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.     

From phage display to dendrimer display: insights into multivalent binding

Phage display is widely used for the selection of target-specific peptide sequences. Presentation of phage peptides on a multivalent platform can be used to (partially) restore the binding affinity. Here, we present a detailed analysis of the effects of valency, linker choice, and receptor density on binding affinity of a multivalent architecture, using streptavidin (SA) as model multivalent receptor. For surfaces with low receptor densities, the SA binding affinity of multivalent dendritic phage peptide constructs increases over 2 orders of magnitude over the monovalent species (e.g., K(d,mono) = 120 μM vs K(d,tetra) = 1 μM), consistent with previous work. However, the affinity of the SA-binding phage presenting the exact same peptides was 16 pM when dense receptor surfaces used for initial phage display were used in assays. The phage affinity for SA-coated surfaces weakens severely toward the nanomolar regime when surface density of SA is decreased. A similarly strong dependence in this respect was observed for dendritic phage analogues. When presented with a dense SA-coated surface, dendrimer display affords up to a 10(4)-fold gain in affinity over the monovalent peptide. The interplay between ligand valency and receptor density is a fundamental aspect of multivalent targeting strategies in biological systems. The perspective offered here suggests that in vivo targeting schemes might best be served to conduct ligand selection under physiologically relevant receptor density surfaces, either by controlling the receptor density placed at the selection surface or by using more biologically relevant intact cells and tissues.     

Phage-Display Based Discovery and Characterization of Peptide Ligands against WDR5

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein–protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.     

Rapid, multiplexed microfluidic phage display

The development of a method for high-throughput, automated proteomic screening could impact areas ranging from fundamental molecular interactions to the discovery of novel disease markers and therapeutic targets. Surface display techniques allow for efficient handling of large molecular libraries in small volumes. In particular, phage display has emerged as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Yet, the process becomes cumbersome and time-consuming when multiple targets are involved. Here we demonstrate for the first time a microfluidic chip capable of identifying high affinity phage-displayed peptides for multiple targets in just a single round and without the need for bacterial infection. The chip is shown to be able to yield well-established control consensus sequences while simultaneously identifying new sequences for clinically important targets. Indeed, the confined parameters of the device allow not only for highly controlled assay conditions but also introduce a significant time-reduction to the phage display process. We anticipate that this easily-fabricated, disposable device has the potential to impact areas ranging from fundamental studies of protein, peptide, and molecular interactions, to applications such as fully automated proteomic screening.     

Chromatographic biopanning for the selection of peptides with high specificity to Pb from phage displayed peptide library

Toxic heavy metal pollution is a global problem occurring in air, soil as well as water. There is a need for a more cost effective, renewable remediation technique, but most importantly, for a recovery method that is selective for one specific metal of concern. Phage display technology has been used as a powerful tool in the discovery of peptides capable of exhibiting specific affinity to various metals or metal ions. However, traditional phage display is mainly conducted in batch mode, resulting in only one equilibrium state hence low-efficiency selection. It is also unable to monitor the selection process in real time mode. In this study, phage display technique was incorporated with chromatography procedure with the use of a monolithic column, facilitating multiple phage-binding equilibrium states and online monitoring of the selection process in search of affinity peptides to Pb²⁺. In total, 17 candidate peptides were found and their specificity toward Pb²⁺ was further investigated with bead-based enzyme immunoassay (EIA). A highly specific Pb²⁺ binding peptide ThrAsnThrLeuSerAsnAsn (TNTLSNN) was obtained. Based on our knowledge, this is the first report on a new chromatographic biopanning method coupled with monolithic column for the selection of metal ion specific binding peptides. It is expected that this monolith-based chromatographic biopanning will provide a promising approach for a high throughput screening of affinity peptides cognitive of a wide range of target species.     

Adsorption behavior of linear and cyclic genetically engineered platinum binding peptides

Recently, phage and cell-surface display libraries have been adapted for genetically selecting short peptides for a variety of inorganic materials. Despite the enormous number of inorganic-binding peptides reported and their bionanotechnological utility as synthesizers and molecular linkers, there is still a limited understanding of molecular mechanisms of peptide recognition of and binding to solid materials. As part of our goal of genetically designing these peptides, understanding the binding kinetics and thermodynamics, and using the peptides as molecular erectors, in this report we discuss molecular structural constraints imposed upon the quantitative binding characteristics of peptides with an affinity for inorganics. Specifically, we use a high-affinity seven amino acid Pt-binding sequence, PTSTGQA, as we reported in earlier studies and build two constructs: one is a Cys-Cys constrained "loop" sequence (CPTSTGQAC) that mimics the domain used in the pIII tail sequence of the phage library construction, and the second is the linear form, a septapeptide, without the loop. Both sequences were analyzed for their adsorption behavior on Pt thin films by surface plasmon resonance (SPR) spectroscopy and for their conformational properties by circular dichroism (CD). We find that the cyclic peptide of the integral Pt-binding sequence possesses single or 1:1 Langmuir adsorption behavior and displays equilibrium and adsorption rate constants that are significantly larger than those obtained for the linear form. Conversely, the linear form exhibits biexponential Langmuir isotherm behavior with slower and weaker binding. Furthermore, the structure of the cyclic version was found to adopt a random coil molecular conformation, whereas the linear version adopts a polyproline type II conformation in equilibrium with the random coil. The 2,2,2-trifluoroethanol titration experiments indicate that TFE has a different effect on the secondary structures of the linear and cyclic versions of the Pt binding sequence. We conclude that the presence of the Cys-Cys restraint affects both the conformation and binding behavior of the integral Pt-binding septapeptide sequence and that the presence or absence of constraints could be used to tune the adsorption and structural features of inorganic binding peptide sequences.     

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.     

RNA architecture dictates the conformations of a bound peptide.

BACKGROUND: The biological function of several viral and bacteriophage proteins, and their arginine-rich subdomains, involves RNA-mediated interactions. It has been shown recently that bound peptides adopt either beta-hairpin or alpha-helical conformations in viral and phage peptide-RNA complexes. We have compared the structures of the arginine-rich peptide domain of HIV-1 Rev bound to two RNA aptamers to determine whether RNA architecture can dictate the conformations of a bound peptide. RESULTS: The core-binding segment of the HIV-1 Rev peptide class II RNA aptamer complex spans the two-base bulge and hairpin loop of the bound RNA and the carboxy-terminal segment of the bound peptide. The bound peptide is anchored in place by backbone and sidechain intermolecular hydrogen bonding and van der Waals stacking interactions. One of the bulge bases participates in U*(A*U) base triple formation, whereas the other is looped out and flaps over the bound peptide in the complex. The seven-residue hairpin loop is closed by a sheared G*A mismatch pair with several pyrimidines looped out of the hairpin fold. CONCLUSIONS: Our structural studies establish that RNA architecture dictates whether the same HIV-1 Rev peptide folds into an extended or alpha-helical conformation on complex formation. Arginine-rich peptides can therefore adapt distinct secondary folds to complement the tertiary folds of their RNA targets. This contrasts with protein-RNA complexes in which elements of RNA secondary structure adapt to fit within the tertiary folds of their protein targets.     

Design of peptide that recognizes double-stranded DNA.

A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.     

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.     

Simultaneous prediction of binding free energy and specificity for PDZ domain–peptide interactions

Interactions between protein domains and linear peptides underlie many biological processes. Among these interactions, the recognition of C-terminal peptides by PDZ domains is one of the most ubiquitous. In this work, we present a mathematical model for PDZ domain–peptide interactions capable of predicting both affinity and specificity of binding based on X-ray crystal structures and comparative modeling with Rosetta. We developed our mathematical model using a large phage display dataset describing binding specificity for a wild type PDZ domain and 91 single mutants, as well as binding affinity data for a wild type PDZ domain binding to 28 different peptides. Structural refinement was carried out through several Rosetta protocols, the most accurate of which included flexible peptide docking and several iterations of side chain repacking and backbone minimization. Our findings emphasize the importance of backbone flexibility and the energetic contributions of side chain-side chain hydrogen bonds in accurately predicting interactions. We also determined that predicting PDZ domain–peptide interactions became increasingly challenging as the length of the peptide increased in the N-terminal direction. In the training dataset, predicted binding energies correlated with those derived through calorimetry and specificity switches introduced through single mutations at interface positions were recapitulated. In independent tests, our best performing protocol was capable of predicting dissociation constants well within one order of magnitude of the experimental values and specificity profiles at the level of accuracy of previous studies. To our knowledge, this approach represents the first integrated protocol for predicting both affinity and specificity for PDZ domain–peptide interactions.     

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.     

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.     

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.     

Accelerated screening of phage-display output with alkaline phosphatase fusions

When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin N-terminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.     

Targeted binding of the M13 bacteriophage to thiamethoxam organic crystals

Phage display screening with a combinatorial library was used to identify M13-type bacteriophages that express peptides with selective binding to organic crystals of thiamethoxam. The six most strongly binding phages exhibit at least 1000 times the binding affinity of wild-type M13 and express heptapeptide sequences that are rich in hydrophobic, hydrogen-bonding amino acids and proline. Among the peptide sequences identified, M13 displaying the pIII domain heptapeptide ASTLPKA exhibits the strongest binding to thiamethoxam in competitive binding assays. Electron and confocal microscopy confirm the specific binding affinity of ASTLPKA to thiamethoxam. Using atomic force microscope (AFM) probes functionalized with ASTLPKA expressing phage, we found that the average adhesion force between the bacteriophage and a thiamethoxam surface is 1.47 ± 0.80 nN whereas the adhesion force of wild-type M13KE phage is 0.18 ± 0.07 nN. Such a strongly binding bacteriophage could be used to modify the surface chemistry of thiamethoxam crystals and other organic solids with a high degree of specificity.     

Context-dependence of the contribution of disulfide bonds to beta-hairpin stability

Incorporation of disulfide bonds to stabilize protein and peptide structures is not always a successful strategy. To advance current knowledge on the contribution of disulfide bonds to beta-hairpin stability, a previously reported beta-hairpin-forming peptide was taken as a template to design a series of Cys-containing peptides. The conformational behavior of these peptides in their oxidized, disulfide-cyclized peptides, and reduced, linear peptides, was investigated on the basis of NMR parameters: NOEs, and 1H and 13C chemical shifts. We found that the effect of disulfide bonds on beta-hairpin stability depends on its location within the beta-hairpin structure, being very small or even destabilizing when connecting two hydrogen-bonded facing residues. When the disulfide bond is linking non-hydrogen-bonded facing residues, we estimated that its contribution to the free-energy change of beta-hairpin folding is approximately -1.0 kcal mol(-1). This value is larger than those reported for most beta-hairpin-stabilizing cross-strand side-chain-side-chain interactions, except for some aromatic-aromatic interactions, in particular the Trp-Trp one, and the cation-pi interaction between Trp and the non-natural methylated Arg/Lys. As disulfide bonds are frequently used to stabilize peptide conformations, our conclusions can be useful for peptide, peptidomimetic, and protein design, and may even extend to other chemical cross-links.     

A minimal peptide scaffold for beta-turn display: optimizing a strand position in disulfide-cyclized beta-hairpins.

Phage display of peptide libraries has become a powerful tool for the evolution of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly understood, and consequently, structure-activity relationships in these molecules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constrained beta-hairpins and assessed their suitability as scaffolds for beta-turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand position. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta-hairpin structure in the disulfide-cyclized peptides. Reexamination of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns and strand-strand interactions make independent, additive contributions to hairpin stability. Significantly, the tryptophan strand substitution is highly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C' ' turn of CD4 on this tryptophan "stem" are highly structured beta-hairpins in water. Thus, we have developed a small, structured beta-turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.     

Mimotopes for Mycotoxins Diagnosis Based on Random Peptides or Recombinant Antibodies from Phage Library

Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.