All D-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries

The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D -amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.     

Successful identification of novel agents to control infectious diseases from screening mixture-based peptide combinatorial libraries in complex cell-based bioassays

Mixture-based peptide synthetic combinatorial libraries (SCLs) represent a valuable source for the development of novel agents to control infectious diseases. Indeed, a number of studies have now proven the ability of identifying active peptides from libraries composed of thousands to millions of peptides in cell-based biosystems of varying complexity. Furthermore, progressing knowledge on the importance of endogenous peptides in various immune responses lead to a regain in importance for peptides as potential therapeutic agents. This article is aimed at providing recent studies in our laboratory for the development of antimicrobial or antiviral peptides derived from mixture-based SCLs using cell-based assays, as well as a short review of the importance of such peptides in the control of infectious diseases. Furthermore, the use of positional scanning (PS) SCL-based biometrical analyses for the identification of native optimal epitopes specific to HIV-1 proteins is also presented.     

One-bead–one-structure combinatorial libraries

Combinatorial libraries employing the one-bead–one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect “coding” methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and “scaffold” libraries, together with a new approach for the discovery of a pentide binding motif using a “library of libraries,” are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries. © 1994 John Wiley & Sons, Inc.     

Extending the coverage of spectral libraries: A neighbor‐based approach to predicting intensities of peptide fragmentation spectra

Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well‐studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor‐based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K‐nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20–60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.     

Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage.

An efficient strategy has been devised for the construction of diverse peptide libraries in bacteriophage vectors. This strategy was used to generate a library of 4 x 10(8) random decapeptide inserts in the pIII protein of bacteriophage fd. A novel method for evaluating the genetic diversity of bacteriophage libraries based on colony hybridization with partially degenerate oligonucleotides has been developed. The decapeptide library was affinity-selected with a previously characterized monoclonal antibody specific for the V3 loop of the gp120 protein of HIV-1. Immunological screening, an efficient technique for the rapid identification of putative binding bacteriophage, is described. Hexapeptide sequences similar to those obtained from affinity selection of a hexapeptide bacteriophage library were obtained from the decapeptide library in all five frames. Immunological screening of 20,000 clones from the two libraries after two rounds of affinity selection rapidly identified antibody-binding sequences; 93% and 86% of the sequences obtained from the hexapeptide and decapeptide libraries, respectively, had IC50 values < or = 10 mM as free peptides.     

Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli

Recent advances in chemical biology and the advantages presented by in vivo screening have highlighted the need for a robust and flexible biologically synthesized small-molecule library. Herein we describe a method for the biosynthesis of cyclic peptide libraries of up to 10(8) members in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS). The method utilizes split-intein chemistry to cyclize randomized peptide sequences. The cyclic peptide library can potentially be of any size and the peptide itself may contain unlimited random residues. However, the library size is limited by the transformation efficiency of E. coli and random residues are generally limited to five, but additional amino acids can be used in the cyclic peptide backbone, varying the structure and ring size of the cyclic peptide. SICLOPPS libraries have been combined with a bacterial reverse two-hybrid system in our labs and used in the identification of inhibitors of several protein-protein interactions. This protocol is expected to take around 3-4 weeks to implement.     

Soluble Combinatorial Libraries: Extending the Range and Repertoire of Chemical Diversity

The generation and use of libraries made up of millions of chemical entities and the ability to identify the active compound in such libraries are at the forefront of a revolution in drug discovery and basic research. Such libraries, when made up of peptide sequences, offer a fundamental, practical advance in the study of interactions between peptides and their biochemical or pharmacological targets. The utility of soluble peptide libraries ranging from three to eight amino acids in length, and made up of mixtures from 361 tripeptides to 200 billion decapeptides, is described. These are readily usable in virtually all in vitro (and even in vivo) assay systems. The examples presented illustrate the utility of soluble peptide libraries for the study of antibody/antigen interactions, the identification of highly active opioid peptides in receptor binding studies using crude rat brain homogenates, and in vivo studies, in which the peptide mixtures making up the library are administered intravenously to determine peptide sequences that affect heart rate and blood pressure. A new class of library is also described, termed a modified peptide library, which is used to determine potent anti-Staphylococcus aureus compounds.     

Green fluorescent protein as a scaffold for intracellular presentation of peptides.

Peptide aptamers provide probes for biological processes and adjuncts for development of novel pharmaceutical molecules. Such aptamers are analogous to compounds derived from combinatorial chemical libraries which have specific binding or inhibitory activities. Much as it is generally difficult to determine the composition of combinatorial chemical libraries in a quantitative manner, determining the quality and characteristics of peptide libraries displayed in vivo is problematical. To help address these issues we have adapted green fluorescent protein (GFP) as a scaffold for display of conformationally constrained peptides. The GFP-peptide libraries permit analysis of library diversity and expression levels in cells and allow enrichment of the libraries for sequences with predetermined characteristics, such as high expression of correctly folded protein, by selection for high fluorescence.     

Phage Peptide Libraries

Filamentous phage particles have been central in the construction of libraries displaying vast numbers of random peptides. These random peptides can be antigenically presented as fusions to coat proteins III and VIII of the phage. The isolation of ligate-reactive phage from an immense background of nonspecific phage is achieved by the biopanning process. Enrichment of reactive phage relative to unreactive phage occurs with alternate rounds of affinity selection to the desired molecular target and amplification of the specifically bound phage. This allows the isolation of rare binding species contained in the phage peptide libraries. Each phage particle contains the information in its genome pertaining to the type of random peptide insert displayed. Hence, the identification of binding motifs displayed on ligate-reactive phage is revealed by sequencing the relevant insert site in the phage genome. Phage peptide libraries have been used to isolate ligands to an array of protein ligates. The libraries have proved particularly effective in defining the binding sites of monoclonal antibodies and to some extent polyclonal sera. The analysis of the peptide insert sequences of a number of different clones of antibody binding phage can reveal a great deal about the nature and restriction of the amino acid residues critical for the antibody–antigen interaction.     

Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach

Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount of HLA molecules, giving a selective force in the binding. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format.     

Phage display of random peptide libraries: applications, limits, and potential

The identification of ligands from large biological libraries by phage display has now been used for almost 15 years. Most of the successful reports on high-affinity ligand identification originated from work with different antibody libraries. In contrast, the progress of applying phage display to random peptide libraries was relatively slow. However, in the last few years several improvements have led to an increasing number of published peptide ligands identified by phage display from such libraries and which exhibited good biological activity and high affinity. This review summarizes the current state and the technical progress of the application of random peptide libraries using filamentous phage for ligand identification.     

Selectide Technology: Bead-Binding Screening

The Selectide process is a random synthetic chemical library method based on the one-bead one-peptide (structure) concept. A "split-synthesis" method is used to generate huge random libraries (10⁶-10⁸). At the end of the synthesis, each bead expresses only one chemical entity (e.g., peptide). The whole library is then tested simultaneously for binding to a specific acceptor molecule of biologic interest. The ligand bead that interacts specifically with the acceptor molecule is then isolated for structure determination. Once a binding motif is identified, a secondary library (based on the motif of the primary screen) is generated and screened under a more stringent condition to identify leads of higher affinity. This process can be applied to both peptide and nonpeptide (small organic) libraries. In the case of nonsequencable structure libraries, the coding principle has to be applied for structure elucidation of positively reacting beads. Coding peptide is synthesized in parallel to the screening structure, and classical Edman degradation (one or multiple-step) is used for structural analysis. To exclude the possibility of interaction of the macromolecular target (e.g., receptor, enzyme, antibody) with the coding structure, a synthetic technique for segregation of the surface (screening structure) and the interior (coding structure) of the beads was developed. The one-bead one-structure process is invaluable in drug discovery for lead identification as well as further optimization of the initial leads. It also serves as an important research tool for molecular recognition.     

Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.     

From Design to Screening: A New Antimicrobial Peptide Discovery Pipeline

Antimicrobial peptides (AMPs) belong to a class of natural microbicidal molecules that have been receiving great attention for their lower propensity for inducing drug resistance, hence, their potential as alternative drugs to conventional antibiotics. By generating AMP libraries, one can study a large number of candidates for their activities simultaneously in a timely manner. Here, we describe a novel methodology where in silico designed AMP-encoding oligonucleotide libraries are cloned and expressed in a cellular host for rapid screening of active molecules. The combination of parallel oligonucleotide synthesis with microbial expression systems not only offers complete flexibility for sequence design but also allows for economical construction of very large peptide libraries. An application of this approach to discovery of novel AMPs has been demonstrated by constructing and screening a custom library of twelve thousand plantaricin-423 mutants in Escherichia coli. Analysis of selected clones by both Sanger-sequencing and 454 high-throughput sequencing produced a significant amount of data for positionally important residues of plantaricin-423 responsible for antimicrobial activity and, moreover, resulted in identification of many novel variants with enhanced specific activities against Listeria innocua. This approach allows for generation of fully tailored peptide collections in a very cost effective way and will have countless applications from discovery of novel AMPs to gaining fundamental understanding of their biological function and characteristics.     

Affinity Selection and Mass Spectrometry-Based Strategies to Identify Lead Compounds in Combinatorial Libraries

The screening of diverse libraries of small molecules created by combinatorial synthetic methods is a recent development which has the potential to accelerate the identification of lead compounds in drug discovery. We have developed a direct and rapid method to identify lead compounds in libraries involving affinity selection and mass spectrometry. In our strategy, the receptor or target molecule of interest is used to isolate the active components from the library physically, followed by direct structural identification of the active compounds bound to the target molecule by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compounds. Once lead compounds have been identified, libraries containing compounds chemically similar to the lead compound can be generated and used to optimize the binding characteristics. These strategies have also been adopted for more detailed studies of protein–ligand interactions.     

Diversity and novelty of the gut microbial community of an herbivorous rodent (Neotoma bryanti)

Mammalian herbivores host diverse microbial communities to aid in fermentation and potentially detoxification of dietary compounds. However, the microbial ecology of herbivorous rodents, especially within the largest superfamily of mammals (Muroidea) has received little attention. We conducted a preliminary inventory of the intestinal microbial community of Bryant’s woodrat (Neotoma bryanti), an herbivorous Muroidea rodent. We collected woodrat feces, generated 16S rDNA clone libraries, and obtained sequences from 171 clones. Our results demonstrate that the woodrat gut hosts a large number of novel microorganisms, with 96% of the total microbial sequences representing novel species. These include several microbial genera that have previously been implicated in the metabolism of plant toxins. Interestingly, a comparison of the community structure of the woodrat gut with that of other mammals revealed that woodrats have a microbial community more similar to foregut rather than hindgut fermenters. Moreover, their microbial community was different to that of previously studied herbivorous rodents. Therefore, the woodrat gut may represent a useful resource for the identification of novel microbial genes involved in cellulolytic or detoxification processes.     

Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides

Cytotoxic CD8(+) T cells recognize the antigenic peptides presented by class I major histocompatibility complex (MHC) molecules. These T cells have key roles in infectious diseases, autoimmunity and tumor immunology, but there is currently no unbiased method for the reliable identification of their target antigens. This is because of the low affinities of antigen-specific T cell receptors (TCR) to their target MHC-peptide complexes, the polyspecificity of these TCRs and the requirement that these TCRs recognize protein antigens that have been processed by antigen-presenting cells (APCs). Here we describe a technology for the unbiased identification of the antigenic peptides presented by MHC class I molecules. The technology uses plasmid-encoded combinatorial peptide libraries and a single-cell detection system. We validated this approach using a well-characterized influenza-virus–specific TCR, MHC and peptide combination. Single APCs carrying antigenic peptides can be detected among several million APCs that carry irrelevant peptides. The identified peptide sequences showed a converging pattern of mimotopes that revealed the parent influenza antigen. This technique should be generally applicable to the identification of disease-relevant T cell antigens.     

Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry

To link cleaved substrates in complex systems with a specific protease, the protease active site specificity is required. Proteomic identification of cleavage sites (PICS) simultaneously determines both the prime- and non-prime-side specificities of individual proteases through identification of hundreds of individual cleavage sequences from biologically relevant, proteome-derived peptide libraries. PICS also identifies subsite cooperativity. To generate PICS peptide libraries, cellular proteomes are digested with a specific protease such as trypsin. Following protease inactivation, primary amines are protected. After incubation with a test protease, each prime-side cleavage fragment has a free newly formed N-terminus, which is biotinylated for affinity isolation and identification by liquid chromatography-tandem mass spectrometry. The corresponding non-prime sequences are derived bioinformatically. The step-by-step protocol also presents a web service for PICS data analysis, as well as introducing and validating PICS peptide libraries made from Escherichia coli.     

Selection of novel ligands from phage display libraries: an alternative approach to drug and vaccine discovery?

Phage display involves the production and screening of large numbers of random peptide sequences of a specific length expressed on the surface of phage particles. This approach provides a powerful tool to probe the molecular basis of many biological processes, including host-parasite interactions. Phage display libraries have been used to study the binding specificity of numerous peptides and protein domains. Practical applications include the identification of peptide sequences that bind with high affinity to antibodies, enzymes or receptors, and that may serve as diagnostics and vaccine or drug candidates. Here, David Jefferies outlines the concept of phage display and summarizes recent developments in the field, with emphasis on those that may be of interest to parasitologists.