3D-Epitope-Explorer (3DEX): localization of conformational epitopes within three-dimensional structures of proteins

Neutralizing antibodies often recognize conformational, discontinuous epitopes. Linear peptides mimicking such conformational epitopes can be selected from phage display peptide libraries by screening with the respective antibodies. However, it is difficult to localize these "mimotopes" within the three-dimensional (3D) structures of the target proteins. Knowledge of conformational epitopes of neutralizing antibodies would help to design antigens able to elicit protective immune responses. Therefore, we provide here a software that allows to localize linear peptide sequences within 3D structures of proteins. The 3D-Epitope-Explorer (3DEX) software allows to map conformational epitopes in 3D protein structures based on an algorithm that takes into account the physicochemical neighborhood of C(alpha)- or C(beta)-atoms of individual amino acids. A given amino acid of a peptide sequence is localized within the protein and the software searches within predefined distances for the amino acids neighboring that amino acid in the peptide. Surface exposure of the amino acids can also be taken into consideration. The procedure is then repeated for the remaining amino acids of the peptide. The introduction of a joker function allows to map peptide mimotopes, which do not necessarily have 100% sequence homology to the protein. Using this software we were able to localize mimotopes selected from phage displayed peptide libraries with polyclonal antibodies from HIV-positive patient plasma within the 3D structure of gp120, the exterior glycoprotein of HIV-1. We also analyzed two recently published peptide sequences corresponding to known conformational epitopes to further confirm the integrity of 3DEX.     

Antigenic and immunogenic phage displayed mimotopes as substitute antigens: applications and limitations

The most exciting potential of phage displayed peptide libraries is to obtain small peptide molecules that mimic an antigen, at least with respect to a particular epitope. In addition to their interest as research tools, such mimotopes could in principle be useful as diagnostic tools or for eliciting antibodies to a predefined epitope. However, the reduction of the phage insert sequence to a short peptide that can compete with the antigenic and in particular with the immunogenic properties of the natural antigen faces considerable difficulties. This review assesses critically the antigenicity of phage displayed peptides as free peptides and in different molecular environments. The difficulties to use mimotopes to induce antibodies that bind to the natural antigen (crossreactive immunogenicity) and the considerable discrepancy between antigenicity and immunogenicity of phage-derived peptides are discussed. Peptides selected with antibodies from phage displayed random peptide libraries have raised considerable expectations as low molecular weight substitutes of the natural antigen. This review will focus on the results of phage displayed random peptide libraries screened with antibodies specific for proteins, carbohydrates and nucleic acids and critically examine how the above expectations have been met.     

MIMOTOPES,CONTINUOUS PARATOPES AND HYDROPATHIC COMPLEMENTARITY: NOVEL APPROXIMATIONS IN THE DESCRIPTION OF IMMUNOCHEMICAL SPECIFICITY

Most antigenic sites of proteins, known as discontinuous epitopes, are made up of residues on different loops that are brought together by the folding of the polypeptide chain. The individual loops are sometimes able, on their own, to bind to the antibody and they are then known as continuous epitopes. The binding sites of antibodies, known as paratopes, are built up from residues on six hypervariable loops known as complementarity determining regions (CDRs). Peptides corresponding to individual CDR loops are often able to bind the antigen and such peptides may be viewed as continuous paratopes. Using random combinatorial peptide libraries, it is possible to obtain peptides that bind to an antiprotein antibody without showing any sequence similarity with any part of the protein. Such epitope mimics are called mimotopes provided they are able also to elicit antibodies that react with the original antigen. The binding activity of mimotopes may partly be due to the phenomenon of hydropathic complementarity between epitope and paratope peptides. Although these concepts are vague in their structural connotation, they are useful for describing the immunological activity of linear peptides.     

Identification of inhibitors of an 80 kDa protease from Trypanosoma cruzi through the screening of a combinatorial peptide library

Two orthogonal peptide combinatorial libraries were screened to discover inhibitors of Tc80 protease, a novel target from Trypanosoma cruzi involved in host cell invasion. These libraries were composed of 15,625 structurally diversified tripeptides, partitioned in 125 mixtures. The screening led to a low micromolar inhibitor which was actually an HF cleavage by-product H-Ipe-D-Tic-D-Glu(S-paratolyl)-OH. IC50 values of several analogous molecules of this hit were determined and are discussed. For the best compounds, conformational analysis revealed a high degree of similarity in shape with a potent prolylendopeptidase inhibitor, SUAM-1221.     

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).     

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.     

Strategies for the construction and use of peptide and antibody libraries displayed on phages

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.     

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.     

Ribosomal synthesis of N-methyl peptides

N-methyl amino acids (N-Me AAs) are a common component of nonribosomal peptides (NRPs), a class of natural products from which many clinically important therapeutics are obtained. N-Me AAs confer peptides with increased conformational rigidity, membrane permeability, and protease resistance. Hence, these analogues are highly desirable building blocks in the ribosomal synthesis of unnatural peptide libraries, from which functional, NRP-like molecules may be identified. By supplementing a reconstituted Escherichia coli translation system with specifically aminoacylated total tRNA that has been chemically methylated, we have identified three N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) that are efficiently incorporated into peptides by the ribosome. Moreover, we have demonstrated the synthesis of peptides containing up to three N-Me AAs, a number comparable to that found in many NRP drugs. With improved incorporation efficiency and translational fidelity, it may be possible to synthesize combinatorial libraries of peptides that contain multiple N-Me AAs. Such libraries could be subjected to in vitro selection methods to identify drug-like, high-affinity ligands for protein targets of interest.     

Bioactive peptides from libraries

New ligands for a variety of biological targets can be selected from biological or synthetic combinatorial peptide libraries. The use of different libraries to select novel peptides with potential therapeutic applications is reviewed. The possible combination of molecular diversity provided by combinatorial libraries and a rational approach derived from computational modeling is also considered. Advantages and disadvantages of different approaches are compared. Possible strategies to bypass loss of peptide bioactivity in the transition from ligand selection to in vivo use are discussed.     

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.     

Approaches to Synthetic Vaccines Design of Epitope-Containing Amphiphilic Peptides Matching the Antigenic Structure in the Native Protein

A general procedure for the design of synthetic vaccines with the retained conformational features of protein antigenic determinants is described. This new concept emerges from detailed studies on the relationship between primary sequence and secondary structure formation of synthetic peptides and takes advantage of the amphiphilic nature of epitope-containing peptide segments in the native protein to accomplish structural modifications. These segments, for example amphiphilic helices or β-sheets, are stabilized by the insertion of secondary structure-inducing amino-acid residues on the hydrophobic part of the peptide without affecting the spatial arrangement of functional residues on the hydrophilic side. The availability of amphiphilic peptides with tailor-made conformational properties, e.g. helices, β-sheets, and, moreover, assemblies of these blocks to structures of higher order (‘folding units’), allows the presentation and stabilization of continuous as well as discontinuous epitopes by this approach. This strategy is exemplified for the case of two discontinuous epitopes taken from lysozyme, which are matched to host molecules with adequate conformational features by the help of computer-assisted molecular modelling. The implications of this new concept for the design of synthetic vaccines are discussed with special emphasis to the important role of peptide synthesis and chemical structure modification.     

Where are the GaPs? A rational approach to monomer acquisition and selection

Gridding and partitioning (GaP) is a computational method for the classification and selection of monomers for combinatorial libraries. The molecules are described in terms of the pharmacophoric groups they contain and where those pharmacophoric groups can be located in three-dimensional space. The approach involves a detailed conformational analysis of each molecule. This conformational analysis is done within a common coordinate frame, thus enabling the monomers to be compared. The use of a partitioned space is central to this particular application as it facilitates the identification of regions of space which are not well represented by existing compounds. Several ways to extend the use of partitioned pharmacophore spaces are described. Applications of the approach in monomer acquisition and in library design are outlined.     

Preferential binding of peptides to graphene edges and planes

Peptides identified from combinatorial peptide libraries have been shown to bind to a variety of abiotic surfaces. Biotic-abiotic interactions can be exploited to create hybrid materials with interesting electronic, optical, or catalytic properties. Here we show that peptides identified from a combinatorial phage display peptide library assemble preferentially to the edge or planar surface of graphene and can affect the electronic properties of graphene. Molecular dynamics simulations and experiments provide insight into the mechanism of peptide binding to the graphene edge.     

The rise, fall and reinvention of combinatorial chemistry

Combinatorial chemistry provides a powerful tool for the rapid creation of large numbers of synthetic compounds. Ideally, these libraries should be a rich source of bioactive molecules, but there is the general feeling that the initial promise of combinatorial chemistry has not yet been realized. In particular, enthusiasm for conducting unbiased (non-structure-guided) screens of large libraries for protein or RNA ligands has waned. A central challenge in this area is to devise methods for the synthesis of chemically diverse, high-quality libraries of molecules with many of the desirable features of natural products. These include diverse functionality, a significant representation of chiral sp(3) centers that provide conformational bias to the molecule, significant skeletal diversity, and good pharmacokinetic properties. However, these libraries must be easy to make from cheap, readily available building blocks, ideally those that would support convenient hit optimization/structure reactivity relationship studies. Meeting these challenges will not be easy. Here I review some recent advances in this area and provide some thoughts on likely important developments in the next few years.     

Immunoassays: biological tools for high throughput screening and characterisation of combinatorial libraries

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.     

Dynamic Amino Acid Side-Chains Grafting on Folded Peptide Backbone**

An efficient strategy for the synthesis of large libraries of conformationally defined peptides is reported, using dynamic combinatorial chemistry as a tool to graft amino acid side chains on a well-ordered 3D (3-dimension) peptide backbone. Combining rationally designed scaffolds with combinatorial side chains selection represents an alternative method to access peptide libraries for structures that are not genetically encodable. This method would allow a breakthrough for the discovery of protein mimetic for unconventional targets for which little is known.     

Multiple Peptide Synthesis Methods and Their Applications. New Synthetic Methods (87)

Rapid developments in the biotechnology of new proteins, as well as advances in immunology and the development of pharmaceuticals based on inhibitors and antagonists, have led to immense demands for synthetic peptides. Simultaneous preparation of 100–150 completely different peptides, having chain lengths of up to 20 amino acids can nowadays be achieved using multiple synthesis methods. The yields and qualities of the peptides so obtained are high enough to permit reliable in vivo and in vitro screening for biological activities. Moreover, it is possible to optimize synthetic conditions and to carry out comparative studies on the secondary structures and conformational mapping of proteins. Special multiple synthesis methods facilitate the epitope mapping of larger peptides for diagnostic purposes and for the development of vaccines based on a few hundreds of free or rod-bound peptides that are useful for immunoassays. Multiple methods of peptide synthesis also enable the preparation of so-called peptide libraries which could comprise hundreds of thousands of peptides, and by which new perspectives for the screening of lead structures will be opened up. Peptide synthesis using a combination of photolabile protecting groups and photolithographic procedures enables the assembling of peptide libraries on small plates for use in miniature immunoassays. Furthermore, lipopeptide-antigen conjugates allow both the preparation of peptide-specific and monoclonal antibodies as well as a complete screening of epitopes of B-, T-helper and T-killer cells. Applications in the areas of AIDS diagnosis, the development of vaccines, and screening for the hormone analogues, demonstrate just some of the possibilities that have been opened up by multiple peptide synthesis methods.     

Molecular forceps from combinatorial libraries prevent the farnesylation of Ras by binding to its carboxyl terminus

INTRODUCTION: Ras is one of the major oncogenes. In order to function properly it has to undergo post-translational processing at its carboxyl terminus. It has been shown that inhibitors of farnesyl transferase, the first enzyme in the processing chain, can suppress the transforming activity of oncogenic Ras. RESULTS: We have identified molecular forceps, branched peptidic molecules, from combinatorial libraries that bind to the carboxyl terminus of Ras and interfere with its farnesylation without inhibiting the farnesyl transferase. The active molecules were selected by a screening against the carboxy-terminal octapeptide of Ras. CONCLUSIONS: The implications of our findings are twofold. First, we demonstrate that it is possible to prevent enzymatic transformations by blocking the enzyme's access to its substrate using a synthetic small molecule to mask the substrate. Second, we show that it is feasible to derive molecules from combinatorial libraries that bind a specific epitope on a protein by selecting these molecules with the isolated peptide epitope.