Ribosomal synthesis of unnatural peptides

Combinatorial libraries of non-biological polymers and drug-like peptides could in principle be synthesized from unnatural amino acids by exploiting the broad substrate specificity of the ribosome. The ribosomal synthesis of such libraries would allow rare functional molecules to be identified using technologies developed for the in vitro selection of peptides and proteins. Here, we use a reconstituted E. coli translation system to simultaneously re-assign 35 of the 61 sense codons to 12 unnatural amino acid analogues. This reprogrammed genetic code was used to direct the synthesis of a single peptide containing 10 different unnatural amino acids. This system is compatible with mRNA-display, enabling the synthesis of unnatural peptide libraries of 10(14) unique members for the in vitro selection of functional unnatural molecules. We also show that the chemical space sampled by these libraries can be expanded using mutant aminoacyl-tRNA synthetases for the incorporation of additional unnatural amino acids or by the specific posttranslational chemical derivitization of reactive groups with small molecules. This system represents a first step toward a platform for the synthesis by enzymatic tRNA aminoacylation and ribosomal translation of cyclic peptides comprised of unnatural amino acids that are similar to the nonribosomal peptides.     

Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries

Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.     

Past and future perspectives of synthetic peptide libraries

Combinatorial preparation and HTS of arrays of compounds have increased the speed of drug discovery. A strong impulse in this field has come by the introduction of the solid phase synthesis method that, through automation and miniaturization, has paved the way to the preparation of large collections of compounds in compact and trackable formats. Due to the well established synthetic procedures, peptides have been largely used to develop the basic concepts of combinatorial chemistry and peptide libraries are still successfully employed in screening programs. However, peptides generally do not fulfil the requirements of low conformational flexibility, stability and bioavailability needed for good drug candidates and peptide leads with high potency and selectivity are often made "druggable" by conversion to more stable structures with improved pharmacological profiles. Such an approach makes the screening of peptide libraries still a valuable tool for drug discovery. We propose here a panoramic review of the most common methods for the preparation and screening of peptide libraries and the most interesting findings of the last decade. We also report on a new approach we follow in our laboratory that is based on the use of "simplified" libraries composed by a minimum number of non-redundant amino acids for the assembly of short peptides. The choice of amino acids is dictated by diversity in lipophilicity, MW, charge and polarity. Newly identified active sequences are then modified by preparing new variants containing analogous amino acids, so that the chemical space occupied by the excluded residues can be explored. This approach offers the advantage of simplifying the synthesis and deconvolution of libraries and provides new active compounds with a molecular size similar to that of small molecules, to which they can be easily converted.     

Inhibition of hIAPP amyloid-fibril formation and apoptotic cell death by a designed hIAPP amyloid- core-containing hexapeptide

The pathogenesis of type II diabetes is associated with the aggregation of the 37-residue human islet amyloid polypeptide (hIAPP) into cytotoxic beta sheet aggregates and fibrils. We have recently shown that introduction of two N-methyl rests in the beta sheet- and amyloid-core-containing sequence hIAPP(22-27), or NFGAIL converted this amyloidogenic and cytotoxic sequence into nonamyloidogenic and noncytotoxic NF(N-Me)GA(N-Me)IL. Here, we show that NF(N-Me)GA(N-Me)IL is able to bind with high-affinity full-length hIAPP and to inhibit its fibrillogenesis. NF(N-Me)GA(N-Me)IL also inhibits hIAPP-mediated apoptotic beta cell death. By contrast, unmodified NFGAIL does not inhibit hIAPP amyloidogenesis and cytotoxicity, suggesting that N-methylation conferred on NFGAIL the properties of NF(N-Me)GA(N-Me)IL. These results support the concept that rational N-methylation of hIAPP amyloid-core sequences may be a valuable strategy to design pancreatic-amyloid diagnostics and therapeutics for type II diabetes.     

DNA libraries for the construction of phage libraries: statistical and structural requirements and synthetic methods

Peptide-based molecular probes identified by bacteriophage (phage) display technology expand the peptide repertoire for in vivo diagnosis and therapy of cancer. Numerous peptides that bind cancer-associated antigens have been discovered by panning phage libraries. However, until now only few of the peptides selected by phage display have entered clinical applications. The success of phage derived peptides essentially depends on the quality of the library screened. This review summarizes the methods to achieve highly homogenous libraries that cover a maximal sequence space. Biochemical and chemical strategies for the synthesis of DNA libraries and the techniques for their integration into the viral genome are discussed in detail. A focus is set on the methods that enable the exclusion of disturbing sequences. In addition, the parameters that define the variability, the minimal numbers of copies per library and the use of alternating panning cycles to avoid the loss of selected hits are evaluated.     

High Speed Development and Synthesis of Novel Small Molecule Libraries

Combinatorial chemistry has produced libraries of millions of compounds in the last decade. Screening of those compounds, unfortunately, has not yet yielded as many new drug candidates as initially expected. Among a number of possible reasons, one is that many libraries combinatorial chemistry produced in the early periods are of the nature of linear, flat, and flexible molecules such as peptides and oligonucleotides, which do not have the desired properties to selectively interact with their targets to yield high quality hits and leads. In order to increase the number of quality hits and leads, rigid, structural featurerich and drug-like compound libraries are highly desirable. Design and development of structural features-rich and natural product-like combinatorial libraries, as well as high speed library production using modern solution and solid phase synthesis techniques such as IRORI's Directed Sorting technology, will be discussed.     

In vitro selection of highly modified cyclic peptides that act as tight binding inhibitors

There is a great demand for the discovery of new therapeutic molecules that combine the high specificity and affinity of biologic drugs with the bioavailability and lower cost of small molecules. Small, natural-product-like peptides hold great promise in bridging this gap; however, access to libraries of these compounds has been a limitation. Since ribosomal peptides may be subjected to in vitro selection techniques, the generation of extremely large libraries (>10(13)) of highly modified macrocyclic peptides may provide a powerful alternative for the generation and selection of new useful bioactive molecules. Moreover, the incorporation of many non-proteinogenic amino acids into ribosomal peptides in conjunction with macrocyclization should enhance the drug-like features of these libraries. Here we show that mRNA-display, a technique that allows the in vitro selection of peptides, can be applied to the evolution of macrocyclic peptides that contain a majority of unnatural amino acids. We describe the isolation and characterization of two such unnatural cyclic peptides that bind the protease thrombin with low nanomolar affinity, and we show that the unnatural residues in these peptides are essential for the observed high-affinity binding. We demonstrate that the selected peptides are tight-binding inhibitors of thrombin, with K(i)(app) values in the low nanomolar range. The ability to evolve highly modified macrocyclic peptides in the laboratory is the first crucial step toward the facile generation of useful molecular reagents and therapeutic lead molecules that combine the advantageous features of biologics with those of small-molecule drugs.     

Synthetic peptides in the diagnosis of systemic autoimmune diseases

Proteins recognized by antibodies from patients with autoimmune diseases have been intensively studied over the two past decades since cDNAs encoding autoantigens have become available. Identity of many of them has been defined, and specific structural motifs or post-translational modifications, which may be important to explain the generation of such antibodies during the autoimmune process, have been pointed out. Immunological analysis of sera from autoimmune patients with recombinant fragments and with short peptides has revealed the presence of dominant epitopes along proteins; some of them are targeted by antibodies from patients with specific diseases or disease subsets. Innovative technologies such as peptide arrays and biosensors as well as the exploitation of large peptides libraries have recently open up new perspectives. Peptides bearing natural modifications, peptide analogues, as well as mimotopes of protein or non-protein antigens (DNA, RNA, sugar) have been developed and might advantageously replace native antigens in routine immunoassays. Although numerous conformational epitopes have not yet been identified, and cannot be identified by the approaches classically used in epitope mapping studies, such peptides and peptide analogues may represent efficient probes to detect the presence of circulating autoantibodies in the serum of autoimmune patients and help for establishing specific and sensitive early diagnostic tests. They may also lead to the design of high-affinity ligands for purifying autoantibodies. These different aspects are discussed and epitope mapping studies of a number of autoantigens (e.g. histones, sn and hnRNP proteins and Ro proteins) are summarized.     

Studying protein-protein interactions using peptide arrays

Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins and peptides. The applications of libraries for detecting and characterizing biological interactions are wide and diverse, including for example epitope mapping, carbohydrate arrays, enzyme binding and protein-protein interactions. Here, we will focus on the use of peptide arrays to study protein-protein interactions. Characterization of protein-protein interactions is crucial for understanding cell functionality. Using peptides, it is possible to map the precise binding sites in such complexes. Peptide array libraries usually contain partly overlapping peptides derived from the sequence of one protein from the complex of interest. The peptides are attached to a solid support using various techniques such as SPOT-synthesis and photolithography. Then, the array is incubated with the partner protein from the complex of interest. Finally, the detection of the protein-bound peptides is carried out by using immunodetection assays. Peptide array screening is semi-quantitative, and quantitative studies with selected peptides in solution are required to validate and complement the screening results. These studies can improve our fundamental understanding of cellular processes by characterizing amino acid patterns of protein-protein interactions, which may even develop into prediction algorithms. The binding peptides can then serve as a basis for the design of drugs that inhibit or activate the target protein-protein interactions. In the current review, we will introduce the recent work on this subject performed in our and in other laboratories. We will discuss the applications, advantages and disadvantages of using peptide arrays as a tool to study protein-protein interactions.     

Identification of enzyme inhibitors from phage-displayed combinatorial peptide libraries

In recent years, there have been a growing number of examples of the successful isolation of peptide ligands for enzymes from phage-displayed combinatorial peptide libraries. These peptides typically bind at or near the active site of the enzymes and can inhibit their activity. We review the literature on peptide ligands that have been isolated for enzymes other than proteases as well as present data on peptide ligands we have identified for E. coli dihydrofolate reductase (DHFR) which bind at, or near, the same site as the known inhibitors methotrexate or trimethoprim. Thus, while the peptide ligand isolated from phage-displayed libraries may not resemble the chemical structure of the normal substrate of the enzyme, the peptide can be used as an inhibitor to evaluate the function of the enzyme or for drug discovery efforts (i.e., as a lead compound for peptidomimetic design or as displaceable probe in high-throughput screens of libraries of small molecules).     

Glycosamino acids: building blocks for combinatorial synthesis-implications for drug discovery

The unique functions of carbohydrates, including energy storage, transport, modulation of protein function, intercellular adhesion, signal transduction, malignant transformation, and viral and bacterial cell-surface recognition, underlie a significant pharmaceutical potential. The development of combinatorial carbohydrate libraries in this important arena has been slow, in contrast to the rapid development of combinatorial synthesis in the area of small-molecule libraries and biopolymers. This is largely as a result of the inherent difficulties presented by this class of polyfunctional compounds. Nevertheless, strategies to cope with these problems have been devised over the past seven years, and combinatorial carbohydrate libraries have appeared. The incorporation of an amino acid moiety into the carbohydrate scaffold generates glycosamino acids, which are attractive building blocks for the preparation of carbohydrate-based libraries because of the well-established automated peptide synthesis. Derivatization as well as homo- and heterooligomerization of glycosamino acids can be used to create novel structures with unique properties. Glycosamino acids are hybrid structures of carbohydrates and amino acids which can be utilized to generate potential glycomimetics and peptidomimetics. The incorporation of glycosamino acids into peptides allows the engineering of carbohydrate-binding sites into synthetic polypeptides, which may also influence the pharmacokinetic and dynamic properties of the peptides. Furthermore, sugar-amino acid hybrids offer a tremendous structural and functional diversity, which is largely unexplored and requires combinatorial strategies for efficient exploitation. This article provides an overview of previous work on glycosamino acids and discusses their use in combinatorial synthesis and drug discovery. Supporting information for this article is available on the WWW under http://www.angewandte.com or from the author.     

Flexizyme‐Mediated Genetic Reprogramming As a Tool for Noncanonical Peptide Synthesis and Drug Discovery

Noncanonical peptides occur frequently in Nature, and often display high bioactivity. However, the lack of tractable systems for the synthesis of diverse libraries of such peptides has thus far hampered their development as drugs. Genetic reprogramming techniques, in which noncanonical amino acids may be incorporated into peptides, have largely removed this limitation. This Concept article outlines the development of these techniques with an emphasis on drug discovery.     

Chemical and Ribosomal Synthesis of Topologically Controlled Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether Formation

Bicyclic and tricyclic peptides have emerged as promising candidates for the development of protein binders and new therapeutics. However, convenient and efficient strategies that can generate topologically controlled bicyclic and tricyclic peptide scaffolds from fully‐unprotected peptides are still much in demand, particularly for those amenable to the design of biosynthetic libraries. In this work, we report a reliable chemical and ribosomal synthesis of topologically controlled bicyclic and tricyclic peptide scaffolds. Our strategy involves the combination of selenoether cyclization followed by disulfide or thioether cyclization, yielding desirable bicyclic and tricyclic peptides. This work thus lays the foundation for developing peptide libraries with controlled topology of multicyclic scaffolds for in vitro display techniques.     

Phage display: selecting straws instead of a needle from a haystack

An increasing number of peptides with specific binding affinity to various protein and even non-protein targets are being discovered from phage display libraries. The power of this method lies in its ability to efficiently and rapidly identify ligands with a desired target property from a large population of phage clones displaying diverse surface peptides. However, the search for the needle in the haystack does not always end successfully. False positive results may appear. Thus instead of specific binders phage with no actual affinity toward the target are recovered due to their propagation advantages or binding to other components of the screening system, such as the solid phase, capturing reagents, contaminants in the target sample or blocking agents, rather than the target. Biopanning experiments on different targets performed in our laboratory revealed some previously identified and many new target-unrelated peptide sequences, which have already been frequently described and published, but not yet recognized as target-unrelated. Distinguishing true binders from false positives is an important step toward phage display selections of greater integrity. This article thoroughly reviews and discusses already identified and new target-unrelated peptides and suggests strategies to avoid their isolation.     

An improved method for the synthesis of cellulose membrane-bound peptides with free C termini is useful for PDZ domain binding studies

SPOT synthesis permits parallel synthesis and screening of thousands of cellulose membrane-bound peptides to study protein-protein interactions in a proteomic context. Recognition of C-terminal residues is one of the most common binding features of PDZ domains. Unfortunately, most solid support-bound peptide libraries lack a free C terminus due to C-terminal fixation on the solid support. To overcome this restriction, we developed a robust methodology based on our previous strategy for generating peptides with authentic C termini. To validate this improved method, we screened a human peptide library of 6223 C termini with the syntrophin PDZ domain. Furthermore, using the same library, new peptide ligands derived from membrane proteins and receptors were found for the ERBIN PDZ domain. Finally, we identified the protein kinase breakpoint cluster region, which is known as a negative regulator of cell proliferation and oncogenic transformation, as an ERBIN ligand.     

Total solid-phase synthesis of marine cyclodepsipeptide IB-01212

A suitable combination of synthetic design, orthogonal protecting groups and coupling reagents was used to complete the first known synthesis of the natural marine cyclodepsipeptide IB-01212. The cyclic, symmetric octapeptide contains two of each of the following residues: L-N,N-Me2Leu, L-Ser, L-N-MeLeu and L-N-MePhe. IB-01212 also features two symmetric ester bonds between the hydroxyl group of Ser and the carboxyl function of the N-MePhe. Total solid-phase syntheses of the product was performed in parallel via three distinct routes: dimerization of heterodetic fragments, linear synthesis, and convergent synthesis. The convergent strategy gave the best results in terms of product yield and purity and is particularly suitable for the large-scale synthesis of IB-01212 and similar peptides.     

Comparison of bacterial and phage display peptide libraries in search of target-binding motif

Genetic engineering allows modification of bacterial and bacteriophage genes, which code for surface proteins, enabling display of random peptides on the surface of these microbial vectors. Biologic peptide libraries thus formed are used for high-throughput screening of clones bearing peptides with high affinity for target proteins. There are reports of many successful affinity selections performed with phage display libraries and substantially fewer cases describing the use of bacterial display systems. In theory, bacterial display has some advantages over phage display, but the two systems have never been experimentally compared. We tested both techniques in selecting streptavidin-binding peptides from two commercially available libraries. Under similar conditions, selection of phage-displayed peptides to model protein streptavidin proved convincingly better.     

An efficient method for the solid-phase synthesis of fluorescently labelled peptides

Fluorescent labelling of peptides is necessary in a wide range of cell biological applications. In the last decade, the application of cell-penetrating molecules has been advanced by the use of peptides, which have proven efficient in aiding nonpermeant molecules to cross the cell membrane. Currently, the development of new cell-penetrating peptides based on the design and synthesis of labelled peptide libraries is becoming critically important. Here we report an improved method for the solid-phase labelling of peptides, mediated by the activation of 5(6)-carboxyfluorescein with PyAOP/ HOAt.     

Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells

BACKGROUND: Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes. RESULTS: Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides. CONCLUSIONS: Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.     

Synthetic peptide arrays and peptide combinatorial libraries for the exploration of protein-ligand interactions and the design of protein inhibitors

Synthetic peptides have a long tradition as molecular tools in biomedical research and drug discovery. The introduction of high-throughput synthesis and screening technologies for synthetic peptides, such as arrays and combinatorial libraries, enabled the large-scale and detailed exploration of protein-ligand interactions, as well as the discovery of novel biologically active peptides. This review summarizes currently available synthetic peptide array and library technologies, in particular mixture-based peptide libraries, which are illustrated by numerous applications in various fields of biomedical research.