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PubMed2003

Current perspectives on established and putative mammalian oligopeptide transporters.

Peptides and peptide-based drugs are increasingly being utilized as therapeutic agents for the treatment of numerous disorders. The increasing development of peptide-based therapeutic agents is largely due to technological advances including the advent of combinatorial peptide libraries, peptide synthesis strategies, and peptidomimetic design. Peptides and peptide-based agents have a broad range of potential clinical applications in the treatment of many disorders including AIDS, hypertension, and cancer. Peptides are generally hydrophilic and often exhibit poor passive transcellular diffusion across biological barriers. Insights into strategies for increasing their intestinal absorption have been derived from the numerous studies demonstrating that the absorption of protein digestion products occurs primarily in the form of small di- and tripeptides. The characterization of the pathways of intestinal, transepithelial transport of peptides and peptide-based drugs have demonstrated that a significant degree of absorption occurs through the role of proteins within the proton-coupled, oligopeptide transporter (POT) family. Considerable focus has been traditionally placed on Peptide Transporter 1 (PepT1) as the main mammalian POT member regulating intestinal peptide absorption. Recently, several new POT members, including Peptide/Histidine Transporter 1 (PHT1) and Peptide/Histidine Transporter 2 (PHT2) and their splice variants have been identified. This has led to an increased need for new experimental methods enabling better characterization of the biophysical and biochemical barriers and the role of these POT isoforms in mediating peptide-based drug transport.

Amino Acid Sequence↗
PubMed2002

Hitting the target: emerging technologies in the search for kinase substrates.

Through phosphorylation, protein kinases can alter the activity, localization, protein association, and stability of their targets. Despite the importance to our understanding of all aspects of cell biology, progress toward identifying bona fide substrates of specific protein kinases has been slow. Traditionally used techniques to identify true kinase substrates, such as genetics, yeast two-hybrid screens, and biochemical purification, are often laborious and unreliable. However, several new approaches have recently been developed and used successfully to identify genuine in vivo substrates of certain protein kinases. These methods include screening for phosphorylation of proteins from phage expression libraries, peptide library screens to determine optimal motifs favored by specific kinases, the use of phospho-motif antibodies, and an approach that uses structurally altered kinases and allele-specific adenosine triphosphate analogs and kinase inhibitors. We describe these approaches and discuss their utility and inherent caveats.

Animals↗
PubMed1990

Peptides on phage: a vast library of peptides for identifying ligands.

We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors. Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage. The vector fAFF1, derived from the tetracycline resistance-transducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene III. A library of 3 x 10(8) recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of beta-endorphin (Tyr-Gly-Gly-Phe). Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody. The striking finding is that all 51 contained tyrosine as the N-terminal residue and that 48 contained glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 microM (Tyr-Gly-Phe-Trp-Gly-Met) to 8.3 microM (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7.1 nM for a known high-affinity ligand (Tyr-Gly-Gly-Phe-Leu). These results show that ligands can be identified with no prior information concerning antibody specificity. Peptide libraries are also likely to be useful in finding ligands that bind to other classes of receptors and in discovering pharmacologic agents.

Amino Acid Sequence↗
PubMedDate not supplied

A new strategy for the preparation of peptide-targeted technetium and rhenium radiopharmaceuticals. The automated solid-phase synthesis, characterization, labeling, and screening of a peptide-ligand library targeted at the formyl peptide receptor.

A new solid-phase synthetic methodology was developed that enables libraries of peptide-based Tc(I)/Re(I) radiopharmaceuticals to be prepared using a conventional automated peptide synthesizer. Through the use of a tridentate ligand derived from N-alpha-Fmoc-l-lysine, which we refer to as a single amino acid chelate (SAAC), a series of 12 novel bioconjugates [R-NH(CO)ZLF(SAAC)G, R = ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, benzyl; Z = Met, Nle] that are designed to target the formyl peptide receptor (FPR) were prepared. Construction of the library was carried out in a multiwell format on an Advanced ChemTech 348 peptide synthesizer where multi-milligram quantities of each peptide were isolated in high purity without HPLC purification. After characterization, the library components were screened for their affinity for the FPR receptor using flow cytometry where the K(d) values were found to be in the low micromolar range (0.5-3.0 microM). Compound 5j was subsequently labeled with (99m)Tc(I) and the product isolated in high radiochemical yield using a simple Sep-Pak purification procedure. The retention time of the labeled compound matched that of the fully characterized Re-analogue which was prepared through the use of the same solid-phase synthesis methodology that was used to construct the library. The work reported here is a rare example of a method by which libraries of peptide-ligand conjugates and their rhenium complexes can be prepared.

Automation↗
PubMedDate not supplied

Identification of a potent peptide deformylase inhibitor from a rationally designed combinatorial library.

Peptide deformylase catalyzes the removal of the N-terminal formyl group from nascent polypeptides during prokaryotic protein synthesis and maturation and is essential for bacterial survival. Its apparent absence from mammalian organisms makes it an attractive target for designing novel antibacterial agents. Based on the substrate specificity of peptide deformylase from Escherichia coli, a focused library of peptide thiols was synthesized on TentaGel resin using a disulfide linkage. Screening of the library against the purified deformylase was carried out in solution phase after the inhibitors were released from the resin with a reducing agent. A potent deformylase inhibitor was obtained from a 750-member library and was further optimized through rational modification into a low nanomolar inhibitor (KI = 15 nM against E. coli deformylase).

Amidohydrolases↗
PubMed2006

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: deconvolution strategy.

Abstract Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.

Biological Assay↗
PubMed1998

An HLA-binding-motif-aided peptide epitope library: a novel library design for the screening of HLA-DR4-restricted antigenic peptides recognized by CD4+T cells.

Susceptibility to a series of autoimmune diseases is strongly associated with particular HLA class II alleles. Identification of T cell clones and antigenic epitopes bound by HLA class II molecules involved in autoimmune diseases is critical to understanding the etiology of these HLA class II-associated diseases. However, establishment of T cell clones in autoimmune diseases is difficult because the antigenic peptides are unknown. Peptide library methods which include all possible peptide sequences offer a potentially powerful tool for the detection of cross-reactive antigenic peptides recognized by T cells. Here, we reduced the number of peptides per mixture by utilizing the known binding motifs of peptides for the HLA-DRB1*0405 molecule and evaluated the effectiveness of this library design. Each library mixture evoked a strong proliferative response in the unprimed peripheral blood lymphocytes (PBL) from HLA-DRB1*0405-positive donors but little or no response in the PBL from HLA-DRB1*0405-negative donors. The library also detected antigenic peptides that activated three antigen-specific T cell lines restricted by HLA-DRB1*0405, with different specificities. The motif-based approach thus presents a powerful method for monitoring T cells in large, heterogeneous T cell populations and is useful for the identification of the mimic peptide epitopes of T cell lines and clones.

Amino Acid Sequence↗
PubMed1995

Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities.

The screening of combinatorial peptide libraries has emerged as an important tool in the discovery of novel substrates or ligands for enzyme and receptor targets. For example, screening linear peptide libraries using streptavidin as a model receptor system has previously identified many low-affinity peptide ligands, all of which contain the common motif His-Pro-Gln (HPQ). We reasoned that constraining the conformational freedom of linear peptides by cyclization in a library would yield peptide ligands of increased affinity. Three different cyclic peptide libraries were constructed in an M13 phage display system as N-terminal pIII protein fusions. The random peptide sequences were flanked by two cysteine residues, which allows efficient disulfide bond formation and cyclization during phage assembly. These cyclic peptide libraries were screened with streptavidin as the model receptor system. Many sequences, all of which contained the motif His-Pro-Gln (HPQ), were discovered, and in the preceding paper, the structures of complexes of streptavidin-bound cyclic and linear peptides are described (Katz, 1995). Analysis of binding kinetics and affinities demonstrated that the conformationally constrained cyclic peptides bound streptavidin with affinities up to 3 orders of magnitude higher than linear peptides identified in previous library screens. These results demonstrate the potential of screening conformationally constrained peptide libraries for high-affinity novel receptor ligands or enzyme substrates.

Amino Acid Sequence↗
PubMed2003

Identification of SNARE complex modulators that inhibit exocytosis from an alpha-helix-constrained combinatorial library.

Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An alpha-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro -reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH2) abolished both Ca2+-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and L-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs.

Amino Acid Sequence↗
PubMed2000

Arginine-based structures are specific inhibitors of cathepsin C. Application of peptide combinatorial libraries.

Novel synthetic peptide inhibitors of lysosomal cysteine proteinase cathepsin C have been designed through the use of soluble peptide combinatorial libraries. The uncovered structural inhibitory module consists of the N-terminal cluster of L-arginine residues. Its modification with D-amino acids or arginine derivatives did not increase the inhibition strength. Inhibitory potency of oligoarginines improves with the elongation of peptide chain reaching a maximum for octa-L-arginine. The oligoarginines specifically interact with the cathepsin C active site as shown by competitive-type inhibition kinetics (Ki approximately 10-5 M) and intrinsic fluorescence measurements. The inhibitory interaction of oligoarginines is established through the specific spatial contact of a net of guanidino groups in the arginine side-chains, as indicated by comparison with inhibitory action of low molecular mass guanidine derivatives (Ki approximately 10-3 M). Nonarginine polyionic compounds cannot mimic the inhibitory effect of oligoarginines. The arginine-based peptide inhibitors were selective towards cathepsin C among other cysteine proteinases tested.

Animals↗
PubMed1997

Characterization of peptides that bind the tumor-associated Thomsen-Friedenreich antigen selected from bacteriophage display libraries.

Peptides with high affinities and specificities for numerous proteins and nucleic acids have been previously identified from random peptide bacteriophage display libraries. Here, random peptide bacteriophage display libraries were used to identify sequences that bound the cancer-associated Thomsen-Friedenreich glycoantigen (T antigen). The T antigen, present on most malignant cells, contains an immunodominant Gal beta1 --> 3GalNAc alpha disaccharide unmasked on the surfaces of most carcinomas. This antigen has been postulated to be involved in tumor cell aggregation and metastasis. Two 15 amino acid random peptide bacteriophage display libraries were affinity selected with glycoproteins displaying T antigen on their surfaces. Sequence analysis revealed that many of the peptides shared homology with sugar recognition sites in several carbohydrate-binding proteins. A comparison of affinity selected sequences from both libraries yielded a common motif (W-Y-A-W/F-S-P) rich in aromatic amino acids. Four peptides, corresponding to the affinity selected sequences, were chemically synthesized and characterized for their carbohydrate recognition properties. The synthetic peptides exhibited high specificities and affinities to T antigen displayed on asialofetuin or conjugated to bovine serum albumin (Kd = 5 nM for MAP-P30 binding to asialofetuin) as well as free T-antigen disaccharide in solution (Kd = 10 microM for MAP-P30, 20 microM for P10). Two peptides, P30 and P10, demonstrated high affinities and specificities for both asialofetuin and T antigen in solution. Iodination of a lone tyrosine residue in each sequence dramatically reduced their abilities to bind T antigen, suggesting that the tyrosine residue plays an important role in carbohydrate recognition. That these peptides are of functional significance is evidenced by the ability of both P30 and P10 to inhibit asialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface of MDA-MB-435 breast carcinoma cells in situ.

Amino Acid Sequence↗
PubMed1993

Acetalins: opioid receptor antagonists determined through the use of synthetic peptide combinatorial libraries.

A synthetic peptide combinatorial library made up of 52,128,400 hexapeptides, each having an acetyl group at the N terminus and an amide group on the C terminus, was screened to find compounds able to displace tritiated [D-Ala2,MePhe4,Gly-ol5]enkephalin from mu opioid receptor binding sites in crude rat brain homogenates. Individual peptides with mu receptor affinity were found using an iterative process for successively determining the most active peptide mixtures. Upon completion of this iterative process, the three peptides with the highest affinity were Ac-RFMWMT-NH2, Ac-RFMWMR-NH2, and Ac-RFMWMK-NH2. These peptides showed high affinity for mu and kappa 3 opioid receptors, somewhat lower affinity for delta receptors, weak affinity for kappa 1 receptors, and no affinity for kappa 2 receptors. They were found to be potent mu receptor antagonists in the guinea pig ileum assay and relatively weak antagonists in the mouse vas deferens assay. These peptides represent a class of opioid receptor ligands that we have termed acetalins (acetyl plus enkephalin).

Amino Acid Sequence↗
PubMed2006

Isolation of targeted AAV2 vectors from novel virus display libraries.

Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.

Animals↗
PubMed1992

The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides.

The systematic preparation of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal peptide sequences has been achieved through the screening in solution of SPCLs, each element of which is composed of more than 100,000 nonsupport-bound peptides in equimolar representation, along with an iterative synthesis and screening process. Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay. This same library was also used to develop highly potent antimicrobial peptides in bacterial growth inhibition assays. A separate non-acetylated SPCL was used to screen and identify high affinity peptide ligands using an opiate radio-receptor binding assay.

Amino Acid Sequence↗
PubMed2002

Positional scanning-synthetic peptide library-based analysis of self- and pathogen-derived peptide cross-reactivity with tumor-reactive Melan-A-specific CTL.

Synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) have recently emerged as a useful tool for the analysis of T cell recognition. This includes identification of potentially cross-reactive sequences of self or pathogen origin that could be relevant for the understanding of TCR repertoire selection and maintenance, as well as of the cross-reactive potential of Ag-specific immune responses. In this study, we have analyzed the recognition of sequences retrieved by using a biometric analysis of the data generated by screening a PS-SCL with a tumor-reactive CTL clone specific for an immunodominant peptide from the melanocyte differentiation and tumor-associated Ag Melan-A. We found that 39% of the retrieved peptides were recognized by the CTL clone used for PS-SCL screening. The proportion of peptides recognized was higher among those with both high predicted affinity for the HLA-A2 molecule and high predicted stimulatory score. Interestingly, up to 94% of the retrieved peptides were cross-recognized by other Melan-A-specific CTL. Cross-recognition was at least partially focused, as some peptides were cross-recognized by the majority of CTL. Importantly, stimulation of PBMC from melanoma patients with the most frequently recognized peptides elicited the expansion of heterogeneous CD8(+) T cell populations, one fraction of which cross-recognized Melan-A. Together, these results underline the high predictive value of PS-SCL for the identification of sequences cross-recognized by Ag-specific T cells.

Antigens, Bacterial↗
PubMed2000

Isolation of leptin-binding peptides from a random peptide phage library.

Leptin plays a role in regulating the body weight in mice. Injection of recombinant mouse leptin expressed in Escherichia coli reduced the food intake and body weight in normal, ob/ob and diet-induced obesity mice. Hyperglycemia, hyperinsulinemia and hypothermia can also be corrected in ob/ob mice after leptin injection. Leptin is a 16-kDa secretory protein comprising 167 amino acids produced in adipose tissue and is secreted to blood stream. In this study, a recombinant mouse leptin was generated and purified from a baculovirus expression system. This protein was used to identify putative ligands using a phage library of random peptides. Three leptin-binding phage clones were found, which were characterized by DNA sequencing and ELISA methods. The amino acid sequences of the reactive peptides are: LAYCSDPVRCLVWWY, MFWISAVSFVDHALV and LVLVLSAFLCCGVG. All three clones bound to recombinant human and mouse leptins. These peptides may be useful tools to study leptin-receptor interaction, food intake and body weight regulation.

Amino Acid Sequence↗