Phagemid encoded small molecules for high throughput screening of chemical libraries

A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.     

Material binding peptides for nanotechnology

Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.     

Phage display of combinatorial peptide libraries: application to antiviral research

Given the growing number of diseases caused by emerging or endemic viruses, original strategies are urgently required: (1) for the identification of new drugs active against new viruses and (2) to deal with viral mutants in which resistance to existing antiviral molecules has been selected. In this context, antiviral peptides constitute a promising area for disease prevention and treatment. The identification and development of these inhibitory peptides require the high-throughput screening of combinatorial libraries. Phage-display is a powerful technique for selecting unique molecules with selective affinity for a specific target from highly diverse combinatorial libraries. In the last 15 years, the use of this technique for antiviral purposes and for the isolation of candidate inhibitory peptides in drug discovery has been explored. We present here a review of the use of phage display in antiviral research and drug discovery, with a discussion of optimized strategies combining the strong screening potential of this technique with complementary rational approaches for identification of the best target. By combining such approaches, it should be possible to maximize the selection of molecules with strong antiviral potential.     

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.     

Application of protein-coupled liposomes to effective affinity screening from phage library

For effective screening by biopanning, we propose a new affinity screening method utilizing protein-coupled liposomes (proteoliposomes) as adsorbents. With multilamellar vesicles (MLVs) composed of dipalmitoylphosphatidylcholine (DPPC): dicetylphosphate (DCP) = 10: 1 (molar ratio), adsorption of nonspecific phage VCSM13 to the liposomes without any blocking was comparable to that on polystyrene tube wall coated with blocking protein. Phages displaying octapeptides specific to an anti-peptide antibody against a peptide antigen (FVNQHLCK) were screened from an octapeptide-displayed phage library by biopanning utilizing liposomes coupled with the antibody (AB-MLVs) or a conventional immunotube coated with the antibody (AB-tube). After four rounds of biopanning, all selected phages displayed homological peptides to the antigen peptide by use of AB-MLVs, while only 15% of the selected phages displayed homological peptides in the conventional biopanning. The octapeptide selected by AB-MLVs against the anti-peptide antibody showed comparable binding affinity, which were determined by the competitive ELISA and an immunoaffinity chromatography, to that of the peptide antigen. Thus, protein-coupled liposomes are useful as adsorbents for screening from combinatorial phage libraries.     

Peptide phage display as a tool for drug discovery: targeting membrane receptors

Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Consequently, peptides derived from library screenings often modulate the target protein's activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. This review discusses the use of phage display to identify membrane receptor modulators with agonistic or antagonistic activities. Because isolating or producing recombinant membrane proteins for use as target molecules in library screening is often impossible, innovative selection strategies such as panning against whole cells or tissues, recombinant receptor ectodomains, or neutralizing antibodies to endogenous binding partners were devised. Prominent examples from a two-decade history of peptide phage display will be presented, focusing on the design of affinity selection experiments, methods for improving the initial hits, and applications of the identified peptides.     

Peptide Ligands Stabilized by Small Molecules

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100‐fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle‐free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X‐ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide–protein complexes and contribute to the high binding affinity.     

High-throughput screening of surface displayed gene products

With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.     

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.     

Identification of small molecule binding sites within proteins using phage display technology

Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.     

Phage display of antibody fragments

In recent years, phage display of peptides and proteins has become a very popular method in oncology, immunology, protein engineering and ligand-receptor studies among others. Antibody fragments, as Fabs or single chain Fv, have been among the first proteins to be displayed on the surface of a filamentous bacteriophage with a procedure initially described in 1990 by McCafferty et al. (Nature, 348, 552-554). From that time, molecular biology techniques have allowed the creation of large repertoires of antibody fragments from antibody V genes, bypassing hybrydoma technology and even immunisation. A large number of phage antibody libraries, from which molecules of the desired functional properties can be rapidly selected, have been built and distributed in many laboratories world-wide. Antibody fragments recovered from phage libraries generally show moderate binding strength; with different systems of biopanning binders can be obtained with dissociation constant ranged between 10-(5) to 10-(8) M. Nevertheless, antibody fragments can be furtherly modified to improve affinity or avidity, respectively by mutating crucial residues of complementarity determining regions or by increasing the number of binding sites making dimeric, trimeric or multimeric molecules. Here, we summarise the latest progress in this field, with particular reference to applications of scFv in the diagnosis and therapy of solid tumours and in the molecular mimicry of viral antigens and membrane receptors. In fact, the production of artificial protein epitopes by phage antibodies is becoming a valid system to overcome problems caused by difficult cloning and low expression of particular recombinant proteins.     

The advantage of being virtual--target-induced adaptation and selection in dynamic combinatorial libraries

Numerical simulations are presented that describe the adaptive behavior of simple dynamic combinatorial libraries (DCLs) upon addition of a target. By studying the effect of various parameters such as the network topology, the initial concentrations, the association constants, and the binding affinities, general characteristics of such systems were derived. It is shown that the adaptation may lead to the amplification of molecules with a high affinity to the target, but only for specific boundary conditions. Furthermore, it is demonstrated that the selection process can be refined by using an evolutionary approach. These results are of importance for the design of selection experiments with DCLs.     

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.     

Protein ligand design: from phage display to synthetic protein epitope mimetics in human antibody Fc-binding peptidomimetics

Phage display is a powerful method for selecting peptides with novel binding functions. Synthetic peptidomimetic chemistry is a powerful tool for creating structural diversity in ligands as a means to establish structure-activity relationships. Here we illustrate a method of bridging these two methodologies, by starting with a disulfide bridged phage display peptide which binds a human antibody Fc fragment (Delano et al. Science 2000, 287, 1279) and creating a backbone cyclic beta-hairpin peptidomimetic with 80-fold higher affinity for the Fc domain. The peptidomimetic is shown to adopt a well-defined beta-hairpin conformation in aqueous solution, with a bulge in one beta-strand, as seen in the crystal structure of the phage peptide bound to the Fc domain. The higher binding affinity of the peptidomimetic presumably reflects the effect of constraining the free ligand into the conformation required for binding, thus highlighting in this example the influence that ligand flexibility has on the binding energy. Since phage display peptides against a wide variety of different proteins are now accessible, this approach to synthetic ligand design might be applied to many other medicinally and biotechnologically interesting target proteins.     

Probing the interaction between peptides and metal oxides using point mutants of a TiO2-binding peptide

An increasing number of peptides with specific binding affinity to inorganic materials are being isolated using combinatorial peptide libraries without prior knowledge about the interaction between peptides and target materials. The lack of understanding of the mechanism and the contribution of constituent amino acids to the peptides' inorganic-binding ability poses an obstacle to optimizing and tuning of the binding affinity of peptides to inorganic materials and thus hinders the practical application of these peptides. Using the phage surface display technique, we previously identified a disulfide-bond-constrained peptide (-CHKKPSKSC-, STB1) cognitive of TiO2. In the present study, the interaction of STB1 with TiO2 was probed using a series of point mutants of STB1 displayed on phage surfaces. Their binding affinity was measured using a quartz crystal microbalance with energy dissipation measurement and compared on the basis of the delta f or delta D values. The three K residues of STB1 were found to be essential and sufficient for phage particle binding to TiO2. One mutant with five K residues showed not stronger but weaker binding affinity than STB1 due to its conformational restriction, as illustrated by molecular dynamics simulation, to align five K residues in a way conducive to their simultaneous interaction with the TiO2 surface. The contextual influence of noncharged residues on STB1's binding affinity was also investigated. Our results may provide insight into the electrostatic interaction between peptides and inorganic surfaces.     

Rapid identification of small binding motifs with high-throughput phage display: discovery of peptidic antagonists of IGF-1 function

A panel of 22 na?ve peptide libraries was constructed in a polyvalent phage display format and sorted against insulin-like growth factor-1 (IGF-1). The libraries were pooled to achieve a total diversity of 4.4 x 10(11). After three rounds of selection, the majority of the phage clones bound specifically to IGF-1, with a disulfide-constrained CX(9)C scaffold dominating the selection. Four monovalently displayed sub-libraries were designed on the basis of these conserved motifs. Sub-library maturation in a monovalent format yielded an antagonistic peptide that inhibited the interactions between IGF-1 and two cell-surface receptors and those between IGF-1 and two soluble IGF binding proteins with micromolar potency. NMR analysis revealed that the peptide is highly structured in the absence of IGF-1, and peptides that preorganize the binding elements were selected during the sorting.     

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.     

Generation of anti-colorectal cancer fab phage display libraries with a high percentage of diverse antigen-reactive clones

A combinatorial Fab phage display library was generated from the antibody variable region genes of each of 2 BALB/c mice immunized with the human colorectal cancer cell lines SW480, SW948, and SW837. These libraries were shown to be diverse by nucleotide sequencing and diagnostic restriction enzyme digestion (fingerprinting) of individual members. The two libraries were combined and selected for binding to a suspension of formaldehyde-fixed human colorectal cancer cells in two successive rounds of selection and phage amplification by infection of bacteria. Analysis of the selected libraries as well as individual library clones by ELISA, showed binding to the cancer cell lines in both formaldehyde-fixed and native forms. Fifty five percent and 94% of library clones were positive for colorectal cancer cell binding after the first and second rounds of selection, respectively. Fingerprinting of individual clones showed the first round selected library to be very diverse and the second round selected library to be of more limited diversity. After absorption with normal human cell types, these anti-cancer selected libraries could be used to develop therapeutic and/or diagnostic agents.     

Crossing borders to bind proteins—a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.