A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

A mini-review and perspective on multicyclic peptide mimics of antibodies

This review gave a brief introduction on recent development in monocyclic and multicyclic peptide mimics of antibodies and provides a perspective on screening and design of multicyclic peptide mimics of antibodies in the future.     

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.     

Short peptides as biosensor transducers

This review deals with short peptides (up to 50 amino acids) as biomimetic active recognition elements in sensing systems. Peptide-based sensors have been developed in recent years according to different strategies. Synthetic peptides have been designed on the basis of known interactions between single or a few amino acids and targets, with attention being paid to the presence of peptide motifs known to allow intermolecular self-organization of the sensing peptides over the sensor surface. Sensitive and sophisticated sensors have been obtained in this way, but the use of designed peptides is limited by severe difficulties in their in silico design. Short peptides from random phage display have been selected in a random way from large, unfocussed, and often preexisting and commercially available phage display libraries, with no design elements. Such peptides often perform better than antibodies, but they are difficult to select when the target is a small molecule because of the need to immobilize it with considerable modifications of its structure. Artificial, miniaturized receptors have been obtained from the reduction of the known sequence of a natural receptor down to a synthesizable and yet stable one. Alternatively, binding sites have been created over a designed, stable peptide scaffold. Short peptides have also been used as active elements for the detection of their own natural receptors: pathogenic bacteria have been detected with antimicrobial and cell-penetrating peptides, but key challenges such as detection of bacteria in real samples, improved sensitivity, and improved selectivity have to be faced. Peptide substrates have been conjugated to fluorescent quantum dots to obtain disposable sensors for protease activity with high sensitivity. Ferrocene–peptide conjugates have been used for electrochemical sensing of protease activity.     

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.     

在肽库中进行小肽筛选的初步研究

以合成六肽ＤＧＧＳＡＡ为模型对在噬菌体肽库中进行小肽筛选做了初步研究。结果表明，含有可形成氢键和离子键残基的小肽能够在噬菌体肽库中进行筛选。并用明显提高了筛选的专一性。     

Phage selection of bicyclic peptides binding Her2

Aberrant expression of the epidermal growth factor receptor Her2 has been implicated in various malignancies including breast cancer. Monoclonal antibodies and an antibody–drug conjugate targeting Her2 have found wide clinical application. Herein, we aimed at developing Her2-specifc ligands based on peptides that have a 100-fold smaller molecular weight than antibodies. Such peptides could potentially offer advantages in the development of ligand–drug conjugates, such as ease of synthesis and conjugation, higher molecule-per-mass ratios, and better tumor penetration. Panning of large bicyclic peptide phage display libraries against Her2 yielded a range of Her2-specific ligands having different formats and binding motifs. Strong sequence similarities among several of the isolated peptides indicated that they interact with Her2 in a specific manner. The best bicyclic peptide obtained after affinity maturation bound Her2 with a K_D of 304 nM. The diverse peptide ligands may offer valuable starting points for the development of high-affinity Her2 binders with potential application for tumor imaging and therapy.     

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.     

Engineered Peptide Macrocycles Can Inhibit Matrix Metalloproteinases with High Selectivity

Matrix metalloproteinases (MMPs) are zinc‐dependent endopeptidases at the intersection of health and disease due to their involvement in processes such as tissue repair and immunity as well as cancer and inflammation. Because of the high structural conservation in the catalytic domains and shallow substrate binding sites, selective, small‐molecule inhibitors of MMPs have remained elusive. In a tour‐de‐force peptide engineering approach combining phage‐display selections, rational design of enhanced zinc chelation, and d ‐amino acid screening, we succeeded in developing a first synthetic MMP‐2 inhibitor that combines high potency (K_i=1.9±0.5 nm ), high target selectivity, and proteolytic stability, and thus fulfills all the required qualities for in cell culture and in vivo application. Our work suggests that selective MMP inhibition is achievable with peptide macrocycles and paves the way for developing specific inhibitors for application as chemical probes and potentially therapeutics.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

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.     

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.     

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.     

A modular assembly strategy for improving the substrate specificity of small catalytic peptides

In contrast to large proteins, small peptide catalysts typically display limited specificity for small molecule substrates. This is presumably a result of the limited opportunities small peptides have to fold in a manner that provides for the formation of an isolated reaction vessel that effectively binds and sequesters substrates from bulk solvent while at the same time catalyzing their transformation. For the preparation of small peptide catalysts that possess improved substrate specificity, we have developed a modular assembly strategy that involves appending phage display-derived substrate binding-domain modules to catalytically active peptide domains. We demonstrate the potential of this strategy with the construction of a small 35-amino acid residue aldolase peptide with improved substrate specificity. The advantages of this approach are that it reduces the demand on the functionalization of the catalytic site and it is modular, therefore making its adaptation to a variety of specificities rapid. The modular assembly strategy studied here may present advantages over exhaustive searches of large random-sequence peptide libraries for peptides with singular function.     

Torsional and Electronic Factors Control the C−H⋅⋅⋅O Interaction

The precise role of non‐conventional hydrogen bonds such as the C?H???O interaction in influencing the conformation of small molecules remains unresolved. Here we survey a series of β‐turn mimetics using X‐ray crystallography and NMR spectroscopy in conjunction with quantum calculation, and conclude that favourable torsional and electronic effects are important for the population of states with conformationally influential C?H???O interactions. Our results also highlight the challenge in attempting to deconvolute a myriad of interdependent noncovalent interactions in order to focus on the contribution of a single one. Within a small molecule that is designed to resemble the complexity of the environment within peptides and proteins, the interplay of different steric burdens, hydrogen‐acceptor/‐donor properties and rotational profiles illustrate why unambiguous conclusions based solely on NMR chemical shift data are extremely challenging to rationalize.     

A Two‐Tailed Phosphopeptide Crystallizes to Form a Lamellar Structure

The crystal structure of a designed phospholipid‐inspired amphiphilic phosphopeptide at 0.8 Å resolution is presented. The phosphorylated β‐hairpin peptide crystallizes to form a lamellar structure that is stabilized by intra‐ and intermolecular hydrogen bonding, including an extended β‐sheet structure, as well as aromatic interactions. This first reported crystal structure of a two‐tailed peptidic bilayer reveals similarities in thickness to a typical phospholipid bilayer. However, water molecules interact with the phosphopeptide in the hydrophilic region of the lattice. Additionally, solid‐state NMR was used to demonstrate correlation between the crystal structure and supramolecular nanostructures. The phosphopeptide was shown to self‐assemble into semi‐elliptical nanosheets, and solid‐state NMR provides insight into the self‐assembly mechanisms. This work brings a new dimension to the structural study of biomimetic amphiphilic peptides with determination of molecular organization at the atomic level.     

On the Role of Chirality in Guiding the Self‐Assembly of Peptides

Homochirality in peptides is crucial in sustaining “like–like” intermolecular interactions that allow the formation of assemblies and aggregates and is ultimately responsible for the resulting material properties. With the help of a series of stereoisomers of the tripeptide F–F–L, we demonstrate the critical role that peptide stereochemistry plays in the self‐assembly of peptides, guided by molecular recognition, and for self‐sorting. Homochiral self‐assemblies are thermally and mechanically more robust compared to heterochiral self‐assemblies. Morphological studies of the multicomponent peptide systems showed that aggregates formed from homochiral peptides possessed a uniform nano‐fibrous structure, whereas heterochiral systems resulted in self‐sorted systems with a heterogeneous morphology. In essence, homochiral peptides form the stronger aggregates, which may be one of reasons why homochirality is preferred in living systems.     

Efficient Phage Display with Multiple Distinct Non‐Canonical Amino Acids Using Orthogonal Ribosome‐Mediated Genetic Code Expansion

Phage display is a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report a system for incorporating non‐canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl‐tRNA synthetase/tRNA pair. We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropene‐containing ncAA (CypK) at diverse sites on a displayed single‐chain antibody variable fragment (ScFv), in response to amber and quadruplet codons. CypK and an alkyne‐containing ncAA are incorporated at distinct sites, enabling the double labeling of ScFv with distinct probes, through mutually orthogonal reactions, in a one‐pot procedure. These advances expand the number of functionalities that can be encoded on phage‐displayed proteins and provide a foundation to further expand the scope of phage display applications.     

Peptides as New Smart Bionanomaterials: Molecular‐Recognition and Self‐Assembly Capabilities

Biomolecules express exquisite properties that are required for molecular recognition and self‐assembly on the nanoscale. These smart capabilities have developed through evolution and such biomolecules operate based on smart functions in natural systems. Recently, these remarkable smart capabilities have been utilized in not only biologically related fields, but also in materials science and engineering. A peptide‐screening technology that uses phage‐display systems has been developed based on this natural smart evolution for the generation of new functional peptide bionanomaterials. We focused on peptides that specifically bound to synthetic polymers. These polymer‐binding peptides were screened by using a phage‐display peptide library to recognize nanostructures that were derived from polymeric structural features and were utilized for possible applications as new bionanomaterials. We also focused on self‐assembling peptides with β‐sheet structures that formed nanoscale, fibrous structures for applications in new bottom‐up nanomaterials. Moreover, nanofiber‐binding peptides were also screened to introduce the desired functionalities into nanofibers without the need for additional molecular design. Our approach to construct new bionanomaterials that employ peptides will open up excellent opportunities for the next generation of materials science and technology.