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PubMed2001

The peptide-specific alloreactive human T cell repertoire varies largely between individuals and is not extended in HLA-A*0205--anti-HLA-A*0201 pairings.

Alloreactive T cells recognize framework or peptide-dependent determinants on foreign MHC molecules. Among the peptide-dependent alloreactive T cells a significant proportion is specific for one particular peptide presented by the allo-MHC molecule as antigen-specific T cells would do. Such alloreactive, peptide-specific T cells are referred to as 'allorestricted'. High-avidity HLA-A*02 allorestricted cytotoxic T lymphocyte (CTL) clones specific for peptide libraries can be generated from HLA-A*02(-) donors. We made use of this technique to study the role of closely related self-HLA molecules on shaping of the alloreactive T cell repertoire. Peripheral blood lymphocytes from HLA-A*0205 individuals were stimulated by HLA-A*0201 targets pulsed with an HLA-A*0201 peptide library. We did not observe a bias towards peptide-specific CTL in the HLA-A*0201-directed alloreactive repertoire of HLA-A*0205 donors as compared to HLA-A*02(-) donors. Comparison of the alloreactive T cell response between two donors having similar HLA haplotypes demonstrated that the allorestricted T cell repertoire is largely different between individuals.

Cell Line, Transformed↗
PubMed2004

A novel method for evaluation and screening of caspase inhibitory peptides by the amino acid positional fitness score.

BACKGROUND: Since caspases are key executioners of apoptosis in cases of severe diseases including neurodegenerative disorders such as Alzheimer's disease and Huntington's disease, and viral infection diseases such as AIDS and hepatitis, potent and specific inhibitors of caspases have clinical potential. A series of peptide inhibitors has been designed based on cleavage sites of substrate proteins. However, these peptides are not necessarily the most potent to each caspase. Moreover, so far, it has proved to be difficult to design potent and specific peptide inhibitors of each caspase from sequence data of known cleavage sites in substrate proteins. We have attempted to develop a computational screening system for rapid selection of potent and specific peptide inhibitors from a comprehensive peptide library. RESULTS: We developed a new method for rapid evaluation and screening of peptide inhibitors based on Amino acid Positional Fitness (APF) score. By using this score, all known peptide inhibitors of each caspases-3,-7,-8, and -9 were rapidly selected in their enriched libraries. In this libraries, there were good correlations between predicted binding affinities of the known peptide inhibitors and their experimental Ki values. Furthermore, a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 was able to be designed by this method. To our knowledge, DNLD is a first reported caspase-3 inhibitory peptide identified by using the computational screening strategy. CONCLUSION: Our new method for rapid screening of peptide inhibitors using APF score is an efficient strategy to select potent and specific peptide inhibitors from a comprehensive peptide library. Thus, the APF method has the potential to become a valuable approach for the discovery of the most effective peptide inhibitors. Moreover, it is anticipated that these peptide inhibitors can serve as leads for further drug design and optimization of small molecular inhibitors.

Algorithms↗
PubMed2006

Screening of alpha-helical peptide ligands controlling a calcineurin-phosphatase activity.

In this paper, we describe an application of 202-membered fluorescently labeled peptide library designed to take an alpha-helix secondary structure. As a proof-of-concept experiment, a calmodulin (CaM)/calcineurin (Cn) pair was chosen to screen alpha-helical peptide ligands that tightly bind to CaM and also control enzymatic functions of Cn. Three peptides were successfully selected from the library by assaying Cn-phosphatase activities and peptide-CaM interactions (dual check process). The strategy using a designed peptide library shows real promise as a peptide-based high-throughput screening system.

Amino Acid Sequence↗
PubMed2002

Autoantibodies to inner ear and endothelial antigens in Cogan's syndrome.

BACKGROUND: Cogan's syndrome is a chronic inflammatory disease of unknown origin, characterised by sensorineural hearing loss, episcleritis, and vasculitis. An autoimmune origin has been suggested but not proven. Our aim was to establish whether or not an autoimmune process is the cause of the disease. METHODS: We used pooled IgG immunoglobulins derived from eight patients with Cogan's syndrome to screen a random peptide library to identify disease relevant autoantigen peptides. Among the identified peptides, one was recognised by all the patients' sera. Antibodies against peptides were affinity purified from patients' sera and used to characterise the autoantigen, to stain human cochlea, and to transfer the features of Cogan's disease into animals. FINDINGS: We identified an immunodominant peptide that shows similarity with autoantigens such as SSA/Ro and with the reovirus III major core protein lambda 1. The peptide sequence shows similarity also with the cell-density enhanced protein tyrosine phosphatase-1 (DEP-1/CD148), which is expressed on the sensory epithelia of the inner ear and on endothelial cells. IgG antibodies against the peptide, purified from the patients' sera, recognised autoantigens and DEP-1/CD148 protein, bound human cochlea, and inhibited proliferation of cells expressing DEP-1/CD148. The same antibodies bound connexin 26, gene mutations of which lead to congenital inner-ear deafness. Furthermore, these antibodies were able to induce the features of Cogan's disease in mice. INTERPRETATION: Our results indicate that Cogan's syndrome is an autoimmune disease, characterised by the presence of autoantibodies able to induce tissue damage on binding of cell-surface molecules present on the sensory epithelia of the inner ear and on endothelial cells.

Animals↗
PubMed1999

Mimotopes of cytolytic T lymphocytes in cancer immunotherapy.

Rapid identification of the cytolytic T lymphocyte (CTL) epitopes that are presented by cancer cells will provide the basis for manipulation of tumor immunity but remains a major challenge in the field. By screening randomly synthesized peptide libraries, mimotopes for tumor-reactive CTLs can be readily constructed and they may be functionally equal to or potentially more potent than natural peptides in stimulating CTL responses; therefore, the peptide library approach provides great promise for design of cancer vaccines and adoptive tumor immunotherapy.

Animals↗
PubMed1999

Identification of novel classes of protein kinase inhibitors using combinatorial peptide chemistry based on functional genomics knowledge.

A discovery approach based on an intramolecular inhibitory mechanism was applied to a prototype calmodulin (CaM)-regulated protein kinase in order to demonstrate a proof-of-principle for the development of selective inhibitors. The overall approach used functional genomics analysis of myosin light chain kinase (MLCK) to identify short autoinhibitory sequences that lack CaM recognition activity, followed by recursive combinatorial peptide library production and comparative activity screens. Peptide 18 (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2), one of several selective inhibitors discovered, has an IC50 = 50 nM for MLCK, inhibits CaM kinase II only at 4000-fold higher concentrations, and does not inhibit cyclic AMP-dependent protein kinase. Analogues of peptide 18 containing conformationally constrained cis-4-aminocyclohexanecarboxylic acid retained affinity and selectivity. The inhibitors add to the armamentarium available for the deconvolution of complex signal transduction pathways and their relationship to homeostasis and disease, and the approach is potentially applicable to enzymes in which the catalytic and regulatory domains are found within the same open reading frame of a cDNA.

Calcium-Calmodulin-Dependent Protein Kinase Type 2↗
PubMed2000

The role of peptides in T cell alloreactivity is determined by self-major histocompatibility complex molecules.

By analyzing T cell responses against foreign major histocompatibility complex (MHC) molecules loaded with peptide libraries and defined self- and viral peptides, we demonstrate a profound influence of self-MHC molecules on the repertoire of alloreactive T cells: the closer the foreign MHC molecule is related to the T cell's MHC, the higher is the proportion of peptide-specific, alloreactive ("allorestricted") T cells versus T cells recognizing the foreign MHC molecule without regard to the peptide in the groove. Thus, the peptide repertoire of alloreactive T cells must be influenced by self-MHC molecules during positive or negative thymic selection or peripheral survival, much like the repertoire of the self-restricted T cells. In consequence, allorestricted, peptide-specific T cells (that are of interest for clinical applications) are easier to obtain if T cells and target cells express related MHC molecules.

Animals↗
PubMed2005

Role of molecular mimicry to HIV-1 peptides in HIV-1-related immunologic thrombocytopenia.

Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the beta3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Ten were molecular mimics with close sequence similarity to HIV-1 proteins nef, gag, env, and pol. Seven were synthesized as 10-mers from their known HIV-1 sequence and found to inhibit anti-GPIIIa49-66-induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated.

Amino Acid Sequence↗
PubMed2002

Peptide-independent folding and CD8 alpha alpha binding by the nonclassical class I molecule, thymic leukemia antigen.

The nonclassical class I molecule, thymic leukemia (TL), has been shown to be expressed on intestinal epithelial cells and to interact with CD8(+) intraepithelial T lymphocytes. We generated recombinant soluble TL (T18(d)) H chains in bacteria as inclusion bodies and refolded them with beta(2)-microglobulin in the presence or absence of a random peptide library. Using a mAb, HD168, that recognizes a conformational epitope on native TL molecules, we observed that protein folds efficiently in the absence of peptide. Circular dichroism analysis demonstrated that TL molecules have structural features similar to classical class I molecules. Moreover, thermal denaturation experiments indicated that the melting temperature for peptide-free TL is similar to values reported previously for conventional class I-peptide complexes. Our results also show that CD8alphaalpha binding is not dependent on either TL-associated peptide or TL glycosylation.

Animals↗
PubMed2003

Analysis of HLA-E peptide-binding specificity and contact residues in bound peptide required for recognition by CD94/NKG2.

The MHC class Ib molecule HLA-E is the primary ligand for CD94/NKG2A-inhibitory receptors expressed on NK cells, and there is also evidence for TCR-mediated recognition of this molecule. HLA-E preferentially assembles with a homologous set of peptides derived from the leader sequence of class Ia molecules, but its capacity to bind and present other peptides remains to be fully explored. The peptide-binding motif of HLA-E was investigated by folding HLA-E in vitro in the presence of peptide libraries derived from a nonameric leader peptide sequence randomized at individual anchor positions. A high degree of selectivity was observed at four of five total anchor positions, with preference for amino acids present in HLA-E-binding peptides from class Ia leader sequences. Selectivity was also observed at the nonanchor P5 position, with preference for positively charged amino acids, suggesting that electrostatic interactions involving the P5 side chain may facilitate assembly of HLA-E peptide complexes. The observed HLA-E peptide-binding motif was strikingly similar to that previously identified for the murine class Ib molecule, Qa-1. Experiments with HLA-E tetramers bearing peptides substituted at nonanchor positions demonstrated that P5 and P8 are primary contact residues for interaction with CD94/NKG2 receptors. A conservative replacement of Arg for Lys at P5 completely abrogated binding to CD94/NKG2. Despite conservation of peptide-binding specificity in HLA-E and Qa-1, cross-species tetramer-staining experiments demonstrated that the interaction surfaces on CD94/NKG2 and the class Ib ligands have diverged between primates and rodents.

Amino Acid Motifs↗
PubMed2006

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81.

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.

Amino Acid Sequence↗
PubMed2004

Multiple binding sites in fibrinogen for integrin alphaMbeta2 (Mac-1).

The leukocyte integrin alphaMbeta2 (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogen providing a key link between thrombosis and inflammation. However, the mechanism by which alphaMbeta2 binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen gammaC domain sequences, P1 (gamma190-202) and P2 (gamma377-395), serve as the alphaMbeta2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding alphaMI-domain of alphaMbeta2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several alphaMI-domain molecules. To localize the alternative alphaMI-domain binding sites, we screened peptide libraries covering the complete sequences of the gammaC and betaC domains, comprising the majority of the D fragment structure, for alphaMI-domain binding. In addition to the P2 and P1 peptides, the alphaMI-domain bound to many other sequences in the gammaC and betaC scans. Similar to P1 and P2, synthetic peptides derived from gammaC and betaC were efficient inhibitors of alphaMbeta2-mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the alphaMI-domain binding depends on basic and hydrophobic residues. These findings establish a new model of alphaMbeta2 binding in which the alphaMI-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin alphaMbeta2.

Algorithms↗
PubMed2004

Peptide mapping of feline immunodeficiency virus by IFN-gamma ELISPOT.

Interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) has become an important tool in studying antigen-specific T lymphocyte responses. Soluble peptides can be used to map T-cell epitopes, providing information that is useful in the design and evaluation of vaccines as well as studies of immunopathogenesis. To date, this assay has not been widely utilized in feline immunodeficiency virus (FIV) research. We have developed a feline IFN-gamma ELISPOT assay and used it to determine FIV-specific T-cell epitopes recognized by infected cats. A panel of 331 peptides, 15 amino acids in length and overlapping by 10 residues was synthesized. The peptide library spanned the FIV structural (Gag), envelope (Env), reverse transcriptase (RT), and open-reading-frame A (OrfA) proteins. Initially, 34 pools, containing 7-10 peptides each were screened by IFN-gamma ELISPOT against peripheral blood mononuclear cells (PBMC) from eight cats chronically infected with the NCSU(1) molecular clone of FIV and four uninfected control cats. Individual peptides from pools recognized by FIV+ cats were then evaluated and optimal peptides were combined into pools representing Gag, Env, RT, and OrfA. A higher percentage of FIV infected cats were identified as responders against the peptide pools when using fresh PBMC as compared to cryopreserved PBMC. In vitro restimulation of cryopreserved PBMC with the peptide pools improved the sensitivity of the assay to similar levels as observed from fresh samples. Individual peptides used in the pools were generally found to stimulate CD8+ T-cells more efficiently than CD4+ T-cells. Comparison of the peptide sequences to representative FIV sequences from clades A-D showed conservation was high among Gag and RT peptides, variable among Env peptides and low for OrfA peptides. The IFN-gamma ELISPOT assay and FIV-specific peptide pools we describe here will be useful in assessing cell-mediated responses to experimental FIV vaccines.

Amino Acid Sequence↗
PubMed2001

Structure-function analysis of a phage display-derived peptide that binds to insulin-like growth factor binding protein 1.

Highly structured, peptide antagonists of the interaction between insulin-like growth factor 1 (IGF-I) and IGF binding protein 1 (IGFBP-1) have recently been discovered by phage display of naïve peptide libraries [Lowman, H. B., et al. (1998) Biochemistry 37, 8870--8878]. We now report a detailed analysis of the features of this turn-helix peptide motif that are necessary for IGFBP-1 binding and structural integrity. Further rounds of phage randomization indicate the importance of residues contributing to a hydrophobic patch on one face of the helix. Alanine-scanning substitutions confirm that the hydrophobic residues are necessary for binding. However, structural analysis by NMR spectroscopy indicates that some of these analogues are less well folded. Structured, high-affinity analogues that lack the disulfide bond were prepared by introducing a covalent constraint between side chains at positions i and i + 7 or i + 8 within the helix. Analogues based on this scaffold demonstrate that a helical conformation is present in the bound state, and that hydrophobic side chains in this helix, and residues immediately preceding it, interact with IGFBP-1. By comparison of alanine scanning data for IGF-I and the turn-helix peptide, we propose a model for common surface features of these molecules that recognize IGFBP-1.

Alanine↗
PubMed2005

Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics.

Epidermal growth factor receptor (ErbB1, EGFR) is overexpressed in a variety of human cancer cells. It has been considered as a rational target for drug delivery. To identify novel ligands with specific binding capabilities to EGFR, we screened a phage display peptide library and found an enriched phage clone encoding the amino acid sequence YHWYGYTPQNVI (designated as GE11). Competitive binding assay and Scatchard analysis revealed that GE11 peptide bound specifically and efficiently to EGFR with a dissociation constant of approximately 22 nM, but with much lower mitogenic activity than with EGF. We showed that the peptides were internalized preferentially into EGFR highly expressing cells, and they accumulated in EGFR overexpressing tumor xenografts after i.v. delivery in vivo. In gene delivery studies, GE11-conjugated polyethylenimine (PEI) vectors were less mitogenic, but still quite efficient at transfecting genes into EGFR highly expressing cells and tumor xenografts. Taken together, GE11 is a potentially safe and efficient targeting moiety for selective drug delivery systems mediated through EGFR.

Cell Line, Tumor↗
PubMed2004

Is phage display technology on target for developing peptide-based cancer drugs?

New tumor targeting agents are required to advance cancer diagnosis and treatment. Bacteriophage (phage) display technology, a molecular genetic means of combinatorial drug discovery, is an emerging approach to identify and improve peptide molecules as pharmaceuticals. Peptides are thought to have clinically desirable benefits over currently used biomolecules, such as antibodies, because of their rapid blood clearance, increased diffusion and tissue penetration, non-immunogenic nature and ease of synthesis. Using phage display, one can rapidly and simultaneously survey billion-clone peptide libraries, resulting in large numbers of "hits". However, only a few lead compounds resulting from the hits historically reach the drug market. Hence determining which peptide may best translate into a useful drug is of particular importance. Examination of successfully marketed drugs has highlighted key features of a winning agent, including low molecular weight, high affinity, stability, solubility, lipophilicity and conformational rigidity. Although peptide modulators of tumor cell function and cancer targeting agents have been developed, the majority of peptide-based drugs reported thus far are immune and cardiac regulators. In this review, we will highlight how phage display has been employed to isolate peptides that target key steps in cancer progression--from tumor growth to metastasis--and how phage display technology can be harnessed to select a priori peptides with inherent features essential for anti-cancer drug efficacy. In 2003, phage display provided us with several novel peptides not only in clinical trials but approved by the FDA for use as therapeutics in a variety of diseases--suggesting that the future looks bright for phage display in anti-cancer drug development.

Animals↗
PubMed2005

Peptide inhibitors of botulinum neurotoxin by mRNA display.

Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs.

Amino Acid Sequence↗