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At least 505 records · Page 28Linked to original sources
PubMed2006

Mimotope of Leptospira from phage-displayed random peptide library is reactive with both monoclonal antibodies and patients' sera.

The study aim was to use random heptapeptide library displayed by bacteriophage T7 for identifying mimotopes from 15 monoclonal antibodies (MAbs) specific to Leptospira spp., and from four leptospirosis patient sera, respectively. The bound phages, selected from fourth round of bio-panning with each antibody, were cloned by plaque isolation and the binding specificity of individual clones were confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. All together 150 phages were selected, mimotope from 86 phages (56.6%) were found to match with protein sequences of Leptospira from GenBank database. The predominant mimotopes were mimotope with sequence LTPCD that found in 27.3%, followed by TPCSK (16%), KSKKSS (4%), KTKRXAS (4%), SSKSYR (3.3%), DPNXNSF (3.3%), KSGRC (2.6%), TLINIF (2%), TPCI (2%), 1.33% each with mimotopes PKKS, PCNTKXTA, and CTKKK, and one phage each (0.66%) with mimotopes PTFGS, TNSKRK, SKSSRC, RSKRIR, VTNNTP, and CSNXSKR. Interestingly, mimotopes LTPCD, TPCSK, and TPCI were found to react with both MAb and patient's sera. The matched proteins from GenBank namely, leptospiral putative outer membrane protein (matched with mimotope PTFGS), thermolysin precursor protein (matched with mimotope TPCIXXGSAS), and hypothetical protein LIC12228 (matched with mimotope CSNXSKR), were found to locate at outer membrane of Leptospira. These phage mimotopes and matched proteins may have potential for further use as diagnostic reagent and immunogen against leptospirosis in the future. The results demonstrate that phage display technique has potential for rapidly identifying phage mimotopes that interact with leptospiral MAbs and patient's sera.

Amino Acid Sequence↗
PubMed2001

Selection of an unnatural peptide library for dsDNA binding.

More and more, nucleic acids have become prime targets in the development of new compounds, able to control gene expression. For the development of new sequence selective dsDNA binding ligands, one can learn a lot from existing models such as, lexitropsins, combilexins and actinomycin D. This analysis, together with the knowledge of the details on protein-DNA interactions, has inspired the assembly of unnatural amino acids in a combinatorial way to generate a dsDNA recognition library. The first selection round has led to the selection of new DNA binding molecules, which may lead on the long run to the discovery of new DNA binding motifs.

Amino Acids↗
PubMed2001

From peptide libraries to optimized nonpeptide ligands in the search for S-farnesyltransferase inhibitors.

A complete 331,776-member library of tetrapeptides made of 24 amino acid building blocks was synthesized robotically on solid phase and subjected to a deconvolution based on the inhibitory potency of the sublibraries in a HPLC assay of the S-farnesyltransferase activity in vitro. One of the non-natural peptide and noncysteine-containing leads Nip-Trp-Phe-His (Nip=p-nitrophenyl-L-alanine) was optimized chemically to give a proteolytically stable pseudopeptide with a 200-fold potency compared with the original lead. The final compound was converted to the C-terminal ethyl ester: p-F-C6H4-CO(CH2)2-CO-Bta-D-Phepsi[CH2NH]His-OEt (Bta = benzothienyl-L-alanine) and shown to behave as a prodrug which was hydrolyzed back to the C-terminal acid following cell penetration. The method confirmed that several structurally original leads can be discovered in large libraries when deconvolution relies upon a highly specific assay and that these leads can be optimized by chemical modification to impart the final compound the desired pharmacological and pharmacokinetic properties.

Alkyl and Aryl Transferases↗
PubMed2005

Combinatorial peptide library binding of mammalian spermatozoa identifies a ligand (HIPRT) in the axin protein: putative identification of a sperm surface axin binding protein and intriguing developmental implications.

The identification of components in cell-cell interactions is an important research goal in reproductive and developmental biology. Such interactions are critical to gamete development, fertilization, implantation and basic development. Several proteins involved with sperm-oocyte interaction and other developmentally important phenomena have been identified. However, these are obviously only a subset of the molecular components involved in such complex cell-cell interactions. One method that has been used to identify binding partners for particular molecular targets is the use of combinatorial libraries accessible on phage surfaces. For the most part, this technique has mainly been applied to screen specific target moieties. However, in some cases whole-cell screening has been attempted. This study describes the first report of screening intact, living mammalian gametes using a proprietary whole-cell combinatorial library binding and analysis protocol. Results from the first screening protocol of mouse spermatozoa strongly identified a putative sperm-binding ligand using proprietary bioinformatic analysis. This amino acid sequence (HIPRT) precisely corresponds with a previously characterized highly conserved protein-protein interaction site in the axin protein. This sequence is found within the binding site for a known sperm surface protein, glycogen synthase kinase-3. This result not only provides proof of the utility of this technique to identify cell surface ligands in mammalian gametes, but it also suggests a potential role for spermatozoa in facilitating developmental axis formation in mammalian embryos.

Amino Acid Sequence↗
PubMed2004

[Screening and identification of human telomerase reverse transcriptase epitopes from random phage display peptide library].

OBJECTIVE: To identify and characterize the epitopes of human telomerase reverse transcriptase (hTERT). METHODS: A random phage displayed dodecapeptide library was screened with anti-hTERT antibody. The selected phage clones were identified by sandwich enzyme-linked immunosorbent assay, anti-hTERT antibody blocking assay and competitive inhibition assay. RESULTS: After 3 rounds of screening, 13 of 24 randomly selected phage clones were identified as positive clones that could specifically bind to anti-hTERT antibody but not to normal mouse IgG. Amino acid sequences deduced from DNA sequences showed 11 different sequences with high percentage of histone and hydrophilic amino acids. CONCLUSION: Eleven epitopes have been obtained that can effectively mimic hTERT, which will be useful for the research of small-molecule inhibitor targeting at hTERT.

Base Sequence↗
PubMed2006

Construction of a small peptide library related to inhibitor OM99-2 and its structure-activity relationship to beta-secretase.

AIM: To develop probes for detecting the binding specificity between beta-secretase and substrate, and provide reliable biological activity data for further researching encircling substrate-based inhibitors. METHODS: To prepare the inhibitors, the hydroxyethylene (HE) segment including P1 and P1'was synthesized after multi-step reactions; the combination of all segments was then completed through solid phase synthesis. Recombinant human beta-secretase ectodomain (amino acid residues 1-460) was expressed as a secreted protein with a C-terminal His tag in insect cells using baculovirus infection, and all compounds were evaluated in this beta-secretase enzyme assay. In order to understand the interaction in detail, the theoretical methods, namely molecular dynamics (MD) simulation and molecular mechanics-generalized-born surface area (MM-GBSA) analysis, were performed on the complex of beta-secretase and OM99-2 to obtain the geometrical and energetical information. RESULTS: We designed and constructed a positional scanning combinatorial library including 16 compounds; all members of the library were synthesized based on HE dipeptide isostere. Structure-activity relationship studies at the P4-P1 and P1' -P4'positions led to the discoveries of P and P'sides binding specificity and potent inhibitors 14, 18, and 22. The binding free energy on the whole system and every residue were compared to the biological assay result. CONCLUSION: The removal of P4' yielded inhibitor 22 (A3 *B2) with high potency; further truncation of P3'gave inhibitor 18 (A3 *B1) with equal activity, implying that the right side of the inhibitors play a less important role and could be easily simplified, while change on the P side may cause substantial results.

Amyloid Precursor Protein Secretases↗
PubMed2002

Identification of novel antifungal nonapeptides through the screening of combinatorial peptide libraries based on a hexapeptide motif.

Four sets of mixture based nonapeptide libraries derived from an antifungal hexapeptide pharmacophore Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH(2) (II) have been synthesized. The three C-terminal positions 7, 8 and 9 were subject to randomization using 19 genetically coded amino acids. They were then screened for their antifungal activity against Candida albicans and Cryptococcus neoformans in order to quantify inhibition at each step of the nonapeptide sublibrary deconvolution. The studies led to the identification of several novel nonapeptides with potent antifungal activity. Two of the nonapeptides exhibited approximately 17-fold increase in the activity in comparison to the lead hexapeptide motif His-D-Trp-D-Phe-Phe-D-Phe-Lys-NH(2) (I) against C. albicans.

Amino Acid Motifs↗
PubMed2002

[Identification of epitope of 22.6 kDa antigen of Schistosoma japonicum with phage-displayed random peptide library].

OBJECTIVE: To identify the epitopes of 22.6 kDa antigen of Schistosoma japonicum (Sj22.6). METHODS: A 12-mer library displayed on pIII of fd phage was used to screen the epitopes of Sj22.6 with the purified multiple clone IgG antibody against the Sj22.6 antigen. Five rounds of biopanning were carried out and fourteen clones from the fifth round biopanning were randomly selected and identified by Western blotting. The mice were immunized with the obtained positive clones and the antibody titers of sera from the mice were detected. The clones which could stimulate the mice to produce anti-Sj22.6 antibodies were sequenced and their amino acid sequences were compared with that of Sj22.6. RESULTS: Six clones selected from the fourteen ones could stimulate the mice to produce anti-Sj22.6 antibodies analysed by Western blotting. The amino acid sequence of one epitope showed high homology with Sj22.6. CONCLUSION: Four epitopes of Sj22.6 were obtained. One may be a structure epitope and the other three were mimic epitopes.

Amino Acid Sequence↗
PubMed2002

Development of novel tumor imaging agents with phage-display combinatorial peptide libraries.

RATIONALE AND OBJECTIVES: Current radiologic methods do not provide sufficient information for unambiguous diagnosis and prognosis of cancer. The present investigation sought to address this deficiency by developing a system for designing novel small molecules targeted against tumor-specific molecules for use as radionuclide imaging agents. MATERIALS AND METHODS: Part of a tumor-specific receptor, purified recombinant epidermal growth factor receptor (EGFR), variant III, extracellular domain (rEGFRvIII-ecd), was used as the target in the selection of EGFRvIII-specific peptide ligands from random peptide bacteriophage (phage) display libraries. After three rounds of screening, phage isolates were tested for binding affinity with an enzyme-linked immunosorbent assay. Positive phage were sequenced, and the peptides were synthesized and tested for binding affinity with a surface plasmon resonance assay. RESULTS: Affinity screening identified 49 peptide-expressing phage that showed enhanced binding to the variant receptor compared with wild-type EGFR. Free peptides from the two phage isolates exhibiting the most favorable binding were tested for target binding. One of these demonstrated a binding affinity for rEGFRvIII-ecd in the 30-nmol/L range. CONCLUSION: These data suggest that phage display libraries may be very useful in the design of novel, high-affinity tumor imaging agents.

Affinity Labels↗
PubMed2003

Mapping alveolar binding sites in vivo using phage peptide libraries.

Targeting lung tissue is nonselective due in part to the lack of specific cell-surface receptors identified on target lung cells. We used in vivo phage display to identify a panel of peptides that can bind selectively to lung epithelial cells with less binding to nonepithelial cells. By direct intratracheal instillation of phage libraries into the lung, we isolated and identified 143 individual phage clones. Three phage clones revealed enhanced binding to the lung in vitro and in vivo. These three identified peptides were synthesized and demonstrated selective binding to epithelial cells in lung tissue versus the control peptide. Further, the peptides specifically bound to freshly isolated type II alveolar epithelial cells compared with Hep2 cells. The results suggest that the airway phage display approach could be exploited for analyzing the molecular diversity in the lower respiratory tract.

Binding Sites↗
PubMed1993

Identification of the amino acids comprising a surface-exposed epitope within the nucleotide-binding domain of the Na+,K(+)-ATPase using a random peptide library.

Monoclonal antibodies that bind native protein can generate considerable information about structure/function relationships, but identification of their epitopes can be problematic. Previously, monoclonal antibody M8-P1-A3 has been shown to bind to the catalytic (alpha) subunit of the Na+,K(+)-ATPase holoenzyme and the synthetic peptide sequence 496-HLLVMK*GAPER-506, which includes Lys 501 (K*), the major site for fluorescein-5'-isothiocyanate labeling of the Na+,K(+)-ATPase. This sequence region of alpha is proposed to comprise a portion of the enzyme's ATP binding domain (Taylor, W. R. & Green, N. W., 1989, Eur. J. Biochem. 179, 241-248). In this study we have determined M8-P1-A3's ability to recognize the alpha-subunit or homologous E1E2-ATPase proteins from different species and tissues in order to deduce the antibody's epitope. In addition the bacteriophage random peptide or "epitope" library, recently developed by Scott and Smith (1990, Science 249, 386-390) and Devlin et al. (Devlin, J. J., Panganiban, L. C., & Devlin, P. E., 1990, Science 249, 404-406), has served as a convenient technique to confirm the species-specificity mapping data and to determine the exact amino acid requirements for antibody binding. The M8-P1-A3 epitope was found to consist of the five amino acid 494-PRHLL-498 sequence stretch of alpha, with residues PRxLx being critical for antibody recognition.

Adenosine Triphosphate↗
PubMed2000

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries.

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.

Alcohol Dehydrogenase↗
PubMed1997

Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.

Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.

Algorithms↗
PubMed2005

Identification of peptide ligands for malignancy- and growth-regulating galectins using random phage-display and designed combinatorial peptide libraries.

Members of the galectin family of endogenous lectins are involved in tumor growth regulation and in establishing characteristics of the malignant phenotype via protein-carbohydrate and protein-protein interactions. To identify peptide ligands with the potential to modulate these tumor-relevant interactions beneficially, complementary screening methods were employed, that is, both phage-display and a combinatorial pentapeptide library with the key YXY tripeptide core. Three representative prototype galectins were selected. The search for high-affinity ligands among phage-displayed random heptamers yielded enrichment after five selection cycles of the nonglycomimetic CQSPSARSC peptide in the case of the chicken homologue of galectin-1 but not the human protein, an indication for specificity. The most active glycomimetic from the combinatorial library of 5832 pentamers was WYKYW. Identification of peptide ligands for galectins with and without glycomimetic properties is thus possible. Our study documents the potential to combine the two library-based approaches for structural optimization of lead peptides.

Animals↗
PubMed1999

Combinatorial chemistry: polymer supported synthesis of peptide and non-peptide libraries.

In recent years, combinatorial chemistry has emerged as a powerful tool for accelerating drug discovery. While industry is rapidly embracing the technology, researchers continue to develop novel library methods including resins, linkers, tagging and deconvolution techniques. Newer strategies involving computer-customized combinatorial libraries offer enormous potential for the design of more "focused" and "smart" chemical libraries with maximal diversity. In addition, miniaturized systems for synthesizing chemical libraries are also being developed, which has made it possible to carry out reactions at submicroliter volumes.

Animals↗
PubMed1995

Oxidative decarboxylation of peptides catalyzed by flavoprotein EpiD. Determination of substrate specificity using peptide libraries and neutral loss mass spectrometry.

The flavoprotein EpiD catalyzes the COOH-terminal oxidative decarboxylation of the lantibiotic precursor peptide EpiA. Variations of the COOH-terminal heptapeptide S1FNSYCC7 of EpiA were used for determining the substrate specificity of EpiD. When Cys7 was replaced by serine, cysteine-amide, homocysteine, or a thioether amino acid residue, no reaction with EpiD was observed. Heptapeptide libraries with one variable amino acid residue at positions 1-7 of the peptide substrate S1FNSYCC7 were incubated with EpiD, and the reaction products were identified by neutral loss mass spectrometry. When the penultimate cysteine residue Cys6 of the substrate peptide was replaced with Ser, Thr, Ala, or Val, the reaction still occurred. Tyr5 could be replaced with other hydrophobic amino acid residues. Mass spectrometry was used to compare the kinetics of the reaction of EpiD with various peptides. Peptide sequencing of the reaction products was performed by tandem mass spectrometry, confirming that the last cysteine residue was modified. The removal of the acid COOH-terminal carboxyl group was confirmed by determination of the isoelectric points of the reaction products. To study the interaction between EpiA and EpiD, EpiA was coupled to N-hydroxysuccinimide-activated Sepharose HiTrap material; EpiD was only retarded under reducing conditions.

Amino Acid Sequence↗
PubMed1995

Combinatorial peptide libraries in drug design: lessons from venomous cone snails.

Many present-day drugs are derived from compounds that are natural products, a traditional source of which is fermentation broths of microorganisms. The venoms of cone snails are a new natural resource of peptides that may have a pharmaceutical potential equivalent to those from traditional sources, particularly for developing drugs that target cell-surface receptors or ion channels. In effect, cone snails have used a combinatorial library strategy to evolve their small, highly bioactive venom peptides. The methods by which the snails have generated thousands of peptides with remarkable specificity and high affinity for their targets may provide important lessons in designing combinatorial libraries for drug development.

Amino Acid Sequence↗
PubMed2003

Partial protection induced by phage library-selected peptides mimicking epitopes of Schistosoma japonicum.

OBJECTIVE: To obtain peptide mimicking epitopes of Schistosoma japonicum (S. japonicum) through screening of a phage peptide library and to test their potential for induction of protection. METHODS: S. japonicum infected sera from Microtus fortis (IMFS) and normal sera from Microtus fortis (NMFS) were used respectively to screen a 12-mers random peptide library by testing the reactivity of anti-S. japonicum serum with the phagotopes. After three rounds of biopanning, the pooled phages were used to immunize mice, after which challenge infection was performed. RESULTS: Of 12 randomly picked clones, 10 clones selected using IMFS and 7 clones selected using NMFS were shown to be antigenic. Significant reduction in adult worms (22.6%) and a high reduction (68.9%) in liver eggs were achieved following immunization with phages screened with IMFS. However, no protection was elicited by those selected with NMFS. CONCLUSION: The results show that the phagotopes are both antigenic and immunogenic, suggesting a potential use of phage displayed peptide as novel vaccines against S. japonicum.

Animals↗