An on-bead assay for the identification of non-natural peptides targeting the androgen receptor-cofactor interaction

An efficient and rapid on-bead screening method was established to identify non-natural peptides that target the Androgen Receptor-cofactor interaction. Binding of the Androgen Receptor ligand binding domain to peptide sequences displayed on beads in a One-Bead-One-Compound format could be screened using fluorescence microscopy. The method was applied to generate and screen both a focussed and a random peptide library. Resynthesis of the peptide hits allowed for the verification of the affinity of the selected peptides for the Androgen Receptor in a competitive fluorescence polarization assay. For both libraries strong Androgen Receptor binding peptides were found, both with non-natural and natural amino acids. The peptides identified with natural amino acids showed great similarity in terms of preferred amino acid sequence with peptides previously isolated from biological screens, thus validating the screening approach. The non-natural peptides featured important novel chemical transformations on the relevant hydrophobic amino acid positions interacting with the Androgen Receptor. This screening approach expands the molecular diversity of peptide inhibitors for nuclear receptors.     

Development of a tactical screening method to investigate the characteristics of functional peptides

Using spot-synthesized peptide arrays, a functional peptide can be screened as a high-binding peptide for a target molecule. We have developed a rational screening method for functional peptides by analyzing the physicochemical rules of high-binding peptide sequences. To screen the peptides simply and strategically, we prepared an exhaustive 4-mer peptide library consisting of 256 peptides (4⁴ = 256) characterized by four physicochemical groups of 20 amino acids: Group 1, non-charged hydrophobic amino acids; Group 2, non-charged hydrophilic amino acids; Group 3, positive-charged hydrophilic amino acids; Group 4, negative-charged hydrophilic amino acids. First, our previous screening data from cell adhesion, bile acid-binding, and nanoparticle-binding peptides were applied to the four-category analysis, and target-specific physicochemical characteristics were obtained. We then prepared an exhaustive 4-mer peptide library using these four physicochemical groups, and screened for high-binding peptides that bind model proteins interleukin-2 and IgG. We obtained individual physicochemical rules for high-binding peptides: group 1 or 4 amino acids in position (P) 1, group 1 in P2 and P4 for IL-2, and group 2 and 3 amino acids at all position for IgG. Therefore, this system, which employs the use of a simple and strategic peptide library, will be useful in the development of functional peptides.     

靶向膜型1基质金属蛋白酶反义肽的虚拟筛选与分子模拟

膜型1基质金属蛋白酶(Membrane type-1 matrix metalloproteinase,MT1-MMP,MMP14)在肿瘤的发生发展及转移中起着重要作用,是肿瘤潜在理想的药物靶标。为了获得MT1-MMP结合肽,我们首先采用生物信息学方法分析MMPs序列,获得MT1-MMP差异大且特异的序列。以此为正义肽靶标,设计反义肽库,然后通过分子对接、分子动力学模拟以及体外细胞实验等多种方法,进行靶向MT1-MMP反义肽的筛选与活性研究。多序列比对确定了位于MT1-MMP环区的特异序列AYIREGHE(简称MT1-loop),并构建1 536条反义肽。经两轮虚拟筛选,选取打分位于前五的反义肽用于后续研究。该五条反义肽与MT1-MMP存在较强的相互作用且能很好地对接于正义肽区域。进一步分析其与MMPs其他家族成员(MMP1-3,MMP7-13,MMP14HPX,MMP16)的亲和力,发现反义肽FVTFPYIR对MT1-MMP具有更强的特异性。分子动力学模拟表明,反义肽FVTFPYIR可能是通过影响受体MT1-MMP的构象稳定性,进而影响其功能活性。体外细胞实验初步确定反义肽FVTFPYIR可选择性地抑制表达MT1-MMP的人成骨肉瘤细胞MG63和乳腺癌MDA-MB-231细胞的增殖。本研究为抗肿瘤反义肽先导药物的研发提供了一种新的思路与途径。     

Computationally assisted screening and design of cell-interactive peptides by a cell-based assay using peptide arrays and a fuzzy neural network algorithm

We developed a method of effective peptide screening that combines experiments and computational analysis. The method is based on the concept that screening efficiency can be enhanced from even limited data by use of a model derived from computational analysis that serves as a guide to screening and combining the model with subsequent repeated experiments. Here we focus on cell-adhesion peptides as a model application of this peptide-screening strategy. Cell-adhesion peptides were screened by use of a cell-based assay of a peptide array. Starting with the screening data obtained from a limited, random 5-mer library (643 sequences), a rule regarding structural characteristics of cell-adhesion peptides was extracted by fuzzy neural network (FNN) analysis. According to this rule, peptides with unfavored residues in certain positions that led to inefficient binding were eliminated from the random sequences. In the restricted, second random library (273 sequences), the yield of cell-adhesion peptides having an adhesion rate more than 1.5-fold to that of the basal array support was significantly high (31%) compared with the unrestricted random library (20%). In the restricted third library (50 sequences), the yield of cell-adhesion peptides increased to 84%. We conclude that a repeated cycle of experiments screening limited numbers of peptides can be assisted by the rule-extracting feature of FNN.     

A novel method for construction of gene fragment library to searching epitopes

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.     

Functional screening of a retroviral peptide library for MHC class I presentation

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.     

Identification of protein-binding peptides by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of peptide beads selected from the screening of one bead-one peptide combinatorial libraries

A fast and inexpensive strategy for the identification of peptide ligands by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of peptide beads screened from one bead-one peptide combinatorial libraries is herein described. Streptavidin was used as the model protein. A combinatorial library of 6561 peptides was synthesized on ChemMatrix resin by the divide-couple-recombine method. 4-Hydroxymethylbenzoic acid was used as the linker and five residues of Gly were incorporated at the C termini to increase the final peptide molecular weight. Positive control peptides with the HPQ motif and negative control peptides without the HPQ motif evidenced that the linker and the five residues of Gly have neither impaired the specific binding nor facilitated unspecific binding. After screening the library, positive beads were isolated and washed with 8M guanidine hydrochloride. The beads were sliced into two or four pieces, deposited onto the stainless steel MALDI sample plate, and treated with ammonia vapor to release the peptides. In addition, 26 beads picked at random from the library were subjected to the same treatment. All samples were analyzed by MALDI-TOF-MS and the peptides were unambiguously identified with very good reproducibility between the bead pieces, thus evidencing the good homogeneity of the bead. All sequences obtained from the screening contained HPQ.     

Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.     

Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries.

We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.     

Applications of topologically segregated bilayer beads in 'one-bead one-compound' combinatorial libraries.

We recently reported the use of a biphasic approach to generate topologically segregated bilayer beads. In generating 'one-bead one-compound' (OBOC) combinatorial libraries, novel encoding methods have been applied to these beads such as the testing library compound and the coding tags residing on the outer layer and inner core of each bead, respectively. In this report, we further exploit these bilayer beads by preparing target bead-libraries with low concentration of random peptides on the outer layer, and full substitution of coding peptides in the bead interior. The low concentration of peptide on the bead surface enables us to greatly increase the stringency of screening so that higher affinity ligands can easily be identified. Full substitution of the inner core of the beads enables us to have enough coding peptides inside the bead for direct microsequencing with Edman chemistry. The biphasic approach of preparing bilayer beads can be carried out at any point during the library construction. Therefore, the nonsequencable or fixed structures of the peptides can be bypassed in the coding tags. As a result, peptide libraries that otherwise cannot be sequenced can now be sequenced, and peptide segments with fixed residues within the libraries can be bypassed so that the microsequencing time can be significantly shortened. Furthermore, peptides with a branch of random sequence in the middle of a fixed peptide chain can be encoded with just the random sequence in the bead interior. We have successfully applied these novel OBOC library concepts in the optimization of cell-surface ligands for a human T-cell leukemia, Jurkat, cell line.     

Angiogenesis induced by novel peptides selected from a phage display library by screening human vascular endothelial cells under different physiological conditions

Angiogenesis is a process modulated by several endogenous vascular growth factors as well as by oxygen conditions. For example VEGF failed to induce useful therapeutic angiogenesis in clinical trials. We used a combinatory phage display peptide library screening on human umbilical endothelial cells under normoxia and hypoxia conditions in order to identify novel peptides that bind endothelial cells. The identified peptides induced angiogenesis as demonstrated by endothelial cell proliferation, migration and tube formation. Injection of peptides into the ears of mice resulted in increased numbers of blood vessels. Peptides did not induce VEGF receptor gene expression indicating a possible VEGF unrelated mechanism.     

Peptides derived from a secretory yeast library restore factor VIII activity in the presence of an inhibitory antibody

The development of autoantibodies against factor VIII represents one of the major complications in the treatment of hemophilia A patients. We have employed a novel library system to obtain peptides that specifically neutralize the interaction between factor VIII and these inhibitors. The random peptides are presented as carboxy-terminal extensions of the eukaryotic initiation factor 5a, an intracellular protein with a molecular mass of 18 kDa. These random peptides formed an unique binding site, as demonstrated by molecular simulations using the computer programs InsightII and GROMACS. The library was screened to identify peptides binding to the murine monoclonal anti-factor VIII antibody ESH8 and to inhibitors derived from patients with factor VIII antibodies. Ten peptides binding to ESH8 were identified. Their specificity was confirmed by displacement assays. Two peptides with the sequences STKTLGRPLHGPAGPVEGGALAGVAEDADLVTAVSGR and YHCKREDLTDRDATCALRQPPQAVRGLGPRVTAVSGR showed the ability to restore the factor VIII activity from 33% up to approximately 90% in functional tests performed in vitro. Three candidates for binding to factor VIII antibodies derived from four different patient's sera were achieved. Three fusion proteins with the peptide sequences PQLGSRRSTTPSLTFQNASWFPAGGPCARSNRG, SGSRQVCKLARSLQPF and WERGRRVGAQVRHARHLVARVLDGAGHQARLTAVNGP bound to inhibitors derived from different patients. Furthermore, two of the obtained fusion proteins with the peptide sequences RHWTALGPAPTHTCADLNYPLLS and WERGRRVGAQVRHARHLVARVLDGAGHQARLTAVNGP did also bind to the monoclonal antibody ESH8. This study demonstrates the potential of this system to identify peptides that inhibit the activity of potent inhibitory antibodies and also shows potential as a method for screening of bioactive peptides.     

De novo identification of binding sequences for antibody replacement molecules

A new in silico method has been developed that automatically identifies peptide sequences that can bind to targets of known three‐dimensional structure. The method is potentially faster and more economical than traditional methods of raising antibodies by means of hybridomas or biopanning technology. The current algorithm creates an initial peptide library that is either completely random or that is constrained by the user. This library represents only a small fraction of possible sequence space and the peptides are created with a specified torsional geometry. The library is used as input to any number of available molecular docking programs and the library is docked and scored. The final rank ordering is then used to create a new library by constraining that library to the sequence conservation pattern deduced from the top N‐scoring peptides in the first round. Successive rounds of screening, scoring, and new library creation ultimately results in the system converging to a final solution set of peptides. To test the method, a family of novel peptides that can bind to, and inhibit the enzyme Deoxyribonuclease I has been discovered. The peptides inhibit the enzyme either alone or when placed into a protein backbone structure as has been confirmed experimentally. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Bacteria-based in vivo peptide library screening using biopanning approach

Traditionally, library screening has been performed to identify biologically active agents including small molecules or peptides that inhibit target proteins or molecules with therapeutic interests. Due to its chemical nature, library screening is usually performed under in vitro environments using purified proteins and molecules. However, active agents identified from in vitro screenings often fail to exhibit biological activities in cells. To overcome this inherent limitation, we have developed an in vivo peptide library screening system that allows for the identification of dissociative inhibitors of protein interactions of interest. The screening is based on the reconstitution of the cI repressor from bacteriophage lambda with high-density expression peptide library and is entirely performed in bacteria cells. Furthermore, to enhance the efficacy and sensitivity of the screening, a multiple-round biopanning approach was employed for amplification and enrichment of positive peptides. Overall, this in vivo screening should provide a fast and efficient tool for identification of biologically active peptide molecules against target protein assembly.     

Panning and identification of a colon tumor binding peptide from a phage display peptide library

Tumor-targeting therapy can be an efficacious way to cure a malignant tumor in clinical trials. Phage display is a molecular diversity technology that allows the presentation of a large number of peptides or proteins on the surface of filamentous phage for various applications. In this study, we report on using phage display to generate peptide libraries that bind to colon cancer tissues. To accomplish this, we developed a screening protocol that contained 3 rounds of in vitro positive panning on colon cancer cells (SW480) and 2 rounds of subtractive screening in vitro on normal human intestinal epithelial cells with a phage display-7 peptide library. After several rounds of panning, both phage titer and recovery efficiency were significantly improved. Through a cell-based enzyme-linked immunosorbent assay, immunofluorescence, in vivo binding assay, immunocytochemical staining, and immunohistochemical staining, peptide CP15 (VHLGYAT) was demonstrated to be the most effective peptide in targeting tumor cells (SW480 and HT29 cells) and tumor tissues but not the normal human intestinal epithelial cells and control colon tissue. These studies suggest that peptide CP15 may be a promising lead candidate in the development of a useful colon tumor diagnostic and targeted drug delivery agent.     

Affinity chromatographic screening of soluble combinatorial peptide libraries.

Affinity chromatography using immobilized S-protein was used for the screening of affinity peptide ligands from two soluble peptide libraries. Peptide library I consisted of octamers with glycine (G) at both termini of each peptide, i.e. GXXXXXXG. The six center positions were constructed using random sequences of six L-amino acids (Y, N, F, E, V, and L). Peptide library II also consisted of octamers but with glycine and valine (V) at both termini of each peptide (GVZZZZVG). The four variable center positions of peptide library II were random sequences of 18 L-amino acids. Peptides that were retained specifically on the immobilized S-protein column were eluted by 2% acetic acid. The peptides in the acid eluate were further separated using reversed-phase HPLC. Each separated peptide fraction was collected and the peptide sequences deconvoluted by mass spectrometry (MS/MS). The screenings of peptide libraries I and II resulted in 12 and 7 affinity peptides, respectively. Eight out of the twelve peptides from peptide library I contained the clear consensus sequence NFEV. Peptide library II resulted in affinity peptides with the sequences GVNFEVVG, GVNFTVVG and GVFFEL(I)VG. The advantages and limitations of affinity chromatography in peptide library screening are discussed.     

Screening of peptide libraries against protective antigen of Bacillus anthracis in a disposable microfluidic cartridge

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×10¹⁰ members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS.     

Production of dehydroamino acid-containing peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.