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.     

Evolution of peptides that modulate the spectral qualities of bound, small-molecule fluorophores

Background: Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo.Results: A panel of Texas red, Rhodamine red, Oregon green 514 and fluorescein binding peptides, termed here ‘fluorettes’, was selected via biopanning of a combinatorial library of 12-mer peptides fused to a minor coat plll protein of the filamentous bacteriophage M 13, The ‘best’ fluorette sequences from each of the groups were subjected to further mutagenesis, followed by a second biopanning to select a new generation of improved fluorettes. Phage were selected that had higher avidity for each fluorophore except Rhodamine red, Of these, peptides were characterized that could specifically and with high affinity bind at least one dye, Texas red, in solution, In addition, the binding of certain peptides to Texas red shifted the peak excitation and/or the emission spectra of the bound dye.Conclusions: Peptides in the context of phage display could readily be selected that could bind to small-molecule fluorophores. The affinities of selected mutant fluorettes could be increased by mutation and further selection, Only a subset of the free peptides could bind free dyes in solution, suggesting that phage context contributed to the selection and ability of certain peptidic regions to independently bind the dyes, Future screens might lead to the creation of other dye-binding peptides with novel characteristics or Texas red derivatives with cross-linking substituents might be designed to increase the utility of the system.     

Small-molecule microarrays as tools in ligand discovery

Small molecules that bind and modulate specific protein targets are increasingly used as tools to decipher protein function in a cellular context. Identifying specific small-molecule probes for each protein in the proteome will require miniaturized assays that permit screening of large collections of compounds against large numbers of proteins in a highly parallel fashion. Simple and general binding assays involving small-molecule microarrays can be used to identify probes for nearly any protein in the proteome. The assay may be used to identify ligands for proteins in the absence of knowledge about structure or function. In this tutorial review, we introduce small-molecule microarrays (SMMs) as tools for ligand discovery; discuss methods for manufacturing SMMs, including both non-covalent and covalent attachment strategies; and provide examples of ligand discovery involving SMMs.     

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.     

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.     

Uracil-directed ligand tethering: an efficient strategy for uracil DNA glycosylase (UNG) inhibitor development

Uracil DNA glycosylase (UNG) is an important DNA repair enzyme that recognizes and excises uracil bases in DNA using an extrahelical recognition mechanism. It is emerging as a desirable target for small-molecule inhibitors given its key role in a wide range of biological processes including the generation of antibody diversity, DNA replication in a number of viruses, and the formation of DNA strand breaks during anticancer drug therapy. To accelerate the discovery of inhibitors of UNG we have developed a uracil-directed ligand tethering strategy. In this efficient approach, a uracil aldehyde ligand is tethered via alkyloxyamine linker chemistry to a diverse array of aldehyde binding elements. Thus, the mechanism of extrahelical recognition of the uracil ligand is exploited to target the UNG active site, and alkyloxyamine linker tethering is used to randomly explore peripheral binding pockets. Since no compound purification is required, this approach rapidly identified the first small-molecule inhibitors of human UNG with micromolar to submicromolar binding affinities. In a surprising result, these uracil-based ligands are found not only to bind to the active site but also to bind to a second uncompetitive site. The weaker uncompetitive site suggests the existence of a transient binding site for uracil during the multistep extrahelical recognition mechanism. This very general inhibitor design strategy can be easily adapted to target other enzymes that recognize nucleobases, including other DNA repair enzymes that recognize other types of extrahelical DNA bases.     

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.     

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 affords peptides that modulate beta-amyloid aggregation

As the population ages, the need to develop methods to understand and intercept the processes responsible for protein aggregation diseases is becoming more urgent. The aggregation of the protein beta-amyloid (Abeta) has been implicated in Alzheimer's Disease (AD); however, whether the toxic species is a large, insoluble aggregate or some lower order form is not yet known. Agents that can modulate the aggregation state of Abeta could resolve this controversy by facilitating our understanding of the consequences of aggregation and its underlying mechanism. To date, however, ligands that bind to specific forms of Abeta have not been identified. To address this deficiency, we tested whether phage display could yield such ligands by screening libraries against Abeta in two different states: monomeric or highly aggregated. Intriguingly, the peptides selected had different effects on Abeta aggregation. Peptides selected for binding to monomeric Abeta did not perturb aggregation, but those selected using highly aggregated Abeta increase the rate of aggregation drastically. The latter also alter the morphology of the resulting aggregate. The ability of a peptide to promote aggregation correlated with its affinity for the N-terminal 10 residues of Abeta. This result indicates that the mechanism by which the peptides influence aggregation is related to their affinity for the Abeta N-terminus. Thus, the identification of compounds that bind to this Abeta section can afford agents that affect aggregation. Moreover, the data suggest that endogenous ligands that interact with the N-terminal region can influence the propensity of Abeta to form aggregates and the morphology of those that form. Our data highlight the utility of phage display for identifying ligands that bind to target proteins in different states, and they indicate that such agents can be used to perturb protein aggregation.     

Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.     

In vivo targeting of organic calcium sensors via genetically selected peptides

A library of constrained peptides that form stable folded structures was screened for aptamers that bind with high affinity to the fluorescent dye Texas red. Two selected clones had binding constants to Texas red of 25 and 80 pM as phage and binding had minimal effects on the fluorescence of Texas red. The peptides interact with distinct but overlapping regions of Texas red. One peptide bound to X-rhod calcium sensors, which share the same core fluorophore as Texas red. These dyes retained calcium sensitivity when bound to the peptide. This peptide was used to label a fusion protein with X-rhod-5F in vivo, and X-rhod sensed changes in calcium locally. Thus, minimal, constrained peptides can functionally bind to environmentally sensitive dyes or other organic agents in biological contexts, suggesting tools for in vivo imaging and analysis.     

Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities

Introduction: WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins. Five classes of WW domains have been annotated to date, where each class is largely defined by the type of peptide ligand selected, rather than by similarities within WW domains. Class I WW domains bind Pro-Pro-Xxx-Tyr containing ligands, and it would be of interest to determine residues within the domains that determine this specificity.Results: Fourteen WW domains selected Leu/Pro-Pro-Xxx-Tyr containing peptides ligands via phage display and were thus designated as Class 1 WW domains. These domains include those present in human YAP (hYAP) and WWP3, as well as those found in ubiquitin protein ligases of the Nedd4 family, including mouse Nedd4 (mNedd4), WWP1, WWP2 and Rsp5. Comparing the primary structures of these WW domains highlighted a set of highly conserved residues, in addition to those originally noted to occur within WW domains. Substitutions at two of these conserved positions completely inhibited ligand binding, whereas substitution at a non-conserved position did not. Moreover, mutant WW domains containing substitutions at conserved positions bound novel peptide ligands.Conclusions: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro-Xxx-Tyr containing peptide ligands. The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro-Xxx-Tyr containing peptide ligands.     

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.     

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.     

Identification of small-molecule inhibitors against human leukocyte antigen-death receptor 4 (HLA-DR4) through a comprehensive strategy

Rheumatoid arthritis (RA) is an autoimmune disease mediated by T-lymphocytes and associated with the human leukocyte antigen-death receptor 4 (HLA-DR4). The HLA-DR4 protein selectively interacts with the antigenic peptides on the cell surface and presents them to the T cell receptor (TCR) on CD4+ T cells. The HLA-DR4-antigen-TCR complex initiates the autoimmune response and eventually causes the chronic inflammation within patients bodies. To inhibit HLA-DR4-restricted T cell activation, an ideal approach is to discover non-T cell stimulating substrates that specifically bind to HLA-DR4. In this paper, a comprehensive structure-based design strategy involved de novo design approach, pharmacophore search, and dock method was presented and applied to "simplify" the known binding peptide ligand of HLA-DR4 and identified specific small-molecule inhibitors for HLA-DR4. The designed three-step strategy successfully identified five nonpeptide ligands with novel scaffolds from a chemical library containing 4 × 10(6) commercially available compounds within a tolerable computing time. The identified five chemicals, BAS-0219606, T0506-2494, 6436645, 3S-71981, and KM 11073, are all non-T cell stimulators and are able to significantly inhibit HLA-DR4-restricted T cell activation induced by type II collagen (CII) 263-272 peptide. IC(50) for the best two potentials, BAS-0219606 and T0506-2494, was 31 and 17 μM, respectively, which is equivalent or better than the known peptide ligands. It is hopeful that they can be used as effective therapeutic means for further treatment of RA patients. In addition, the comprehensive strategy presented in this paper exhibited itself to be an effective flow line from peptide ligands to small-molecule inhibitors and will have applications to other targets.     

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.     

Challenges in optimizing a prostate carcinoma binding peptide, identified through the phage display technology

The transfer of peptides identified through the phage display technology to clinical applications is difficult. Major drawbacks are the metabolic degradation and label instability. The aim of our work is the optimization of DUP-1, a peptide which was identified by phage display to specifically target human prostate carcinoma. To investigate the influence of chelate conjugation, DOTA was coupled to DUP-1 and labeling was performed with 111In. To improve serum stability cyclization of DUP-1 and targeted D-amino acid substitution were carried out. Alanine scanning was performed for identification of the binding site and based on the results peptide fragments were chemically synthesized. The properties of modified ligands were investigated in in vitro binding and competition assays. In vivo biodistribution studies were carried out in mice, carrying human prostate tumors subcutaneously. DOTA conjugation resulted in different cellular binding kinetics, rapid in vivo renal clearance and increased tumor-to-organ ratios. Cyclization and D-amino acid substitution increased the metabolic stability but led to binding affinity decrease. Fragment investigation indicated that the sequence NRAQDY might be significant for target-binding. Our results demonstrate challenges in optimizing peptides, identified through phage display libraries, and show that careful investigation of modified derivatives is necessary in order to improve their characteristics.     

Selection of phage-displayed antibodies with high affinity and specificity by electrophoresis in microfluidic devices

A method development aimed for high-throughput and automated antibody screening holds great potential for areas ranging from fundamental molecular interactions to the discovery of novel disease markers, therapeutic targets, and monoclonal antibody engineering. Surface display techniques enable efficient manipulation of large molecular libraries in small volumes. Specifically, phage display appeared as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Here, we present a phage-selection microfluidic device wherein electrophoresis was performed under two orthogonal electric fields through an agarose gel functionalized with the respective antigen. This microdevice was capable of screening and sorting in a single round high-affinity phage-displayed antibodies against virus glycoproteins, including human immunodeficiency virus-1 glycoprotein 120 or the Ebola virus glycoprotein (EBOV-GP). Phages were differentially and laterally swept depending on their antigen affinity; the high-affinity phages were recovered at channels proximal to the application site, whereas low-affinity phages migrated distal after electrophoresis. These experiments proved that the microfluidic device specifically designed for phage-selection is rapid, sensitive, and effective. Therefore, this is an efficient and cost-effective method that allowed highly controlled assay conditions for isolating and sorting high-affinity ligands displayed in phages.     

Synthetic ligands discovered by in vitro selection

The recognition and catalytic properties of biopolymers derive from an elegant evolutionary mechanism, whereby the genetic material encoding molecules with superior functional attributes survives a selective pressure and is propagated to subsequent generations. This process is routinely mimicked in vitro to generate nucleic-acid or peptide ligands and catalysts. Recent advances in DNA-programmed organic synthesis have raised the possibility that evolutionary strategies could also be used for small-molecule discovery, but the idea remains unproven. Here, using DNA-programmed combinatorial chemistry, a collection of 100 million distinct compounds is synthesized and subjected to selection for binding to the N-terminal SH3 domain of the proto-oncogene Crk. Over six generations, the molecular population converges to a small number of novel SH3 domain ligands. Remarkably, the hits bind with affinities similar to those of peptide SH3 ligands isolated from phage libraries of comparable complexity. The evolutionary approach has the potential to drastically simplify and accelerate small-molecule discovery.     

Targeting of phage display vectors to mammalian cells

Phage display libraries offer a strategy to isolate peptide ligands to target proteins and to define potential interaction sites between proteins. Recent studies have indicated a novel utility for phage display in that bacteriophage engineered to express peptide ligands to specific cell surface receptors are internalized by mammalian cells. Thus, reporter genes such as green fluorescent protein and lacZ harbored in the phage genome can be delivered to mammalian cells using targeting peptides displayed on the surface of phage. There is also the possibility to generate novel types of peptide libraries expressed intracellularly using a phage capable of inducing expression of its coding genes in human cells.