of the invention are provided in the following tables. 
     
       
         
           
               
             
               
                 TABLE1  
               
               
                   
               
               
                 Peptide sequences identified from screening with 
               
               
                 linear 12-mer Phage Display Library, where all 
               
               
                 peptides are written from N-terminus to C-terminus  
               
               
                 and have a free N-terminus and an amidated C-terminus. 
               
               
                 Linear 12-mer Peptide 
               
               
                 Sequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 ADWYHWRSHSSS 
                 GLHTSATNLYLH 
                 LETRLGTSPGHT 
                 SPDVTRWPYWVI 
               
               
                 AKDTHTGRMTNW 
                 GLPHAREDYLDL 
                 LPSDYRSRWADP 
                 SSSVTPVSALHG 
               
               
                 ANVKAFFSTTQV 
                 GLVNLMRPWHLL 
                 MDLGYVEGSARV 
                 SVLSYSVAYSDS 
               
               
                 APFPSVALKVPL 
                 GMLQHALVPKVW 
                 NGYHPPGFLAPE 
                 SWFSDWDLELHA 
               
               
                 ASPVNYRDMFSR 
                 GPFVNLRSHFES 
                 NHLSTPVWSITG 
                 SWHHGDGPIWYG 
               
               
                 ATNAKRTPNTRI 
                 GQSEHHMRVASF 
                 QCFNSVCLHTNP 
                 TGVYWTQLNADS 
               
               
                 ATWARVDAIARA 
                 GSAPLLTVDTSK 
                 QDIQTTPPKFSV 
                 TQAIDDIIIGRI 
               
               
                 CLNDSLYCQGMP 
                 GTGLVTLPRLTV 
                 QFDYMRPANDTH 
                 TTVDYFRKVWVV 
               
               
                 CPLFDSVRCTSK 
                 GTTWVATAGKLI 
                 QGRIDLYGFLSH 
                 TYTANDLHLADL 
               
               
                 DLMPHKRMIELV 
                 GVGLTTNVTRAG 
                 QITSSHVWDMGH 
                 VEAKCCFSMHKT 
               
               
                 DRWVARDPASIF 
                 HHHFVTHPAWVL 
                 QLATLHKLSGPT 
                 VFARGQYDAPHP 
               
               
                 EFHPMPDGFRSR 
                 HKHWSTPEFLSS 
                 QSHYDSHLAMLV 
                 VGSNLRLLHQWK 
               
               
                 EGNWFLSFHAST 
                 HKTDLWMTNTIK 
                 QVDNTSSIERLR 
                 VHWDFRQWWQPS 
               
               
                 FAPALSNPPLRD 
                 HRDPHSALTRSW 
                 RKVKRRPRVSNL 
                 VPKSVDNTFYTP 
               
               
                 FGRMAWTPMAPM 
                 HTSSLWHLFRST 
                 SAGNSANWLLHW 
                 VSHRSTANFIGS 
               
               
                 FHFPLGMHSRDE 
                 HVVTSSKTAGPA 
                 SFPHFTLRAYAS 
                 VSNFTKPHKPKA 
               
               
                 FIPFDPMSMRWE 
                 IGRTVPHQDFAR 
                 SGVYKVAYDWQH 
                 WHPRYVVSPLQY 
               
               
                 FYLPPNASYMSR 
                 IPLGRDGGSYQR 
                 SINGLLSNSHGS 
                 YAQVYSNHGSRI 
               
               
                 GDSGLVESHRNV 
                 ITGLGSGSSTST 
                 SLDGAGAALRTS 
                 YIGEMDTLPIST 
               
               
                 GDVSDVWTAANN 
                 KHFPLGMEYLVT 
                 SLTVPYLTSISD 
                 YPDKITWQAPWL 
               
               
                 GGGLGSLHETSM 
                 KPGDTAMHYFPP 
                 SNSIDKVNRPIN 
                 YSLRSDFLPFAT 
               
               
                 GGRRCRIKNCYA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE2  
               
               
                   
               
               
                 Peptide sequences identified from screening with linear 7-mer 
               
               
                 Phage Display Library, where all peptides are written from 
               
               
                 N-terminus to C-terminus and have free a N-terminus and an 
               
               
                 amidated C-terminus. 
               
               
                 Linear 7-mer Peptide 
               
               
                 Sequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 ADRFQAL 
                 GKDYMGY 
                 LPGSEQR 
                 SADYSAR 
                 VDSRYHP 
               
               
                 AGKPFHF 
                 GMWHLPQ 
                 LPKMYSQ 
                 SEHNGTQ 
                 VHPLKLI 
               
               
                 AHGRSRG 
                 GNVGSVR 
                 LPNSAYV 
                 SEVYPQK 
                 VLTRCCG 
               
               
                 AHTDWFN 
                 GQSEKHL 
                 LPTGHFL 
                 SFFEQVH 
                 VPIYHLT 
               
               
                 AIDFARN 
                 GSFWHHN 
                 LPVRLDW 
                 SFRIGPA 
                 VPVWALT 
               
               
                 ALQPQKH 
                 GSPDSEF 
                 LTLGLPY 
                 SFSQNLH 
                 VSGFRID 
               
               
                 ALSYSRG 
                 GVHREQI 
                 LVMHSEN 
                 SFVSMPE 
                 VSQRTEP 
               
               
                 APKPIKL 
                 GVMNHTF 
                 LVPSDKL 
                 SHENFTS 
                 VSRANEG 
               
               
                 APTPGNV 
                 HGGVRLY 
                 MAPTHSI 
                 SHGTWTP 
                 VSRDTPQ 
               
               
                 AQYVAVG 
                 HIARLSY 
                 MIRGTTV 
                 SLIAHYQ 
                 VTNTPWP 
               
               
                 ASLSKYS 
                 HLFTTGV 
                 MMVLRNQ 
                 SNMSHAT 
                 VTSPYAF 
               
               
                 ATYGNLW 
                 HLKHSLL 
                 MPDMTRQ 
                 SPWQYTN 
                 VYPGPSY 
               
               
                 AVRGYEW 
                 HLNQQNH 
                 NDLMNRA 
                 SQNFVRE 
                 WDPRVNV 
               
               
                 AYDDWFW 
                 HMGKLNR 
                 NDRLHTR 
                 SSDVPYL 
                 WGRISHV 
               
               
                 DISRMAT 
                 HVMTKAL 
                 NIGQDMH 
                 SSLRIPV 
                 WPTHYLV 
               
               
                 DSVETKP 
                 HVRHYSD 
                 NIVSRES 
                 SSNQFHQ 
                 WQEHRDQ 
               
               
                 DTALHSL 
                 HYIDFRW 
                 NLRLPYI 
                 STKTLPA 
                 WQWPARV 
               
               
                 DVMMPRH 
                 IPFSFTG 
                 NSIYQAW 
                 STVKYID 
                 WSLSELH 
               
               
                 ETALIAA 
                 IRIAEPM 
                 NSYDVQA 
                 SWTALGP 
                 WSWGEQK 
               
               
                 FPAWFSA 
                 ISTPYIG 
                 NTAVPLG 
                 TDEIKLL 
                 YGGAALQ 
               
               
                 FPITYDF 
                 KCCYTLP 
                 NTVANNY 
                 TGFLVNV 
                 YMLDSTM 
               
               
                 FSTTHPD 
                 KFYAHLD 
                 PRLPRTR 
                 TMQNIPN 
                 YNISVNK 
               
               
                 FSYSFQH 
                 KLSMQHR 
                 QLAVAPS 
                 TQTVLGD 
                 YPFFSSM 
               
               
                 FVRIHDV 
                 KPPPTLD 
                 QLKWYHA 
                 TSQYLMI 
                 YPWFIRA 
               
               
                 GASESYL 
                 KTALALE 
                 QQTNWSL 
                 TTLLTVS 
                 YPWWNTL 
               
               
                 GFGYNVQ 
                 KVKKRPD 
                 RHDIRKT 
                 TTQVLEA 
                 YQWELYS 
               
               
                 GGGHLSR 
                 LAQSSIQ 
                 RPTAHMA 
                 TVNFKLY 
                 YSEPAVT 
               
               
                 GHRVRFP 
                 LIQGTSL 
                 RTYPREK 
                 TVSPRFL 
                 YSGASTL 
               
               
                 GHYISAN 
                 LMPSYPR 
                 SAAWNKS 
                 TWSLDYP 
                 YYNTTPN 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE3  
               
               
                   
               
               
                 Peptide sequences identified from screening with cyclic 
               
               
                 7-mer Phage Display Library, where all peptides are written 
               
               
                 from N-terminus to C-terminus, have free a N-terminus and 
               
               
                 an amidated C-terminus and are cyclized through a disulfide 
               
               
                 bridge through two cysteine residues by a disulfide bond. 
               
               
                 Cyclic 7-mer Peptide 
               
               
                 Sequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 CAGHNRDRC 
                 CIAARHMNC 
                 CSEGLLNTC 
                 CSGWQVRMC 
               
               
                 CAKSPMNCC 
                 CILLPDKCL 
                 CSEHNLQTC 
                 CSHMEYPRC 
               
               
                 CASKSTHDC 
                 CKAALTRWC 
                 CNMQITKGC 
                 CSISSLTHC 
               
               
                 CDAMIGKSC 
                 CKDHVTRVC 
                 CNPEHNNHC 
                 CSNHRIMSC 
               
               
                 CDFIMGITC 
                 CKHLLGENC 
                 CNPTHYRSC 
                 CSQLPWYSC 
               
               
                 CDGHDQSLC 
                 CKLTTQMMC 
                 CNQNASHYC 
                 CSSPFPEFC 
               
               
                 CDHPHKQQC 
                 CKMSMLHNC 
                 CNQTAARVC 
                 CSSVTDRWC 
               
               
                 CDHTYTNKC 
                 CKSMMRLNC 
                 CNRWHHLEC 
                 CSTNSHSRC 
               
               
                 CDKFHELQC 
                 CKTLQPWTC 
                 CNSFGVSMC 
                 CSVGTNFQC 
               
               
                 CDNIMTPVC 
                 CLDIFSSSC 
                 CNTGSPYEC 
                 CTERTSTEC 
               
               
                 CDQMWHTSC 
                 CLHGDVAYC 
                 CNTTEAASC 
                 CTGKNAPKC 
               
               
                 CDRTISNKC 
                 CLKLGEKWC 
                 CPFWLSGHC 
                 CTILMKILC 
               
               
                 CEDLTTLSC 
                 CLKNQSDQC 
                 CPRDLGTDC 
                 CTKSLAHTC 
               
               
                 CEGQRWMQC 
                 CLNSSQPSC 
                 CPVALSTKC 
                 CTLRDSPHC 
               
               
                 CELGTVQSC 
                 CLRTSNPAC 
                 CPVISNGSC 
                 CTNANHYFC 
               
               
                 CFGQGTLQC 
                 CLVSQHTDC 
                 CQGNPSLRC 
                 CTNTNTAIC 
               
               
                 CFNMFSRVC 
                 CLWSTGATC 
                 CQHLRGLLC 
                 CTPGHTNRC 
               
               
                 CGDGSQRTC 
                 CMAPDSRVC 
                 CQMQLRSAC 
                 CTPSFSKIC 
               
               
                 CGGGPLYMC 
                 CMARYMSAC 
                 CQNWISRFC 
                 CTQMNDSFC 
               
               
                 CGHSNLSNC 
                 CMERMSLRC 
                 CQPRNLNNC 
                 CTQSSAMSC 
               
               
                 CGLKALKEC 
                 CMGFSNMSC 
                 CQYETPRYC 
                 CTVRTSADC 
               
               
                 CGNSSLNRC 
                 CMSTGLSSC 
                 CRGATPMSC 
                 CVGMQSNTC 
               
               
                 CGYSSFNRC 
                 CMSWSLQRC 
                 CRINPMSNC 
                 CVNLQKDMC 
               
               
                 CHDLNGSMC 
                 CNAKHHPRC 
                 CRSANIYTC 
                 CVPILEGTC 
               
               
                 CHNEGNRAC 
                 CNENIVHHC 
                 CRSATHSAC 
                 CVPMQDHTC 
               
               
                 CHNRVPLMC 
                 CNFLYSWTC 
                 CRSQSGSNC 
                 CVQMPAHSC 
               
               
                 CHPVSGQKC 
                 CNHDATHTC 
                 CSDARSPKC 
                 CYAFNYPHC 
               
               
                 CHSDANSIC 
                 CNIIHHQTC 
                 CSGPGINLC 
                 CYGNVTNTC 
               
               
                 CHYNAHRTC 
                 CYVSKNNSC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE4  
               
               
                   
               
               
                 Peptide sequences selected/identified from screening with 
               
               
                 Bicyclic Phage Display Library A and B, where all peptides are 
               
               
                 written from N-terminus to C-terminus, have a free N-terminus 
               
               
                 and an amidated C-terminus and are cyclized through three 
               
               
                 cysteine residues withTBMB (tribromomethylbenzene). 
               
               
                 Bicyclic PeptideSequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 CAAHQYCWTSC 
                 CLTAHCPQSISC 
                 CQLNMCTSANNC 
                 CSYLCEPAQHVC 
               
               
                 CAEQSCIFNLC 
                 CLTQPCNNPRPC 
                 CQLQLFCQTRTC 
                 CTAPGNCSQLC 
               
               
                 CALLIDCQYPLC 
                 CLVPCTQYVC 
                 CQPRHCIHSTVC 
                 CTDSCPPQSC 
               
               
                 CALSCHQVSLC 
                 CMRACVMQFDC 
                 CQPSTSCLIQRC 
                 CTGNCVSSVGC 
               
               
                 CAPLCGHRVPQC 
                 CNDVTKLCSQFC 
                 CQPTPTCGWTC 
                 CTLEICRSQLGC 
               
               
                 CARACQFGAC 
                 CNLSCTSQTLEC 
                 CQQFGQCSQFSC 
                 CTLNCNSGFQRC 
               
               
                 CDHQCGDHLC 
                 CNLSPCLLPPQC 
                 CQQRYSCFTNC 
                 CTLTQCSLSKAC 
               
               
                 CDQELCRELTSC 
                 CNPPHICQNPKC 
                 CQQYNCVPVGRC 
                 CTPLCTPQHVC 
               
               
                 CDQGDCHQKINC 
                 CNQRPPFCLVRC 
                 CQSFSCGQRLSC 
                 CTQLCTASPFSC 
               
               
                 CDVPCVAQYIC 
                 CPAVLSCTAEQC 
                 CQSLECAMRAHC 
                 CTQQCPSSVC 
               
               
                 CEKKYCTQQLPC 
                 CPDQCQFSSC 
                 CQSPSLCMGLPC 
                 CTQVPCTPYQGC 
               
               
                 CEMLQSCQQDWC 
                 CPDTCQAAFFLC 
                 CQSPWCQRLDLC 
                 CTRDCPSQAHC 
               
               
                 CETRGCYQRFRC 
                 CPESCLDLQWC 
                 CQSQDHCFHKDC 
                 CTRVCSSSQLYC 
               
               
                 CFKQNCSQSRSC 
                 CPMALCSQGATC 
                 CQTDVCQRTIC 
                 CTSSLCQLSVLC 
               
               
                 CFQLCPSVDFC 
                 CPPQRRCTAFAC 
                 CQYNDCDMLHC 
                 CTTSCVKSSIC 
               
               
                 CGAQGCFGVQSC 
                 CPQLSCPSGGSC 
                 CRAACNPFIC 
                 CTVPTCSQSLRC 
               
               
                 CGGGICRTHNC 
                 CPQPQPCLRTSC 
                 CREVTCHHLQC 
                 CVGQEPCLSYTC 
               
               
                 CGQVCNQKVC 
                 CPQSSCQGLRLC 
                 CRPQECAQHVC 
                 CVKSCGQSVC 
               
               
                 CHRQLCSPSEC 
                 CPTMTVCQHPRC 
                 CRQAYCSNLLLC 
                 CVNSCSSLKC 
               
               
                 CHRTPCSLPTTC 
                 CPTSACMQQSGC 
                 CRSETCAYQDC 
                 CVNSLCTLPSQC 
               
               
                 CIARDCWQGFSC 
                 CPVPQCDPKKLC 
                 CRSTPCQNQLEC 
                 CVPSQWCYAQRC 
               
               
                 CIEQPACPNIFC 
                 CQAGVICLQQVC 
                 CSHQCRSSELLC 
                 CVPTCSRSGC 
               
               
                 CIPIKRCNDQLC 
                 CQAVCQLGPC 
                 CSKQHTCVSPVC 
                 CVQLTCEYLYAC 
               
               
                 CISKCTSVAQSC 
                 CQDGQCPRNC 
                 CSLITQCGGVGC 
                 CVWQGCALNWRC 
               
               
                 CISLQQLCIRAC 
                 CQDLCGQMVC 
                 CSMGMCALPWQC 
                 CVYTSCVQSLTC 
               
               
                 CISRVGCQNPMC 
                 CQFHIGCYSNC 
                 CSNICLAKPHC 
                 CWEQACSQEC 
               
               
                 CLPSCQHAEIC 
                 CQHPCKSTVPNC 
                 CSQSNCVKAC 
                 CWRSCPKGYC 
               
               
                 CLRESACSKQC 
                 CQISHCQNMIIC 
                 CSSHVICNSNSC 
                 CYAQRCGVTGC 
               
               
                 CLRNCDYVQPPC 
                 CQLINLCHDFLC 
                 CSSREQCMITVC 
                 CYESCRVQSALC 
               
               
                 CLSQFCVIDC 
                 CQLISCTGGLQC 
                 CSTLDRCYQLC 
                 CYHVRPCSSQLC 
               
               
                 CLSSGCSAQDLC 
                 CQLLCVQSSSEC 
                 CSWYKCFNQPSC 
                 CYMPCGQSVVC 
               
               
                 CYQSPCPSGLC 
                 CYNQRSSCAMSC 
                 CYSGCGNLQGC 
               
               
                   
               
            
           
         
       
     
     The present invention provides methods of treating bacterial infections, and related disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated. 
     The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). 
     As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. 
     As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. 
     The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which bacterial infections may be implicated. 
     In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with a bacterial infection, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject&#39;s disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment. 
     Therapeutics 
     Peptide antibiotics of the invention are useful for the treatment of bacterial infections (e.g., infections associated with  Aerobacter, Aeromonas, Acinetobacter, Agrobacterium, Bacillus, Bacteroides, Bartonella, Bortella, Brucella, Calymmatobacterium, Campylobacter, Citrobacter, Clostridium, Cornyebacterium, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Hafnia, Helicobacter, Klebsiella, Legionella, Listeria, Morganella, Moraxella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Treponema, Xanthomonas, Vibrio , or  Yersinia ). In some embodiments the infection is a  Staphylococcus  or  Enterococcus  infection. In other embodiments, the infection is a  B. subtilis, E. coli, E. faecalis  or  S. aureus  infection. In other embodiments, the infection is methicillin-resistant  S. aureus  (MRSA), low level vancomycin resistant  E. faecium , or high level vancomycin resistant  E. faecium.    
     For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Treatment may be accomplished directly, e.g., by treating the subject with peptides of the invention. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections which provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of an anti-pathogenic agent in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington&#39;s Pharmaceutical Sciences by E. W. Martin. The amount of the agent (e.g., a peptide antibiotic) to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the type of disease and extensiveness of the disease. Generally, amounts will be in the range of those used for other agents used in the treatment of other bacterial diseases, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that inhibits bacterial proliferation. For example, for systemic administration a compound is administered typically in the range of 0.1 ng-10 g/kg body weight. 
     The administration of a peptide for the treatment of an infection may be by any suitable means that results in a concentration of the therapeutic peptide that, combined with other components, is effective in ameliorating, reducing, or stabilizing a bacterial infection. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams &amp; Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). 
     Pharmaceutical compositions according to the invention may be formulated to release the active peptide substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a bacterial infection. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level. 
     Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes. 
     The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. 
     EXAMPLES 
     Phage display has proven to be a useful tool for identifying antimicrobial peptides by screening molecular targets, such as replication/cell division enzymes and host-pathogen virulence factors, or whole bacterial cells. 13-14  Several reports detail the use of phage display for the isolation of peptides with antibacterial activity against different Gram-positive and Gram-negative bacteria. 15-18  Mirror image phage display as a concept was pioneered by Kim and co-workers to select ‘D-peptides’ from screening experiments. 19  Briefly, phage display peptide libraries are panned against the D-enantiomer of the target of interest. This results in the selection of L-peptide ligands that bind specifically to the D-target. By symmetry arguments, if the antipode of the ligand (D-peptide) is chemically synthesized, it will bind specifically to the native L-target with the same affinity. This technology has been successfully used to discover D-peptide inhibitors of HIV entry into host cells, the p53-MDM2 complex, VEGF, stabilization of amyloid beta (Aβ) peptide as well as D-peptide ligands for the cell-surface carbohydrates. 20-24  Identifying D-peptide ligands for several different target molecules using mirror image phage display has gained popularity in the recent past. Martin and co-workers recently reported the identification of lipid II binding lipopeptide ligands with D-amino acids against vancomycin-resistant bacteria. 25    
     We describe here the discovery and characterization of peptide antibiotics composed of D-amino acids using mirror image phage display that are effective against  S. aureus , MRSA and VRE that display high proteolytic and metabolic stability, and exhibit little to no toxicity. We accomplished this goal by screening 7-12 residue peptides (linear, cyclic and bicyclic) against enantiomers of (i) a derivative of the β-lactam antibiotic cephalosporin that mimics a high energy conformation of the D-Ala-D-Ala terminus of peptidoglycan structures in bacterial cell walls; and (ii) the cell wall crosslinking pentapeptide precursor. The new peptide scaffolds described here and variants thereof are useful as antibiotics. 
     Example 1: Design and Synthesis of Target Molecules for Phage Display Screening 
     Vancomycin, the bedrock of antibiotics in the ICU, targets the final step of bacterial cell wall biosynthesis, and inhibits the transpeptidase crosslinking reaction by binding to the D-alanyl-D-alanine terminal end of the pentapeptide unit of the un-crosslinked peptidoglycan (PG). The vancomycin:D-Ala-D-Ala complex is held together by five hydrogen bonds with a reasonably tight binding affinity (K d ˜1 μM). 26  It is important that target molecules used for phage display screening maintain a desired conformation during affinity selection. Tipper and Strominger proposed that penicillin, a β-lactam antibiotic, mimics a high energy conformation of D-Ala-D-Ala, and thereby inhibits the cross-linking of bacterial cell walls by irreversibly binding the active site of the peptidoglycan transpeptidase enzyme ( FIGS. 1 and 26 ). 27  We therefore hypothesized that using a β-lactam might enable selection of high-affinity binders. The enantiomer of cephalosporin was incorporated in the construct Cep-1 as a structurally rigid mimic of L-Ala-L-Ala ( FIG. 9 ) using a slightly modified version of a previously reported synthetic approach starting from 6-aminopenicillanic acid. 28-29  In addition, we also incorporated the enantiomer of the pentapeptide precursor of the bacterial cell wall (D-Ala-L-γ-Glu-D-Lys-L-Ala-L-Ala) into a short linear peptide target (Ala-2) which was chemically assembled using solid-phase peptide synthesis (as shown below) using t-Boc chemistry. Both target molecules were coupled to a polyethyleneglycol (PEG) linker, and a biotin moiety was attached to the N-terminus as a handle for immobilization on streptavidin beads during affinity selections. It is known that there is no significant difference in binding of vancomycin to UDP-MurNAc-L-Ala-D-γ-Glu-L-Lys-D-Ala-D-Ala versus to the pentapeptide precursor alone lacking the glycan. 30-31  The carbohydrate backbone that harbors the pentapeptide precursors during biosynthesis was therefore omitted from the target molecules. Another short peptide, incorporating α-Glu instead of γ-Glu in the peptide backbone (Ala-3) (shown below) was also prepared to interrogate how an extended backbone compound alters affinity selections. 
     Example 2: Panning Against Vancomycin-Sensitive Bacteria 
     Phage display allowed us to access diverse peptide libraries composed of up to ˜10 9  members ( FIG. 30 ). Commercially available linear and cyclic phage display (Ph.D.) libraries, Ph.D.-12, Ph.D.-7 and Ph.D.-C7C, that display peptides with randomized residues fused to the pIII minor coat protein of M13 phage, were used in initial screenings. Affinity selections were performed using two different strategies, a high yield selection and a more stringent one. This results in identification of tight binders, as well as elevating the chance of finding molecules with moderate binding. In the stringent selections, 25 ng of the target molecules in each round were subjected to four rounds of biopanning, whereas 5 μg of the target molecules was used in the first round, and then decreased to 2 μg, 500 ng, and 100 ng in the successive rounds of high yield selections. After four subsequent rounds, 50 phage clones were randomly picked from each screening for DNA sequencing to identify the peptide sequences. These sequences revealed the short hydrophobic motifs, “TTL”, “I/L-S/T”, “Q/N-S/T”, “LQ”, “G-S/T/V”, “G-x-S/T”, “L-K/R”, “R-L/V”, “VLS”, “N/Q-K/R; and several consensus sequences were found. Four peptides with identical sequences were enriched in independent affinity selections using Cep-1 and Ala-2 in spite of their structural differences ( FIG. 2 ), and slight differences in their binding affinities were observed as judged by phage-ELISA. Selection of identical sequences from these panning experiments is consistent with specific binding interactions between L-Ala-L-Ala and both target molecules Cep-1 and Ala-2. Additionally, peptides selected from screenings using Ala-2 and Ala-3 resulted in similar sequences albeit with different binding affinities in phage-ELISA ( FIG. 27 ). Negative selections as controls were also included, and enrichment factors suggest that the selection was successful in favor of target-specific phage and not of background. While iterative affinity selections yielded several binders for each target molecule, the same short motifs were conserved in the case of both Ala-2 and Cep-1 further confirming that the screenings resulted in identification of peptide ligands that specifically bind to the target molecules. To determine candidates for further investigation, we used the results of a phage-ELISA assay for each phage clone, and peptides were chosen based on the amplitude of the ELISA signals as well as frequency of appearance. These select sequences were synthesized in D-enantiomeric form by solid-phase peptide synthesis (SPPS). 
     Example 3: Bicyclic Peptide Ligands 
     We also explored more constrained peptide ligands by using phage display employing bicyclic peptide libraries as described by Heinis and Winter ( FIG. 3 ). 32  These libraries are expressed as linear peptides containing three cysteine residues on the pIII minor coat protein of phage M13, and are later reacted with 1,3,5-tris(bromomethyl)benzene (TBMB) to obtain bicyclic scaffolds. Bicyclic peptides mimic the complementarity-determining regions of antibodies and are able to bind to their targets tighter than their linear or mono-cyclic counterparts. 33-37  Bicyclic peptide libraries maintain diversity of members by incorporation of different loop lengths in the phage pool. Affinity selections were carried out using two bicyclic peptide libraries (libraries A and B) using the high yield strategy where 5 μg of target was used in the first round, and decreased to 2 μg in the second round. Phage titers (Tables 7-9) from the second round of selection were three orders of magnitude higher in number than the negative selection indicating that specific binders were present in the phage pool. Short peptide motifs similar to those obtained from commercial phage display libraries were observed from both target molecules, Cep-1 and Ala-2, namely “TTS”, “Q-R/K/L”, “SQL”, “VSQL”, “GQV”, “SV”, “SSV”, “T/P-Q”, “YQ-S/L”, “QRL” in addition to repeating bicyclic peptide sequences. 
     Example 4: Antimicrobial Activity of D-Peptides 
     The L-peptides identified from five different phage display libraries were catalogued as described previously and selected sequences were synthesized in D-enantiomeric form by SPPS for further investigation in several biological assays. Peptides were assembled as C-terminal amides to mimic peptides displayed on the coat protein of phage particles. Antibiotic susceptibility of several Gram-positive strains (with one Gram-negative strain as control) was tested in presence of the synthetic D-peptides, and assessed according to a broth micro-dilution protocol described by National Committee for Clinical Laboratory Standards (NCCLS) with slight modifications specific for peptide antibiotics as described by Hancock. 38  Each test was conducted in triplicate with vancomycin and melittin serving as control peptides. Table 5 only lists the antibacterial activity of constructs having a minimal inhibitory concentration (MIC) value of &lt;256 g/ml among the 170 D-peptides that were identified through screening of five different phage display libraries. Some of the D-peptides (linear 12-mer, cyclic 7-mer, and bicyclic) showed higher activity with MIC values in the 8 to 32 μg/ml range, while others had moderate activity with MIC values in the 64 to 128 μg/ml regime, and several showed no antibacterial activity (MIC value of &gt;256 μg/ml). Positive antibiotic action was only observed against Gram-positive strains as expected (Table 5). The most active D-peptide, a bicyclic construct P14, displayed promising antibacterial action against  S. aureus  and MRSA with MIC values of 8 μg/ml and 32 μg/ml, respectively. In addition, it was also active against two vancomycin-resistant Enterococci strains, vanB (MIC=32 μg/ml) and vanA (MIC=128 m/ml); in contrast to vancomycin that has no activity against vanA. Another potent bicyclic structure P15 showed similarly high activity as P14 for vancomycin-sensitive bacterial strains, while it had moderate effect on the low-level vancomycin-resistant strain and no activity on the high-level one. Other phage display derived D-peptides showed MICs ranging from 8 μg/ml to 128 μg/ml against  S. aureus  and MRSA but not for vancomycin-resistant strains. The phage-ELISA results of selected active peptides are shown in  FIG. 4 a    compared to a control peptide that contains the streptavidin specific binding motif “HPQ”. None of the four identical sequences identified from both Cep-1 and Ala-2 screens showed antibacterial activity. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Antibiotic activity of D-peptides against vancomycin-sensitive bacteria 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 MIC (μg/ml) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 ID 
                 Lib 
                 Sequence 
                 
                   E. 
                   coli 
                   a 
                 
                 
                   B. 
                   subtilis 
                   b 
                 
                 
                   S. 
                   aureus 
                   c 
                 
                 MRSA d   
                 
                   E. 
                   faecalis 
                   e 
                 
                 VRE f   
                 VRE g   
               
               
                   
               
               
                 P1 
                   
                 Vancomycin 
                 &gt;256 
                  0.5 
                  0.5 
                    0.5 
                    1 
                    32 
                 &gt;256 
               
               
                 P2 
                   
                 Melettin 
                    32 
                  2   
                   2   
                     4   
                     8 
                    32 
                    32 
               
               
                 P3 
                 L12 
                 VGSNLRLLHQWK 
                 &gt;256 
                  32   
                  32   
                    64   
                    32 
                 &gt;256 
                 &gt;256 
               
               
                 P4 
                 L12 
                 VHWDFRQWWQPS 
                 &gt;256 
                  32   
                  32   
                    64   
                    32 
                   128 
                 &gt;256 
               
               
                 P5 
                 L7 
                 KCCYTLP 
                 &gt;256 
                 128   
                 128   
                 &gt;256   
                 &gt;256 
                 &gt;256 
                 &gt;256 
               
               
                 P6 
                 L7 
                 VLTRCCG 
                 &gt;256 
                 128   
                 128   
                 &gt;256   
                 &gt;256 
                 &gt;256 
                 &gt;256 
               
               
                 P7 
                 L7 
                 TGFLVNV 
                 &gt;256 
                  32   
                  64   
                 &gt;256   
                 &gt;256 
                 &gt;256 
                 &gt;256 
               
               
                 P8 
                 L7 
                 HYIDFRW 
                 &gt;256 
                  32   
                  62   
                    64   
                    64 
                 &gt;256 
                 &gt;256 
               
               
                 P9 
                 C7C 
                 CLKLGEKWC 
                 &gt;256 
                  32   
                  64   
                    64   
                    64 
                 &gt;256 
                 &gt;256 
               
               
                 P10 
                 C7C 
                 CRGATPMSC 
                 &gt;256 
                 128   
                 128   
                   128   
                   256 
                 &gt;256 
                 &gt;256 
               
               
                 P11 
                 C7C 
                 CSISSLTHC 
                 &gt;256 
                  32   
                  32   
                    64   
                    32 
                    64 
                 &gt;256 
               
               
                 P12 
                 C7C 
                 CLWSTGATC 
                 &gt;256 
                  64   
                  64   
                    64   
                   128 
                 &gt;256 
                 &gt;256 
               
               
                 P13 
                 C7C 
                 CDNIMTPVC 
                 &gt;256 
                  64   
                  64   
                    64   
                   128 
                 &gt;256 
                 &gt;256 
               
               
                 P14 
                 BC-A 
                 CQTDVCQRTIC 
                 &gt;256 
                  8   
                  8   
                    32   
                    32 
                    32 
                   128 
               
               
                 P15 
                 BC-B 
                 CSLITQCGGVGC 
                 &gt;256 
                  8   
                  8   
                    32   
                    16 
                   128 
                 &gt;256 
               
               
                   
               
               
                 Lib = phage display peptide library 
               
               
                 MIC = minimum inhibitory concentration 
               
               
                   a   E.   coli  ATCC 25992 
               
               
                   b   B.   subtilis  ATCC 6633 
               
               
                   c   S.   aureus  ATCC 6538 
               
               
                   d Methicillin-resistant  S.   aureus  (MRSA) ATCC 43300 
               
               
                   e   E.   faecalis  ATCC 29121 
               
               
                   f low level vancomycin resistant  E.   faecium  (vanB) ATCC 51299 
               
               
                   g high level vancomycin resistant  E.   faecium  (vanA) ATCC 51559 
               
               
                 The bactericidal activity of an antimicrobial agent against a particular organism is related to its mechanism of action. 39  Briefly, agents that disrupt the cell wall or cell membrane, or interfere with essential bacterial enzymes, are likely to be bactericidal, whereas those agents that inhibit ribosome function and protein synthesis tend to be bacteriostatic. The bacteria killing kinetics of P14 and P15 was interrogated by a “time-kill” assay and compared to vancomycin to assess the mode of action. These “time-kill” assays showed time-dependent reductions in the number of colony-forming units per mL (cfu/ml) by P14 and P15. Completely killing of bacterial cells was observed in 24 h, similar to vancomycin. While both macrocyclic peptides exhibited similar killing kinetics, peptide P14 exhibited a modestly faster rate than vancomycin against  S.   aureus  (Fig. 4b). 
               
            
           
         
       
     
     Example 5: Mechanism of Action of Potent Bicyclic Peptides 
     We next examined the effect of P14 and P15 on cell wall biosynthesis in live bacteria. Blockade of this biosynthetic pathway causes accumulation of the water soluble intracellular intermediate lipid II precursor UDP-MurNAc-L-Ala-D-γ-Glu-L-Lys-D-Ala-D-Ala. 40  This intermediate is detectable after extraction from the cytosol in response to antibiotic administration by chromatographic methods. 
     Incubation of  S. aureus  with P14 and P15 at 10-fold MIC led to accumulation of UDP-MurNAc-pentapeptide at levels similar to vancomycin as has been previously reported ( FIGS. 4 c  and 4 d   ). This result suggests that the action mechanism of the two bicyclic constructs is similar to vancomycin and is the outcome of cell wall biosynthesis inhibition. 
     Example 6: Phage Selection Against Vancomycin-Resistant Strains 
     Since the first report of vancomycin-resistant  Staphylococcus aureus  (VRSA) in the US in 2002, the threat of vancomycin being rendered ineffective has become real. More concerning is the possibility that vancomycin resistance may spread to multi-drug resistant pathogenic bacteria, such as methicillin resistant  S. aureus  (MRSA). Bacteria exhibiting vancomycin resistance have a modified cell wall precursor pentapeptide, where some of the terminal D-alanine residues are substituted with D-lactate. This substitution results in an estimated 1000-fold reduced binding affinity for vancomycin due to the loss of one hydrogen bond and introduction of a repulsive interaction. 41  Several approaches have been tried to overcome vancomycin-resistance including modification of vancomycin itself, utilizing combinatorial libraries to identify binders of the D-Ala-D-Lac precursor, and the design of vancomycin-inspired semi-synthetic glycopeptide derivatives and increasing affinity by use of dimeric vancomycin. 42-60    
     We synthesized the enantiomer of the pentapeptide precursor of vancomycin-resistant bacterial cell walls (D-Ala-L-γ-Glu-D-Lys-L-Ala-L-Lac) with a PEG linker and a biotin group to use as the target molecule in phage screening (Lac-4, as shown below). Both commercial phage display and bicyclic libraries were used in the selections. All commercial phage libraries yielded sequences with short hydrophobic peptide motifs, “TTL”, “I/L-S/T”, “Q/N-S/T”, “LQ”, “GQS”, “G-S/V”, “G-X-S”, “L-K/R”, “RV”, “VLS”, “Q/N-K/R” similar to previous screenings with target molecules Cep-1 and Ala-2. Target molecules Ala-2 and Lac-4 resulted in selection of several identical peptide sequences with different binding affinities by phage-ELISA ( FIG. 5 ). Since short pentapeptide precursors for vancomycin-sensitive and resistant strains differ only in a single residue (Ala to Lac), selection of similar peptide sequences was to be expected. It also suggests that these peptides might have binding interactions with entire pentapeptide precursor rather than solely with the D-Ala-D-Ala terminal end. 
     In addition, screening of Lac-4 against bicyclic peptide libraries resulted in short hydrophobic peptide motifs, “GQV”, “QL”, “SSV”, “T/P-Q”, “YQ-S/L”, “QRL” as previously observed in Cep-1 and Ala-2. These observations are in the agreement with the findings of Liskamp and co-workers where ligands binding to D-Ala-D-Lac had a significant number of polar amino acids, mostly glutamine and serine residues. 44  None of the D-peptides identified from screenings of five different peptide libraries showed significantly high antibacterial activity for vancomycin-resistant bacteria, whereas one D-peptide, P18, had moderate activity for the low-level vancomycin-resistant bacterium (Table 6). On the other hand, six D-peptides had moderate activity for  S. aureus , MRSA, and  E. faecalis  strains with MIC values ranging from 32 to 128 μg/ml. 
     
       
         
           
               
             
               
                 TABLE6  
               
             
            
               
                   
               
               
                 Antibiotic activity of D-peptides against  
               
               
                 vancomycin-resistant bacteria 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 MIC(μg/ml) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 
                   E. 
                 
                 
                   B. 
                 
                 
                   S. 
                 
                   
                 
                   E. 
                 
                   
                   
               
               
                 ID 
                 Lib 
                 Sequence 
                 
                   coli 
                   a 
                 
                 
                   subtilis 
                   b 
                 
                 
                   aureus 
                   c 
                 
                 MRSA d   
                 
                   faecalis 
                   e 
                 
                 VRE f   
                 VRE g   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 P1 
                   
                 Vancomycin 
                 &gt;256 
                 0.5 
                 0.5 
                 0.5 
                 1 
                 32 
                 &gt;256 
               
               
                   
               
               
                 P2 
                   
                 Melittin 
                 32 
                 2 
                 2 
                 4 
                 8 
                 32 
                 32 
               
               
                   
               
               
                 P8 
                 L7 
                 HYIDFRW 
                 &gt;256 
                 32 
                 32 
                 64 
                 64 
                 &gt;256 
                 &gt;256 
               
               
                   
               
               
                 P9 
                 C70 
                 CLKLGEKWC 
                 &gt;256 
                 32 
                 64 
                 64 
                 64 
                 &gt;256 
                 &gt;256 
               
               
                   
               
               
                 P10 
                 C7C 
                 CRGATPMSC 
                 &gt;256 
                 128 
                 128 
                 128 
                 256 
                 &gt;256 
                 &gt;256 
               
               
                   
               
               
                 P16 
                 BC-A 
                 COYNDCDMLHC 
                 &gt;256 
                 64 
                 64 
                 128 
                 128 
                 &gt;256 
                 &gt;256 
               
               
                   
               
               
                 P17 
                 BC-A 
                 CLSQFCVIDC 
                 &gt;256 
                 128 
                 128 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
               
               
                   
               
               
                 P18 
                 BC-A 
                 CGGGICRTHNC 
                 &gt;256 
                 16 
                 16 
                 32 
                 128 
                 128 
                 &gt;256 
               
               
                   
               
               
                 P19 
                 BC-B 
                 CTRVCSSSQLYC 
                 &gt;256 
                 128 
                 128 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
               
               
                   
               
               
                 P20 
                 BC-B 
                 CGAQGCFGVQSC 
                 &gt;256 
                 32 
                 32 
                 256 
                 256 
                 256 
                 &gt;256 
               
               
                   
               
               
                 Lib = phage display peptide library 
               
               
                 MIC= minimum inhibitory concentration 
               
               
                   a   E. Coli  ATCC25992 
               
               
                   b   B. subtilis  ATCC6633 
               
               
                   c   S. aureus  ATCC6538 
               
               
                   d Methicillin-resistant  S. aureus  (MRSA) ATCC43300 
               
               
                   e   E. faecali s ATCC29121 
               
               
                   f low level vancomycin resistant  E. faecium  (vanB) ATCC51299 
               
               
                 ghigh level vancomycin resistant  E. faecium  (vanA) ATCC51559 
               
            
           
         
       
     
     Example 7: D-Peptides Discovered in this Study are not Toxic 
     Toxicity is a major problem in the development of new antibacterials that target the bacterial cell wall. 61  The lytic activity of D-peptides on human red blood cells (hRBC) was evaluated by a hemolysis assay along with melittin and vancomycin as positive and negative controls respectively. Only minimal hemolytic activity (less than 10%) was observed with D-peptides even at high concentrations ( FIGS. 6 a , 6 b   , and  28 ) as compared to the natural antimicrobial peptide, melittin. The nontoxic nature of the two most active D-peptides, P14 and P15, was also confirmed on mammalian (HeLa) cells as judged by using a colorimetric MTT assay. While many natural and designed antimicrobial peptides are cytotoxic for mammalian cells, the bicyclic D-peptides described here did not exhibit any toxicity on HeLa cells even at high concentrations and were comparable to observations with vancomycin ( FIG. 6 c   ). To catalog relative activities of D-peptides against bacteria and erythrocytes, therapeutic index (TI) values were calculated. Higher TI numbers indicate that the select D-peptides were more active against bacteria relative to their cytotoxic action on erythrocytes ( FIG. 7 ). 
     Example 8: Stability of Potent D-Peptides 
     Peptides suffer from low metabolic stability as they are rapidly degraded by hydrolytic action of proteases and therefore are typically not orally bioavailable. Our approach to this problem obviates the use of L-amino acids, with the expectation that D-peptides would be refractory to protease action. We tested stability of three potent bicyclic peptides in human serum and pancreatin (simulated intestinal fluid containing digestive enzymes) by incubating 1 mg/ml of peptides separately in human blood and pancreatin solution for 24 h. RP-HPLC was employed to monitor the level of each active D-peptide remaining at various time points ( FIG. 29 ). None of these peptides were digested even after a 24 h incubation. Pancreatin is a proteolytic mixture of digestive enzymes including amylases, lipases, and proteases (e.g. trypsin); we reasoned that degradation resistance to this protease cocktail would strengthen our working hypothesis that results in more stable peptide therapeutics. D-amino acid oxidase is the only known mammalian enzyme that metabolizes D-peptides in the kidney. 62  Despite the refractory nature of our constructs towards degradation by pancreatin, detailed pharmacological studies will be required to determine whether or not gastrointestinal administration in preclinical models or ultimately, oral use in patients is a viable possibility. Bicyclic peptide scaffolds composed of D-amino acids proffer orally available and metabolically stable molecules that can be used as antibiotics. For every class of antibiotic introduced into human clinical use, it has been found that resistant organisms are selected for, and eventually become abundant in human infections. The need therefore remains to create platforms that can effectively generate new lead compounds that can then be subjected to traditional medicinal chemistry approaches to yield molecular entities that are effective as therapeutics in practice.  S. aureus  is especially notorious for its ability to develop resistance to antibiotics, and therefore novel strategies are urgently needed to tackle this global public health threat. Here, we report the discovery of peptide antibiotics composed of D-amino acids active against  S. aureus , Methicillin-resistant  S. aureus  and vancomycin-resistant Enterococci with excellent antibacterial activity with MICs in the range of 8 μg/ml to 32 μg/ml. In addition, these compounds show minimal to no toxicity to both mammalian and red blood cells. Short peptides with high metabolic stability that target bacterial cell wall precursors with higher or equal affinities than vancomycin in drug sensitive and resistant bacterial strains are exciting and promising platforms for the development of new therapeutics. Initially, we investigated peptide antibiotics active against vancomycin-sensitive bacteria using five different peptide libraries (linear, monocyclic and bicyclic). Target design for affinity selections of vancomycin-sensitive bacteria was based on the enantiomer of the pentapeptide precursor of bacterial cell wall of  S. aureus ; a conformationally locked construct, Cep-1, and a flexible structure construct, Ala-2. Biopanning of peptide libraries resulted in identification more than a hundred L-peptide ligands that specifically bind to target molecules with different affinities as judged by phage-ELISA experiments compared to streptavidin binding peptide containing an “HPQ” motif. The peptides identified from these screens (7-12 mer peptides) shared short hydrophobic motifs that were similar. Peptides were then synthesized in D-enantiomeric form by SPPS and evaluated for antibacterial activities against several strains. The most potent peptides identified (P14 and P15) during affinity selections against vancomycin-sensitive bacteria using Cep-1 and Ala-2 as targets, were from phage display of bicyclic libraries. Bicyclic peptide P14 showed high antibacterial activity not only against  S. aureus , MRSA and Enterococci but also against two vancomycin-resistant Enterococci strains vanB and vanA. The other bicyclic peptide P15 had similar inhibitory activity on vancomycin-sensitive bacteria whereas it was less active against vancomycin-resistant strains. The minimum inhibitory concentrations (MIC) values of these un-optimized antibacterials from target-based screenings are in the 16-32 μg/ml have been deemed sufficient for primary hit compounds. 63  We envision that by modifications guided by computational efforts and by use of noncanonical side chains in the scaffolds, the activities and pharmacological properties can be rapidly improved. 64    
     We subsequently employed this strategy to target vancomycin-resistant bacteria by using Lac-4 as the bait molecule in phage display screenings. Among the identified peptides, bicyclic peptide P18 showed excellent activity against vancomycin-sensitive bacteria and moderate activity against the vancomycin-resistant strain, vanB. None of identified peptides displayed high activity against both vanB and vanA type vancomycin-resistant Enterococci. The three most potent bicyclic peptides, P14, P15 and P18, neither caused lysis of erythrocytes nor showed significant toxicity against mammalian cells (HeLa) at concentrations up to 256 μg/ml, suggesting that they are promising candidates for further elaboration. Since peptide antibiotics identified here composed of D-amino acids, all of them showed high stability in human serum and also in the presence of protease cocktail pancreatin. 
     Example 9: Materials and Methods 
     Synthesis of Construct Cep-1 Containing an Enantiomeric Cephalosporin Scaffold 
     Construct Cep-1 containing an enantiomeric cephalosporin scaffold was synthesized using the procedure outlined in  FIG. 9 . 
     Synthesis of 2S,5R,6R)-6-(1,3-dioxoisoindolin-2-yl)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic Acid (1) 
     
       
         
         
             
             
         
       
     
     To a vigorously stirred solution of 6-aminopenicillanic acid (3.00 g, 13.9 mmol) and Na 2 CO 3  (1.47 g, 13.9 mmol) in water (20 ml) was added N-carboethoxyphthalimide (3.05 g, 13.9 mmol). The mixture was stirred at room temperature for 2 h and then washed with CH 2 Cl 2  (3×5 ml). The aqueous layer was mixed with a fresh portion of CH 2 Cl 2  (20 ml) and acidified during vigorous stirring with 1 M HCl (40 ml). The phases were separated and the extraction was completed with two additional portions of CH 2 Cl 2  (2×5 ml). The combined organic extracts were washed with water (2×10 ml) and satd. brine (5 ml). The organic layer was dried over MgSO 4 , and evaporated in vacuo to afford compound 1 (2.30 g, 45%) as an off-white solid which was used in the next step without further purification.  1 H NMR (CDCl 3 , 500 MHz) δ 7.89 (dddd, 2H, J=8.5, 5.5, 3.0, 1.0 Hz), 7.77 (dddd, 2H, J=8.5, 5.5, 3.0, 1.0 Hz), 5.69 (dd, 1H, J=4.0, 1.0 Hz), 5.59 (dd, 1H, J=4.0, 1.0 Hz), 4.71 (s, 1H), 1.85 (s, 3H), 1.62 (s, 3H). 
     Synthesis of (2S,5R,6R)-benzyl-6-(1,3-dioxoisoindolin-2-yl)-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (2) 
     
       
         
         
             
             
         
       
     
     To a stirred solution of compound 1 (2.3 g, 76.64 mmol) in DMF (12 ml) was added triethylamine (0.93 ml, 6.64 mmol) followed by benzyl bromide (0.99 ml, 8.3 mmol). The mixture was stirred at room temperature for 6 h and then poured into vigorously stirred ice water (55 ml). The resulting suspension was extracted with CHCl 3  (3×25 ml), organic layers were combined and washed with satd. NaHCO 3  (3×15 ml), water (3×15 ml), and saturated brine (15 ml). The organic phase was dried over MgSO 4  and evaporated in vacuo to obtain the crude compound as light yellow oil. The compound was purified by flash chromatography on silica (60% hexanes/EtOAc) to afford compound 2 (1.87 g, 60%) as a white solid.  1 H NMR (CDCl 3 , 300 MHz) δ 7.88 (dd, 2H, J=5.4, 3.0 Hz), 7.77 (dd, 2H, J=5.4, 3.0 Hz), 7.42-7.37 (m, 5H), 5.68 (d, 1H, J=3.9 Hz), 5.59 (d, 1H, J=3.9 Hz), 4.70 (s, 1H), 4.51 (s, 2H), 1.80 (s, 3H), 1.44 (s, 3H). 
     Synthesis of 2S,5R,6S)-benzyl-6-(1,3-dioxoisoindolin-2-yl)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylate (3) 
     
       
         
         
             
             
         
       
     
     To a solution of ester 2 (1.87 g, 4.28 mmol) in CH 2 Cl 2  (16 ml) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.042 ml, 0.28 mmol) and the mixture was stirred at room temperature for 90 min. The solution was washed with 1 M NH 4 Cl (2×10 ml), water (3×10 ml), and satd. brine (5 ml). The organic phase was dried over MgSO 4  and evaporated in vacuo to afford a white foam. It was purified by flash chromatography on silica (60% hexanes/EtOAc) to afford compound 3 (1.87 g, 64%) as a white foam.  1 H NMR (CDCl 3 , 500 MHz) δ 7.90 (dd, 2H, J=5.5, 3.0 Hz), 7.77 (dd, 2H, J=5.5, 3.0 Hz), 7.41-7.34 (m, 5H), 5.57 (d, 1H, J=2.0 Hz), 5.40 (d, 1H, J=2.0 Hz), 5.22 (d, 2H, J=2.0 Hz), 4.66 (s, 1H), 1.64 (s, 3H), 1.43 (s, 3H). 
     (2S,5S,6S)-benzyl-6-(1,3-dioxoisoindolin-2-yl)-3,dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (5) 
     
       
         
         
             
             
         
       
     
     A solution of compound 3 (0.050 g, 0.114 mmol) in CCl 4  (2 ml) was treated with equimolar amount of sulfuryl chloride (0.114 mmol, 1 M solution in CCl 4 ) by stirring at 0° C. for 30 min. The solvents were evaporated in vacuo to give a mixture of the trans ds, cis ds (desired) and SM in a ratio of ca. 3:1:1.2 as a yellow foam. The foam was dissolved in THF (2 ml) and treated with anhydrous SnCl 2  (0.0167 g, 0.088 mmol). The mixture was stirred at room temperature for 2 h and then the solvent was evaporated in vacuo. The residual oil was dissolved in EtOAc (5 ml) and washed with water (3×5 ml) and satd. brine (5 ml). The organic phase was dried over MgSO 4  and evaporated in vacuo to give a white foam. It was purified by flash chromatography on SiO 2  (100% CH 2 Cl 2 -94% CH 2 Cl 2 /EtOAc) to afford compound 5 (0.005 g, 30% over two steps) as a white foam.  1 H NMR (CDCl 3 , 300 MHz) δ 7.88 (dd, 2H, J=5.4, 3.0 Hz), 7.75 (dd, 2H, J=5.4, 3.0 Hz), 7.48-7.34 (m, 5H), 5.62 (d, 1H, J=3.9 Hz), 5.31 (d, 2H, J=6.0 Hz), 5.26 (d, 2H, J=3.9 Hz), 4.02 (s, 1H), 1.70 (s, 3H), 1.69 (s, 3H). 
     Benzyl(2S,4S,5S,6S)-3,3-dimethyl-7-oxo-6-phthalimido-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4-oxide (6) 
     
       
         
         
             
             
         
       
     
     Compound 5 (0.115 g, 0.263 mmol) was dissolved in acetone (5.0 ml), cooled to −78° C., and treated with a stream of ozone. The solvent was evaporated in vacuo to give a white foam compound 6 (0.132 mg, quant. yield) that was used without purification.  1 H NMR (CDCl 3 , 500 MHz) δ 7.91 (dd, 2H, J=5.5, 3.0 Hz), 7.80 (dd, 2H, J=5.5, 3.0 Hz), 7.48-7.35 (m, 5H), 5.92 (d, 1H, J=4.8 Hz), 5.37 and 5.30 (ABq, 2×1 H, J=12.0 Hz), 4.56 (d, 1H, J=4.8 Hz), 4.30 (s, 1H), 1.61 (s, 3H), 1.55 (s, 3H). 
     (6S,7S)-benzyl-7-(1,3-dioxoisoindolin-2-yl)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (7) 
     
       
         
         
             
             
         
       
     
     A stirred solution of sulfoxide compound 6 (0.132 g, 0.292 mmol) and anhydrous p-toluenesulfonic acid (pTSA, 0.0068 g, 0.0394 mmol) in THF (15 ml) was heated at 100° C. for 2 h. The reaction mixture was cooled to room temperature, diluted with EtOAc (5 ml) and washed with water (4×5 ml), satd. NaHCO 3  (3×5 ml), and satd. brine (2 ml). The organic layer was dried over MgSO 4  and evaporated in vacuo to yield a brown foam which was subjected to purification using flash chromatography (100% CH 2 Cl 2  to 98% CH 2 Cl 2 /EtOAc) to afford compound 7 (0.059 g, 52%) as a white foam.  1 H NMR (CDCl 3 , 300 MHz) δ 7.89 (dd, 2H, J=5.5, 3.0 Hz), 7.78 (dd, 2H, J=5.5, 3.0 Hz), 7.46-7.32 (m, 5H), 5.73 (d, 1H, J=4.3 Hz), 5.31-5.21 (m, 2H), 5.11 (d, 1H, J=4.3 Hz), 3.73 and 3.01 (q, 2×1 H, J=15.5 Hz), 2.33 (s, 3H). 
     (6S,7S)-benzyl-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylate (8) 
     
       
         
         
             
             
         
       
     
     To a solution of compound 7 (0.0478 g, 0.110 mmol) at −78° C. in THF (2.0 ml) was added 1 M N 2 H 4  solution in THF (0.220 ml, 0.220 mmol). The reaction mixture stirred at for 30 min without cold bath. The phthalhydrazide complex was decomposed by addition of 1 M HCl (0.28 ml) and the solvents were evaporated in vacuo. The residual oil was dissolved, with vigorous stirring, in water (2.0 ml) and the insoluble phthalhydrazide was removed by filtration. The aqueous filtrate was layered with EtOAc (2.0 ml) and basified with satd. NaHCO 3  to pH 8. The two phase mixture was separated, and extracted with EtOAc (2×2 ml). The combined organic extracts were washed with water (3×2 ml) and satd. brine (1 ml), dried over MgSO 4 , and evaporated in vacuo to give a thick yellow oil which was purified by flash chromatography (100% CH 2 Cl 2  to 70% CH 2 Cl 2 /EtOAc) to afford compound 8 (0.021 g, 40%) as a white foam.  1 H NMR (CDCl 3 , 500 MHz) δ 7.43-7.30 (m, 5H), 5.27 (d, 2H, J=4.1 Hz), 4.91 (d, 1H, J=4.9 Hz), 4.70 (d, 1H, J=4.9 Hz), 3.52 and 3.21 (ABq, 2×1 H, J=18.4 Hz), 2.11 (s, 3H). 
     (6S,7S)-benzyl-7-(poly(ethylene glycol)ether-2-(biotinylamino)ethane)-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylate (9) 
     
       
         
         
             
             
         
       
     
     To a solution of compound 8 (0.007 g, 0.023 mmol) and NHS-PEG 12 biotin (0.025 g, 0.026 mmol) at 0° C. in DMF (0.5 ml) was added DIPEA (12 ml, 0.069 mmol). The reaction mixture was stirred at 0° C. for 24 h and the solvents were evaporated in vacuo to give a thick yellow oil which was purified four times by RP-HPLC (rt=16.5 min, C18 semi-prep column, 30-40% B; A=98% H 2 O/Acetonitrile; B=99% Acetonitrile/H 2 O) to afford compound 9 as a white solid.  1 H NMR (CDCl 3 , 500 MHz) δ 7.43-7.35 (m, 5H), 6.72 (bs, 1H), 5.81 (q, 1H, J=4.5 Hz), 5.28 (dd, 2H, J=20.0, 12.5 Hz), 4.98 (d, 1H, J=5.0 Hz), 4.55-4.50 (m, 1H), 4.32-4.36 (m, 1H), 3.77 (dd, 2H, J=11.5, 5.0 Hz), 3.68-3.65 (m, 47H), 3.59-3.57 (m, 2H), 3.50 and 3.25 (ABq, 2×1 H, J=18.5 Hz), 3.47-3.44 (m, 2H), 3.19-3.15 (m, 1H), 2.93 (dd, 1H, J=12.5, 5.0 Hz), 2.74 (d, 1H, J=12.5 Hz), 2.62-2.57 (m, 1H), 2.26-2.22 (m, 2H), 2.16 (s, 3H), 1.71-1.64 (m, 4H), 1.50-1.43 (m, 2H). 
     (6S,7S)-benzyl-7-(poly(ethylene glycol)ether-2-(biotinylamino)ethane)-3-methyl-8-oxo-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic Acid (10) (Cep-1) 
     
       
         
         
             
             
         
       
     
     To an ice-bath cooled solution of compound 9 (0.172 gr, 0.152 mmol) and anisole (198 μl, 1.82 mmol) in CH 2 Cl 2  (3 ml) was added a cold solution of AlCl 3  (0.122 gr, 0.91 mmol) in MeNO 2 . The ice-bath was removed, and the reaction mixture was stirred for 8 h in room temperature. The reaction mixture was diluted with EtOAc (9 ml), and washed with 1 M HCl (3×9 ml) and sat. brine (10 ml). The combined aqueous extracts were washed with Et 2 O (10 ml), acidified to pH 1 with 1 M HCl, and re-extracted with EtOAc (3×10 ml). The combined organic layers were washed with satd. brine (10 ml), dried over MgSO 4 , and evaporated in vacuo to give compound 10 (Cep-1) which was purified by RP-HPLC (rt=16.5 min, C18 semi-prep column, 20-40% B; A=98% H 2 O/Acetonitrile; B=99% Acetonitrile/H 2 O).  1 H NMR (D 2 O, 500 MHz) δ 5.81 (q, 1H, J=4.5 Hz), 5.49 (dd, 2H, J=20.0, 12.5 Hz), 4.98 (d, 1H, J=5.0 Hz), 4.51-4.448 (m, 1H), 4.29-4.33 (m, 1H), 3.77 (dd, 2H, J=11.5, 5.0 Hz), 3.62-3.58 (m, 47H), 3.59-3.57 (m, 2H), 3.50 and 3.25 (ABq, 2×1 H, J=18.5 Hz), 3.46-3.43 (m, 2H), 3.24-3.19 (m, 1H), 2.89 (dd, 1H, J=12.5, 5.0 Hz), 2.68 (d, 1H, J=12.5 Hz), 2.57-2.5 (m, 1H), 2.2-2.19 (m, 2H), 1.8 (s, 3H), 1.65-1.5 (m, 4H), 1.33-1.28 (m, 2H).  13 C NMR (D 2 O, 125 MHz) δ C  19.8, 25.2, 28.3, 28.5, 29, 34.6, 36.5, 36.7, 39.8, 40.1, 55.6, 56.5, 58.3, 60.5, 62.2, 66.4, 66.7, 69.4, 70.5, 70.8, 123.4, 126.7, 164.7, 165.1, 170.4, 175.6, 177.3 
     Synthesis of Enantiomeric Derivatives of Pentapeptide Cell Wall Precursor of  S. aureus  (Ala-2 and Ala-3) 
     
       
         
         
             
             
         
       
     
     Synthesis of the enantiomeric pentapeptide cell wall precursor (D-Ala-L-γ-Glu-D-Lys-L-Ala-L-Ala) from  S. aureus  (Ala-2) attached to a biotin moiety via linker is shown above. Enantiomer of pentapeptide precursor of  S. aureus  with α-Glu instead of γ-Glu harboring an extended backbone, Ala-3, was also synthesized as a control peptide in phage display selection experiments. 
     D-ala-L-γ-Glu-D-lys-L-Ala-L-Ala, and D-ala-L-α-Glu-D-lys-L-Ala-L-Ala were synthesized manually using the in-situ neutralization protocol for t-Boc (tert-Butoxycarbony) chemistry on 4-Hydroxymethyl-phenylacetamidomethyl (PAM) resin (0.2 mmol scale, 0.85 mmol/g, Chemimpex). Boc-protected amino acids (0.8 mmol) were coupled to resin in DMF with 0-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) (0.72 mmol) and N,N, diisopropylethyl amine (DIEA) (1.2 mmol) for 15 min. N-Boc group were removed by treatment of neat trifluoroacetic acid (TFA) for 1 min twice. After coupling of last amino acid residue, Biotin-PEG4-NHS (2 eq.) linker was coupled to each pentapeptide overnight with DIEA (4 eq.). Peptides were cleaved from PAM-resin by treatment with HF/anisole (9:1 v/v) at 0° C. for 2 h, and then precipitated with cold diethyl ether. Lyophilized crude peptides were purified by reversed-phase HPLC on a C18 column using a linear gradient of increasing concentration of solvent B in solvent A (solvent A: water/acetonitrile/TFA, 98/2/0.1; solvent B: acetonitrile/water/TFA, 99/1/0.1). Purity of peptides were determined by analytical RP-HPLC on a C18 column (5 μm, 4×250 mm) using a linear gradient (5 to 20% solvent B in solvent A) over 30 min at a flow rate of 1 ml min-1. MALDI-TOF mass spectra was recorded using alpha-cyano 4-hydroxycinnaminic acid as matrix. Ala-2, calcd [M+H]+, 962.46, obsd, 962.121; Ala-3, calcd [M+H]+, 962.46, obsd, 962.121. 
     Synthesis of Enantiomer of Cell Wall Precursor of Vancomycin-resistant Enterococci (Lac-4) 
     
       
         
         
             
             
         
       
     
     Synthesis of the enantiomer of cell wall analogue of vancomycin-resistant Enterococci (VRE) is shown above. a Synthetic scheme of Lac-4 starting with coupling of L-Lac lithium salt on 2-chlorotrityl resin, and followed by solid-phase peptide synthesis by Fmoc-chemistry. b Structure of Lac-4 used in phage display screening. 
     D-Ala-L-γ-Glu-D-Lys-L-Ala-L-Lac, Lac-4, was synthesized manually by SPPS using standard Fmoc-chemistry (N-(9-fluorenyl)methoxycarbonyl) on 2-chlorotrityl chloride resin (0.2 mmol scale, 0.8 meq/g, ChemImpex). 1.5 mmol L-Lac lithium salt was coupled directly to 2-chlorotritiyl chloride resin in DMSO:DCM (1:1) overnight. Following day, the next residue Fmoc-L-Ala-OH (1.5 mmol) in DMF was coupled overnight to resin-complex by adding DIC (1.5 mmol) and catalytic amount of DMAP (0.08 mmol). The following amino acids were coupled using standard Fmoc-chemistry, where 0.8 mmol amino acid in DMF was activated by Azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) (0.72 mmol) and DIEA (1.2 mmol), and coupled to peptide-resin for 30 min. After coupling of last amino acid, Biotin-PEG 4 -NHS linker (2 eq.) was coupled to peptide-resin overnight. Biotinylated peptide was cleaved from resin by acidic cleavage cocktail containing TFA/Tri-isopropylsilane (TIPS)/H 2 O (95/2.5/2.5) for 3 hours. TFA was evaporated under flow of nitrogen, and the cleaved peptide was precipitated with ice-cold diethyl ether three times. Lyophilized crude peptide was purified by reversed-phase HPLC on a C18 column using a linear gradient of increasing concentration of solvent B in solvent A (solvent A: water/acetonitrile/TFA, 98/2/0.1; solvent B: acetonitrile/water/TFA, 99/1/0.1). Purity was determined by analytical RP-HPLC on a C18 column (5 μm, 4×250 mm) using a linear gradient (10 to 45% solvent B in solvent A) over 30 min at a flow rate of 1 ml min −1 . MALDI-TOF mass spectras were recorded using alpha-cyano 4-hydroxycinnaminic acid as matrix. Lac-4, calcd [M+H] + , 963.46, obsd, 963.11 
     Phage Selection with Linear and Cyclic Libraries. 
     Ph.D.-12, Ph.D.-7 and Ph.D.-C7C Phage Display (Ph.D.) peptide libraries were purchased from New England Biolabs (NEB #E8110S, #E8100S and #E8120S). In the first round of selection, target molecules were incubated with 100-fold diluted (10 11  pfu/ml) phage libraries in TBS buffer containing 0.1% Tween-20 for 30 min by agitation in room temperature. The phage/target molecule was then captured on streptavidin-coated magnetic beads (NEB #S1420S) by incubation for 30 min at room temperature, washed six times with TBS containing 0.1% Tween-20. The phage were then eluted by incubation with 0.2 M Glycine-HCl (pH 2.2) for 15 min. After neutralization with 1.0 M Tris-HCl (pH 9.1), phages were propagated by infection of  E. coli  ER2738 cells that were grown to an optical density of 0.5 at 600 nm (OD 600 ) by a 4.5 h incubation at 37° C. at 250 rpm. The infected cells were harvested by centrifugation, plated on Luria-Bertani/tetracycline plates, incubated at 37° C. overnight, and used to produce phage for the next round of selection. Three additional selection rounds were performed with higher concentration of Tween-20, ranging from 0.1% to 0.5% and by use of streptavidin and neutravidin-coated magnetic beads in alternating rounds. The more stringent selection was carried out using 25 ng of target molecule in each round while the high yield selection was performed with reduced amount of target molecule (5 mg, 2 mg, 500 ng, and 100 ng, respectively) in each round. Negative selection was also performed at the second, third and fourth rounds of biopanning to assess enrichment during each round by using solely neutravidin-coated magnetic beads with phage library not containing target molecule in the second and the fourth rounds, and using solely streptavidin-coated magnetic beads with phage library not containing the target molecule in the third round. 
     Blue/White Plaque Assays for Phage Titering. 
     After each round of biopanning, the number of phage in each unamplified and amplified eluate was determined by blue/white plaque assay for plaque forming units as in NEB protocol. A single colony from re-streaked host cell ( E. coli  ER2738) was inoculated in LB (10 μg/ml tetracycline) medium at 37° C. by shaking at 250 rpm till cells growth to mid-log phase, OD 600  of 0.5. Meanwhile, serial ten-fold dilutions of phage (10 2  to 10 4  serial dilutions for unamplified eluates, and 10 8  to 10 11  serial solution for amplified eluates) in LB medium were prepared. Then, 10 μl of each phage dilutions were infected with 200 μl of host cell  E. coli  culture (OD 600  of 0.5), and spread onto LB medium/X-gal/IPTG plates (Teknova, San Diego) at 37° C. overnight. Subsequently, blue plaques appeared as a result of cleavage of XGal by β-galactosidase encoded by the lacZ gene in the M13 phage construct in the presence of inducer IPTG on LB medium/X-gal/IPTG plates, and were counted and phage titers were calculated as plaque forming unit (pfu/ml). 
     Plaque Purification for DNA Sequencing and Phage-ELISA. 
     A single colony of  E. coli  ER2537 was inoculated overnight in 10 ml of LB (10 mg/ml tetracycline) medium at 37° C./250 rpm, and then diluted 100-fold in LB medium for plaques to be tested. Randomly picked single well-separated blue plaques from LB/IPTG/Xgal plates were transferred into 1 ml of diluted  E. coli  culture, and amplified by incubation for 4.5 h at 37° C./250 rpm. Following this, the amplified phage plaque culture was centrifuged at 14,000 rpm for 2×1 min at room temperature. The upper 600 μl of the supernatant was then mixed with 600 μl of 50% glycerol in a new microcentrifuge tube. Individual phage stocks were kept at −20° C. till further use for phage-ELISA experiments and DNA sequencing. 
     Phage ELISA. 
     Plates (96-well, Nunc Maxisorp #44-2404-21, Fisher Scientific) were coated with 100 μg/ml streptavidin solution overnight at 4° C. Following this, the coated wells were washed 4× with TBS containing 0.5% Tween-20, blocked with 0.1 M NaHCO 3  (pH 8.6), 5 mg/ml BSA for 2 h in a refrigerator (4° C.), and washed 6× with TBS containing 0.5% Tween-20. Biotinylated target molecules (200 pmol) were added into each well and allowed to bind for 2 h at room temperature followed by incubation with amplified phage for 2 h at room temperature. After washing 10×, a 1:5000 diluted Horseradish Peroxidase-conjugated anti-M13 monoclonal antibody solution (GE Healthcare, #27-9421-01) was added to each well, followed by incubation for 1 h with agitation at room temperature. The absorbance of each well was recorded at 405 nm using a microplate reader after incubation with ABTS substrate solution (azino-bis(3-ethylben-zothiazole) sulfonic acid), pH 4, (Sigma Aldrich, #A-1888) with 30% H 2 O 2 . Streptavidin and BSA coated wells as well as empty wells were used as negative controls for binding specificity by carrying out the same protocols without use of target molecules. 
     Bicyclic Phage Library Production and Amplification. 
     Each phage library was amplified till to reach OD 600  of 0.1 overnight by vigorous shaking at 250 rpm/30° C. in 2×YT/chloramphenicol medium. The amplified phage library was purified by precipitation with 1/4 volume of ice-cold 20% (w/v) PEG8000/2.5 M NaCl in an ice-bath for 1 h, and purified phage were centrifuged at 8500 rpm for 45 min at 4° C. to obtain phage pellets. The phage pellets were then suspended in the reaction buffer (20 mM NH 4 HCO 3 , 5 mM EDTA, pH 8.0) for 30 min, centrifuged at 4000 rpm for 15 min at 4° C., and the supernatant containing phage particles was used to assess the number of phage recovered. To PEG-purified phage solution, tris(2-carboxyethyl)phosphine (TCEP) was added to get 1 mM final concentration and incubated for 1 h in a hot water bath at 42° C. to reduce cysteine disulfide bridges in phage proteins. This mixture was cooled for 5 min in an ice-bath, then centrifuged at 4000 rpm at 4° C. until the volume was reduced to 1 ml with Vivaspin-20 100,000 PES filter tubes (Sartorius Stedim, #VS2042). All reduced phage particles were recovered by washing with reaction buffer twice and excess TCEP solution was washed away. The volume of TCEP-reduced phage was adjusted to 8 ml with ice-cold degassed reaction buffer, and incubated with 100 μM 1,3,5-tris(bromomethyl)benzene (TBMB) in acetonitrile for 1 h in a hot water bath at 30° C. for bicyclization of phage library. Then, bicyclic phage libraries were purified by precipitation with 20% (w/v) PEG8000/2.5 M NaCl solution on ice-bath for 30 min. Phage pellets were suspended in 1 ml blocking buffer (TBS buffer with 1% (w/v) BSA and 0.1% (v/v) Tween-20) to use for phage display selection at 4° C. 
     Phase Selection with Bicyclic Phase Display Libraries. 
     Biotinylated target molecules (5 mg) dissolved in binding buffer (10 mM TBS, 150 mM NaCl, pH 7.4) were incubated with streptavidin-coated magnetic beads (SA-beads) for 30 min. Post incubation, excess biotinylated target molecules were washed away, and target-coupled SA-beads were added to a blocking buffer (TBS buffer with 1% (w/v) BSA and 0.1% (v/v) Tween-20) at room temperature for 2 h. After washing target-coupled SA-beads three times, overnight pre-blocked bicyclic phage library were added and allowed to stand for 1 h at room temperature by slow agitation. The unbound phage were washed away, eluted with 0.2 M Glycine-HCl (pH 2.2) for 10 min and neutralized with 1.0 M Tris-HCl (pH 8.0). To an overnight-grown  E. coli TG 1 culture was added in 2×YT medium at 37° C./250 rpm till OD 600  reached 0.4, and the eluted phage were then added to the culture for amplification at 37° C. After this step, the cells were pelleted by centrifugation at 4000 rpm for 5 min at 4° C., and re-suspended in 2×YT medium, then plated on 2×YT/30 μg/ml chloramphenicol agar plates at 37° C. overnight. The following day, cells on the agar plates were harvested with 2×YT medium/20% glycerol stored at −80° C. to use in the next round of selection. The second round of selection was performed by using neutravidin-coated magnetic beads instead of streptavidin in order to prevent the enrichment of streptavidin-specific peptide binders. The amount of target molecules used were limited to 2 mg (from 5 mg) to increase stringency. Control selections were performed at each round without using target molecules to calculate enrichment factors. 
     Synthesis of Linear D-Peptides. 
     Peptides were chemically synthesized by SPPS using Fmoc-chemistry on Rink-4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink Amide) (0.1 mmol scale, 0.33 meq/g, ChemImpex). Amino acids were coupled by addition of 0.8 mmol Fmoc-protected amino acid, 0.72 mmol HATU, and 1.2 mmol DIEA in 2 ml DMF for 45 min. Peptides were cleaved from the resin under reducing conditions (90% TFA, 2.5% ethanedithiol, 2.5% phenol, 2.5% thioanisole, 2.5% H 2 O). TFA was evaporated under flow of nitrogen, and the cleaved peptides were precipitated in dry-ice cold diethylether three times, and centrifuged. Crude peptides were dried under high vacuum, dissolve in 50% acetonitrile/water and lyophilized. Lyophilized crude peptides were purified by reversed-phase HPLC on a C18 column using linear gradient of increasing concentration of solvent B in solvent A (from 10% to 45%). The purity of peptides was confirmed by analytical RP-HPLC, and MALDI-TOF mass spectras were recorded using alpha-cyano 4-hydroxycinnaminic acid as matrix. 
     Synthesis of Cyclic D-Peptides. 
     After synthesis and purification of linear peptides using the above described protocol, cyclization of peptides was achieved by air oxidation to yield disulfide bonds. Purified peptides (5 mg) were dissolved in 5 ml of oxidation buffer (20 mM NH 4 CO 3 , pH 8) and incubated overnight while exposed to air. The resultant oxidized peptides were lyophilized and purity was confirmed by analytical RP-HPLC and MWs were confirmed using MALDI-TOF mass spectroscopy. 
     Synthesis of Bicyclic D-Peptides. 
     Linear D-peptides having three cysteines were synthesized by SPPS using standard Fmoc-chemistry on Rink Amide resin (0.1 mmol scale, 0.33 meq/g). After cleavage of linear peptides in reducing cleavage cocktail, crude peptides were lyophilized. 1 mM solution of the crude peptides was dissolved in 0.1 M NH 4 HCO 3 , pH 8, and reacted by addition of 1.2 mM 1,3,5-tris-(bromomethyl)benzene (TBMB) in acetonitrile for 1 h in hot water bath at 30° C. This solution was lyophilized and the crude peptides were purified by RP-HPLC using a linear gradient from 10 to 60% solvent B over 40 min and purity was confirmed by analytical RP-HPLC and MWs were determined with MALDI-TOF mass spectroscopy that all showed +114 Da peaks indicative of bicyclic products. 
     Minimum Inhibitory Concentration (MIC). 
     MICs were determined according to a 96-well microdilution protocol outlined by the Clinical and Laboratory Standard Institute (CLSI) guideline with modifications for peptide antibiotics reported by Wiegand et al. 35  Two-fold serial peptide dilutions were performed to go from 2560 μg/ml to 5 μg/ml in0.2% BSA and 0.01% acetic acid buffer in 96-well polypropylene plates (COSTAR, #3879). Dilution of bacterial suspensions (1:200) of different bacterial strains that were grown until 0.5 McFarland standard were prepared to obtain a final inoculum concentration of 8×10 5  CFU/ml in Mueller-Hinton broth. Bacterial inoculum (900 were cultured with 10 μl of two-fold serial dilution solutions of peptides at 37° C. for 18 h. MICs were determined visually on the basis of turbidity as the lowest concentration inhibiting bacterial growth following CLSI guideline. MH broth without antibiotic and bacterial suspension was used as a growth control, as well as vancomycin and melittin were used as control peptide antibiotics in each assay. Each assay was repeated for  E. coli  ATCC 25992,  B. subtilis  ATCC 6633,  S. aureus  ATCC 6538, MRSA ATCC 43300,  E. faecalis  ATCC 29121, VRE ATCC 51299, VRE ATCC 51559 in triplicate at three independent days. 
     Time-Kill Kinetic Assays. 
     The bacteria-killing kinetics of peptides with  S. aureus  was measured at 5×MICs, and vancomycin was used as a positive control. An overnight culture of bacterial cells was re-suspended in fresh MH broth at density of 10 7  cells/ml, and cultured with peptides at 37° C. Viable colony forming units (CFUs) were counted by performing tenfold serial dilutions of the aliquot sample at intervals of 0, 1, 2, 4, 8, 12, and 24 hours after inoculation, and plated on MHB agar plates. Time-kill curves were plotted as Log 10  (cfu ml −1 ) versus time functions. 
     Mammalian Cytotoxicity (MTT) Assay. 
     Cervical cancer cells (HeLa) were cultured in Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) (Invitrogen #11995-073) supplemented with 10% bovine calf serum at 37° C., 5% CO 2 , and seeded (5×10 3  cells/well) in 96-well plate flat-bottomed plate for 24 h before the assay. Peptides ranging in concentration from 0n/ml to 128 μg/ml in DMEM were incubated with HeLa cells at 37° C. for 24 h. After washing the cells with PBS, the cells were subjected to the standard yellow tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay protocol at final concentration of 0.5 mg/ml for 4 h in a humidified 5% CO 2  atmosphere at 37° C. MTT solution was then aspirated, and the purple formazan crystals were resuspended in DMSO by incubation for 5 min at 37° C. The absorbance at 540 nm was then read using a microplate reader. Viability control (100% viability) was assigned to samples with only HeLa cells and DMEM media, 0% viability was assigned to samples with 1% added Triton-X-100 and no peptide. Experiments were performed as triplicates at different days. 
     Hemolytic Activity. 
     Fresh red human blood cells (hRBC) were washed and centrifuged at 3000 rpm for 5 min at 4° C. with PBS until a clear solution was obtained. Two-fold serial dilutions of each peptide in PBS from 0.5 μg/ml to 256 μg/ml were incubated with 0.25% (v/v) hRBC suspension at 37° C. for 1 h, and then centrifuged for 5 min at 3500 rpm in 96-well plate. 50 μl of the supernatant from each well was diluted with 50 μl pure water, and the OD at 415 nm was measured using microplate reader. Melittin at the same concentrations (200 μg/ml for complete lysis) was used as a positive control. Percent hemolysis was calculated according to the equation: 
     
       
         
           
             
               Percent 
               ⁢ 
               
                   
               
               ⁢ 
               hemolysis 
             
             = 
             
               
                 
                   ( 
                   
                     
                       
                         OD 
                         415 
                       
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       peptide 
                     
                     - 
                     
                       
                         OD 
                         415 
                       
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       buffer 
                     
                   
                   ) 
                 
                 
                   ( 
                   
                     
                       
                         OD 
                         415 
                       
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       complete 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       hemolysis 
                     
                     - 
                     
                       
                         OD 
                         415 
                       
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       buffer 
                     
                   
                   ) 
                 
               
               × 
               100 
             
           
         
       
     
     where complete hemolysis was defined as the average hemolysis of all wells containing a final concentration of melittin ranging from 50 to 400 μg/ml. Experiments were performed as triplicates at different days. 
     Protease Stability Assay. 
     Pancreatin (Sigma, 100 μg/ml solution in PBS) was added to peptide solutions to give a final concentration of 1 mg/ml peptide and 50 μg/ml of pancreatin, and maintained at 37° C. Aliquots of samples (15 μl) were quenched with 4% (v/v) TFA for 5 min at selected time points (0, 15, 30, 60, 120, 180, 240, 360 min and overnight). Samples were analyzed using RP-HPLC immediately to assess the proteolytic activity. 
     Serum Stability Assay. 
     Human serum was prepared by centrifuging the coagulated human blood at 3000 rpm for 15 min to get a clear supernatant. Peptide solutions (1 mg/ml) were diluted ten-fold in human serum, and incubated at 37° C. Aliquots from serum/peptide solution were taken at different time points, 0, 5, 30, 60, 120, 240 min and overnight, and quenched with 4% TFA at 4° C. for 15 min, and centrifuged for 15 min at 13,000 rpm. The supernatant was analyzed using RP-HPLC immediately. 
     Accumulation of UDP-N-acetylmuramyl-pentapeptide precursor from  S. aureus  Accumulation of peptidoglycan precursor of  S. aureus  (ATCC 6633) was analyzed in the presence of peptide antibiotics. Bacteria were grown at 37° C. till OD 600  of 0.5 was attained. Ten times MICs of peptide antibiotics were added per ml of grown bacterial culture and allowed to stand for 1 h. After cooling the cells on ice, they were harvested by centrifugation at 4° C. for 15 min. Bacterial cells were suspended in distilled water, and then slowly stirred into boiling water for 15 min. The suspension was allowed to cool first at room temperature, then on ice. After centrifugation at 4° C. for 1 h, the supernatant was lyophilized. The lyophilized sample was dissolved in water, and pH was adjusted to 2 by addition of 20% H 3 PO 4 . The white precipitate was removed by centrifugation, and the supernatant was immediately subjected to RP-HPLC analysis and MWs were assessed by MALDI-TOF mass spectroscopy. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Phage titers using Cep-1 during phage display screening 
               
               
                 with bicyclic peptide libraries including negative selections. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Round 1 
                 Round 2 
                 Enrichment 
               
               
                   
               
               
                 Library A 
                 N.S. 
                  2.8 × 10 4   
                  1.6 × 10 4   
                 — 
               
               
                   
                 Cep-1 
                  1.9 × 10 4   
                  3.6 × 10 7   
                 1.9 × 10 3   
               
               
                 Library B 
                 N.S. 
                 1.30 × 10 4   
                 3.50 × 10 3   
                 — 
               
               
                   
                 Cep-1 
                 2.75 × 10 5   
                 1.25 × 10 6   
                  36 × 10 2   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Phage titers using Ala-2 during phage display screening 
               
               
                 with bicyclic peptide libraries including negative selections. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Round 1 
                 Round 2 
                 Enrichment 
               
               
                   
               
               
                 Library A 
                 N.S. 
                 1.8 × 10 3   
                  1.1 × 10 2   
                 — 
               
               
                   
                 Ala-2 
                 4.7 × 10 4   
                  3.0 × 10 4   
                 7.5 × 10 3   
               
               
                 Library B 
                 N.S. 
                 2.6 × 10 4   
                 1.10 × 10 5   
                 — 
               
               
                   
                 Ala-2 
                 2.0 × 10 5   
                 1.75 × 10 5   
                 1.6 × 10 3   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Phage titers of Lac-4 during phage display screening  
               
               
                 with bicyclic peptide libraries including negative selections. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Round 1 
                 Round 2 
                 Enrichment 
               
               
                   
               
               
                 Library A 
                 N.S. 
                 1.3 × 10 4   
                 1.1 × 10 4   
                 — 
               
               
                   
                 Lac 
                   6 × 10 4   
                 4.6 × 10 6   
                 4.1 × 10 2   
               
               
                 Library B 
                 N.S. 
                 3.1 × 10 3   
                 1.6 × 10 4   
                 — 
               
               
                   
                 Lac 
                 1.7 × 10 4   
                 3.9 × 10 6   
                 2.4 × 10 2   
               
               
                   
               
            
           
         
       
     
     REFERENCES 
     
         
         1. Levy, S. B.; Marshall, B., Antibacterial resistance worldwide: causes, challenges and responses.  Nat. Med.  2004, 10, S122-S129. 
         2. Plata, K.; Rosato, A. E.; Wegrzyn, G.,  Staphylococcus aureus  as an infectious agent: overview of biochemistry and molecular genetics of its pathogenicity.  Acta Biochim. Pol.  2009, 56, 597-612. 
         3. Li, J. W. H.; Vederas, J. C., Drug Discovery and Natural Products: End of an Era or an Endless Frontier?  Science  2009, 325, 161-165. 
         4. Livermore, D. M. et al., Discovery research: the scientific challenge of finding new antibiotics.  J. Antimicrob. Chemother.  2011, 66, 1941-1944. 
         5. Theuretzbacher, U., Resistance drives antibacterial drug development.  Curr. Opin. Pharmacol.  2011, 11, 433-438. 
         6. Walsh, C. T.; Wencewicz, T. A.,  Antibiotics: Challenges, Mechanisms, Opportunities.  1st Edition ed.; ASM Press: 2016. 
         7. Borek, F.; Stupp, Y.; Fuchs, S.; Sela, M., Relation between optical configuration and immunogenicity of synthetic polypeptides.  Biochem. J.  1965, 96, 577-&amp;. 
         8. Dintzis, H. M.; Symer, D. E.; Dintzis, R. Z.; Zawadzke, L. E.; Berg, J. M., A comparison of the immunogenicity of a pair of enantiomeric proteins.  Proteins  1993, 16, 306-308. 
         9. Gill, T. J.; Doty, P.; Gould, H. J., Synthetic antigens composed exclusively of L- or D-amino acids. I. Effect of optical configuration on immunogenicity of synthetic polypeptides in mice.  Nature  1963, 197, 746-&amp;. 
         10. Janeway, C. A.; Sela, M., Synthetic antigens composed exclusively of L- or D-amino acids. I. Effect of optical configuration on immunogenicity of synthetic polypeptides in mice.  Immunology  1967, 13, 29-&amp;. 
         11. Pappenheimer, J. R.; Dahl, C. E.; Karnovsky, M. L.; Maggio, J. E., Intestinal-absorption and excretion of octapeptides composed of D-amino-acids.  Proc. Natl. Acad. Sci. U.S.A  1994, 91, 1942-1945. 
         12. Pappenheimer, J. R.; Karnovsky, M. L.; Maggio, J. E., Absorption and excretion of undegradable peptides: Role of lipid solubility and net charge.  J. Pharmacol. Exp. Ther.  1997, 280, 292-300. 
         13. Sidhu, S. S., Phage display in pharmaceutical biotechnology.  Curr. Opin. Biotechnol.  2000, 11, 610-616. 
         14. Wilson, D. R.; Finlay, B. B., Phage display: applications, innovations, and issues in phage and host biology.  Can. J. Microbiol.  1998, 44, 313-329. 
         15. Barbu, E. M.; Ganesh, V. K.; Gurusiddappa, S.; Mackenzie, R. C.; Foster, T. J.; Sudhof, T. C.; Hook, M., beta-Neurexin Is a Ligand for the  Staphylococcus aureus  MSCRAMM SdrC.  PLoS Pathog.  2010, 6. 
         16. Huang, W. Z.; Zhang, Z.; Palzkill, T., Design of potent beta-lactamase inhibitors by phage display of beta-lactamase inhibitory protein.  J. Biol. Chem.  2000, 275, 14964-14968. 
         17. Soykut, E. A.; Dudak, F. C.; Boyaci, I. H., Selection of staphylococcal enterotoxin B (SEB)binding peptide using phage display technology.  Biochem. Biophys. Res. Commun.  2008, 370, 104-108. 
         18. Yang, G.; Cheng, H. C.; Liu, C.; Xue, Y. N.; Gao, Y. P.; Liu, N. L.; Gao, B.; Wang, D. P.; Li, S. R.; Shen, B. F.; Shao, N. S., Inhibition of  Staphylococcus aureus  pathogenesis in vitro and in vivo by RAP-binding peptides.  Peptides  2003, 24, 1823-1828. 
         19. Schumacher, T. N. M.; Mayr, L. M.; Minor, D. L.; Milhollen, M. A.; Burgess, M. W.; Kim, P. S., Identification of D-peptide ligands through mirror-image phage display.  Science  1996, 271, 1854-1857. 
         20. Eckert, D. M.; Malashkevich, V. N.; Hong, L. H.; Can, P. A.; Kim, P. S., Inhibiting HIV-1 entry: Discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket.  Cell  1999, 99, 103-115. 
         21. Klussmann, S.; Nolte, A.; Bald, R.; Erdmann, V. A.; Furste, J. P., Minor-image RNA that binds D-adenosine.  Nat. Biotechnol.  1996, 14, 1112-1115. 
         22. Kozlov, I. A.; Mao, S. L.; Xu, Y.; Huang, X. F.; Lee, L.; Sears, P. S.; Gao, C. S.; Coyle, A. R.; Janda, K. D.; Wong, C. H., Synthesis of solid-supported mirror-image sugars: A novel method for selecting receptors for cellular-surface carbohydrates.  ChemBioChem  2001, 2, 741-746. 
         23. Liu, H. M.; Funke, S. A.; Willbold, D., Transport of Alzheimer Disease Amyloid-beta-Binding D-Amino Acid Peptides across an In Vitro Blood-Brain Barrier Model.  Rejuv. Res.  2010, 13, 210-213. 
         24. Wiesehan, K.; Buder, K.; Linke, R. P.; Patt, S.; Stoldt, M.; Unger, E.; Schmitt, B.; Bucci, E.; Willbold, D., Selection of D-Amino-Acid peptides that bind to Alzheimer&#39;s disease amyloid peptide A beta(1-42) by mirror image phage display.  ChemBioChem  2003, 4, 748-753. 
         25. Hart, P.; Wood, T. M.; Tehrani, K.; van Harten, R. M.; Sleszynska, M.; Rebollo, I. R.; Hendrickx, A. P. A.; Willems, R. J. L.; Breukink, E.; Martin, N. I., De novo identification of lipid II binding lipopeptides with antibacterial activity against vancomycin-resistant bacteria.  Chem. Sci.  2017, 8, 7991-7997. 
         26. Williamson, M. P.; Williams, D. H.; Hammond, S. J., Interactions of Vancomycin and Ristocetin with peptides as a model for protein-binding.  Tetrahedron  1984, 40, 569-577. 
         27. Tipper, D. J.; Strominger, J. L., Mechanism of action of penicillins—A proposal based on their structural similarity to acyl-D-alanyl-D-alanine.  Proc. Natl. Acad. Sci. U.S.A  1965, 54, 1133-+. 
         28. Fekner, T.; Baldwin, J. E.; Adlington, R. M.; Jones, T. W.; Prout, C. K.; Schofield, C. J., Syntheses of (6S)-cephalosporins from 6-aminopenicillanic acid.  Tetrahedron  2000, 56, 6053-6074. 
         29. Fekner, T.; Baldwin, J. E.; Adlington, R. M.; Schofield, C. J., Syntheses of (6S,7S)- and (6S,7R)-deacetoxycephalosporanic acids from 6-aminopenicillanic acid.  Chem. Commun.  1996, 1989-1990. 
         30. Popieniek, P. H.; Pratt, R. F., A fluorescent ligand for binding-studies with glycopeptide antibiotics of the vancomycin class.  Anal. Biochem.  1987, 165, 108-113. 
         31. Popieniek, P. H.; Pratt, R. F., Kinetics and mechanism of binding of specific peptides to vancomycin and other glycopeptide antibiotics.  J. Am. Chem. Soc.  1991, 113, 2264-2270. 
         32. Heinis, C.; Rutherford, T.; Freund, S.; Winter, G., Phage-encoded combinatorial chemical libraries based on bicyclic peptides.  Nat. Chem. Biol.  2009, 5, 502-507. 
         33. Angelini, A.; Cendron, L.; Chen, S. Y.; Touati, J.; Winter, G.; Zanotti, G.; Heinis, C., Bicyclic Peptide Inhibitor Reveals Large Contact Interface with a Protease Target.  ACS Chem. Biol.  2012, 7, 817-821. 
         34. Baeriswyl, V.; Calzavarini, S.; Gerschheimer, C.; Diderich, P.; Angelillo-Scherrer, A.; Heinis, C., Development of a Selective Peptide Macrocycle Inhibitor of Coagulation Factor XII toward the Generation of a Safe Antithrombotic Therapy.  J. Med. Chem.  2013, 56, 3742-3746. 
         35. Baeriswyl, V.; Rapley, H.; Pollaro, L.; Stace, C.; Teufel, D.; Walker, E.; Chen, S. Y.; Winter, G.; Tite, J.; Heinis, C., Bicyclic Peptides with Optimized Ring Size Inhibit Human Plasma Kallikrein and its Orthologues While Sparing Paralogous Proteases.  ChemMedChem  2012, 7, 1173-1176. 
         36. Diderich, P.; Heinis, C., Phage selection of bicyclic peptides binding Her2 . Tetrahedron  2014, 70, 7733-7739. 
         37. Urech-Varenne, C.; Radtke, F.; Heinis, C., Phage Selection of Bicyclic Peptide Ligands of the Notch1 Receptor.  ChemMedChem  2015, 10, 1754-1761. 
         38. Wiegand, I.; Hilpert, K.; Hancock, R. E. W., Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.  Nat. Protoc.  2008, 3, 163-175. 
         39. Griffith, R. S., Vancomycin Use—An Historical Review.  J. Antimicrob. Chemother.  1984, 14, 1-5. 
         40. Dai, D.; Ishiguro, E. E., MurH, a new genetic-locus in  Escherichia Coli  involved in cell-wall peptidoglycan biosynthesis.  J. Bacteriol.  1988, 170, 2197-2201. 
         41. Walsh, C. T.; Fisher, S. L.; Park, I. S.; Prahalad, M.; Wu, Z., Bacterial resistance to vancomycin: Five genes and one missing hydrogen bond tell the story.  Chem. Biol.  1996, 3, 21-28. 
         42. Allen, N. E.; Hobbs, J. N.; Nicas, T. I., Inhibition of peptidoglycan biosynthesis in vancomycin-susceptible and -resistant bacteria by a semisynthetic glycopeptide antibiotic.  Antimicrob. Agents Chemother.  1996, 40, 2356-2362. 
         43. Bischoff, D.; Pelzer, S.; Bister, B.; Nicholson, G. J.; Stockert, S.; Schirle, M.; Wohlleben, W.; Jung, G.; Sussmuth, R. D., The biosynthesis of vancomycin-type glycopeptide antibiotics—The order of the cyclization steps.  Angew. Chem .- Int. Edit.  2001, 40, 4688-+. 
         44. Chamorro, C.; Liskamp, R. M. J., Selection of synthetic receptors capable of sensing the difference in binding of D-Ala-D-Ala or D-Ala-D-Lac ligands by screening of a combinatorial CTV-based library.  Tetrahedron  2004, 60, 11145-11157. 
         45. Chiosis, G.; Boneca, I. G., Selective cleavage of D-Ala-D-Lac by small molecules: Re-sensitizing resistant bacteria to vancomycin.  Science  2001, 293, 1484-1487. 
         46. Crane, C. M.; Boger, D. L., Synthesis and Evaluation of Vancomycin Aglycon Analogues That Bear Modifications in the N-Terminal D-Leucyl Amino Acid.  J. Med. Chem.  2009, 52, 1471-1476. 
         47. Griffin, J. H.; Linsell, M. S.; Nodwell, M. B.; Chen, Q. Q.; Pace, J. L.; Quast, K. L.; 
       
    
     Krause, K. M.; Farrington, L.; Wu, T. X.; Higgins, D. L.; Jenkins, T. E.; Christensen, B. G.; Judice, J. K., Multivalent drug design. Synthesis and in vitro analysis of an array of vancomycin dimers.  J. Am. Chem. Soc.  2003, 125, 6517-6531.
     48. Isomura, S.; Ashley, J. A.; Wirsching, P.; Janda, K. D., Antibody-catalyzed cleavage of the D-Ala-D-lac depsipeptide: An immunological approach to the problem of vancomycin resistance.  Bioorg. Med. Chem. Lett.  2002, 12, 861-864.   49. Jones, R. N.; Barret, M. S.; Erwin, M. E., In vitro activity and spectrum of LY333328, a novel glycopeptide derivative. Antimicrob.  Agents Chemother.  1997, 41, 488-493.   50. Ma, N. C.; Jia, Y. X.; Liu, Z. S.; Gonzalez-Zamora, E.; Bois-Choussy, M.; Malabarba, A.; Brunati, C.; Zhu, J., Design and synthesis of macrocycles active against vancomycin-resistant enterococci (VRE): the interplay between D-Ala-D-Lac binding and hydrophobic effect.  Bioorg. Med. Chem. Lett.  2005, 15, 743-746.   51. Maffioli, S. I.; Ciabatti, R.; Romano, G.; Marzorati, E.; Preobrazhenskaya, M.; Pavlov, A., Synthesis and antibacterial activity of alkyl derivatives of the glycopeptide antibiotic A40926 and their amides.  Bioorg. Med. Chem. Lett.  2005, 15, 3801-3805.   52. Nakama, Y.; Yoshida, O.; Yoda, M.; Araki, K.; Sawada, Y.; Nakamura, J.; Xu, S.; Miura, K.; Maki, H.; Arimoto, H., Discovery of a Novel Series of Semisynthetic Vancomycin Derivatives Effective against Vancomycin-Resistant Bacteria.  J. Med. Chem.  2010, 53, 2528-2533.   53. Pavlov, A. Y.; Berdnikova, T. F.; Olsufyeva, E. N.; Lazhko, E. I.; Malkova, I. V.; Preobrazhenskaya, M. N.; Testa, R. T.; Petersen, P. J., Synthesis and biological-activity of derivatives of glycopeptide antibiotics eremomycin and vancomycin nitrosated, acylated or carbamoylated at the N-terminal.  J. Antibiot.  1993, 46, 1731-1739.   54. Rao, J. H.; Whitesides, G. M., Tight binding of a dimeric derivative of vancomycin with dimeric L-Lys-D-Ala-D-Ala.  J. Am. Chem. Soc.  1997, 119, 10286-10290.   55. Xie, J.; Pierce, J. G.; James, R. C.; Okano, A.; Boger, D. L., A Redesigned Vancomycin Engineered for Dual D-Ala-D-Ala and D-Ala-D-Lac Binding Exhibits Potent Antimicrobial Activity Against Vancomycin-Resistant Bacteria.  J. Am. Chem. Soc.  2011, 133, 13946-13949.   56. Xing, B. G.; Yu, C. W.; Ho, P. L.; Chow, K. H.; Cheung, T.; Gu, H. W.; Cai, Z. W.; Xu, B., Multivalent antibiotics via metal complexes: Potent divalent vancomycins against vancomycin-resistant enterococci.  J. Med. Chem.  2003, 46, 4904-4909.   57. Xu, R.; Greiveldinger, G.; Marenus, L. E.; Cooper, A.; Ellman, J. A., Combinatorial library approach for the identification of synthetic receptors targeting vancomycin-resistant bacteria.  J. Am. Chem. Soc.  1999, 121, 4898-4899.   58. Yao, N. H.; Liu, G.; He, W. Y.; Niu, C. Q.; Carlson, J. R.; Lam, K. S., Solid-phase synthesis and antibacterial evaluations of N-demethylvancomycin derivatives.  Bioorg. Med. Chem. Lett.  2005, 15, 2325-2329.   59. Yarlagadda, V.; Akkapeddi, P.; Manjunath, G. B.; Haldar, J., Membrane Active Vancomycin Analogues: A Strategy to Combat Bacterial Resistance.  J. Med. Chem.  2014, 57, 4558-4568.   60. Zhanel, G. G.; Calic, D.; Schweizer, F., New lipoglycopeptides: a comparative review of dalbavancin, oritavancin and telavancin (vol 70, pg 859, 2010).  Drugs  2011, 71, 526-526.   61. Zasloff, M., Antimicrobial peptides of multicellular organisms.  Nature  2002, 415, 389-395.   62. Meister, A.,  Biochemistry of the amino acids.  2d ed. ed.; New York, Academic Press: New York, 1965.   63. Boman, H. G., Antibacterial peptides: basic facts and emerging concepts.  J. Intern. Med.  2003, 254, 197-215.   64. Meng, H.; Kumar, K., Antimicrobial activity and protease stability of peptides containing fluorinated amino acids.  J. Am. Chem. Soc.  2007, 129, 15615-15622.   

     Enumerated Embodiments 
     It shall be understood that the preceding examples are intended to demonstrate how the compositions and methods of the disclosure may be practiced, and are not intended to be limiting as to the scope of the disclosed subject matter. 
     The subject matter of the present disclosure is also exemplified by the following enumerated embodiments: 
     Embodiment 1. An isolated peptide comprising an amino acid sequence comprising, consisting of, or having at least 85% identity to the amino acid sequence of a peptide of any of Tables 1-6.
 
Embodiment 2. The peptide of embodiment 1, wherein each amino acid in the peptide is a D-amino acid.
 
Embodiment 3. The peptide of embodiment 1 or 2, wherein the peptide comprises one or more sequence motifs selected from TTL, I/L-S/T, Q/N-S/T, LQ, G-S/T/V, G-X-S/T, G-X-S, L-K/R, R-L/V, RV, VLS, N/Q-K/R, GQS, G-S/V, GQV, QL, SSV, T/P-Q, YQ-S/L, and QRL, wherein each X represents any D- or L-amino acid.
 
Embodiment 4. The peptide of embodiment 3, wherein each X represents any D-amino acid.
 
Embodiment 5. The peptide of any one of embodiments 1-4, wherein the peptide comprises an amino acid sequence selected from VGSNLRLLHQWK, VHWDFRQWWQPS, KCCYTLP, VLTRCCG, TGFLVNV, HYIDFRW, CLKLGEKWC, CRGATPMSC, CSISSLTHC, CLWSTGATC, CDNIMTPVC, CQTDVCQRTIC, CSLITQCGGVGC, CQYNDCDMLHC, CLSQFCVIDC, CGGGICRTHNC, CTRVCSSSQLYC, and CGAQGCFGVQSC.
 
Embodiment 6. The peptide of any one of embodiments 1-5, wherein the peptide is linear, cyclic, or bicyclic.
 
Embodiment 7. An isolated polynucleotide encoding a peptide of embodiment 1.
 
Embodiment 8. A vector comprising the isolated polynucleotide of embodiment 7.
 
Embodiment 9. A host cell comprising the vector of embodiment 8.
 
Embodiment 10. A method of treating a bacterial infection in a subject, said method comprising administering to said subject a therapeutically-effective amount of a peptide of any one of embodiments 1-6.
 
Embodiment 11. The method of embodiment 10, wherein said subject is a human.
 
Embodiment 12. The method of embodiment 10 or 11, wherein said subject has an antibiotic resistant infection.
 
Embodiment 13. The method of any one of embodiments 10-12, wherein said subject has a chronic infection.
 
Embodiment 14. The method of any one of embodiment 10-13, wherein the said bacteria belongs to the genus  Staphylococcus  or  Enterococcus.  
 
Embodiment 15. The method of any one of embodiments 10-14, wherein the infection is a  B. subtilis, E. coli E. faecalis , or  S. aureus  infection.
 
Embodiment 16. A method of treating Methicillin-resistant  S. aureus  (MRSA), low level vancomycin resistant  E. faecium , or high level vancomycin resistant  E. faecium  said method comprising administering to said mammal a therapeutically-effective amount of a peptide of any one of embodiments 1-6.
 
     OTHER EMBODIMENTS 
     From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. 
     The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. 
     All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.