Peptide research最新文献

Granulocyte/macrophage colony stimulating factor (GM-CSF) is both a hematopoietic growth factor and a cytokine implicated in inflammatory disease. The development of GM-CSF antagonist peptides corresponding to the GM-CSF native sequence should allow their modification into higher affinity analogs, but this is hampered by the low affinity of linear peptides. To adequately evaluate such low affinity peptides, the use of several independent assays should allow specific versus nonspecific inhibitors to be distinguished. In this study, inhibition of GM-CSF-dependent cell growth, inhibition of GM-CSF binding and immunologic cross-reactivity between GM-CSF-derived peptides and native protein by neutralizing antibodies have been used to evaluate peptide analogs with potential bioactivity. The GM-CSF sequence was divided into 6 peptides ranging in size from 15-24 amino acids. Antisera were raised to these peptides in mice and assayed for immunologic cross-reactivity. 4/6 anti-peptide antisera bound GM-CSF on ELISA and 3/6 on immunoprecipitation. Antisera to two of the peptides (corresponding to residues 17-31 and 96-112) inhibited GM-CSF-dependent cellular proliferation in two cell lines, with one peptide derived from residues 17-31 demonstrating inhibition of GM-CSF binding and direct biological inhibitory activity. A peptide that did not elicit native GM-CSF reactive antibodies, corresponding to residues 54-78, was recognized by two neutralizing monoclonal antibodies. It exhibited inhibition of GM-CSF binding and direct biological antagonist activity. These studies implicate two sites in mediating GM-CSF biological activity, and indicate that biological antagonists can be developed based on these sites.

Reduction of methionine sulfoxide with TiCl4/NaI is very rapid for simple methionine-containing peptides. The utility of this oxido/reduction system has been evaluated for three model peptides that contain oxidation/reduction-sensitive components such as a disulfide bond and/or a tryptophan residue. Completely specific reduction of methionine sulfoxide without some reduction of the disulfide bond was not possible with TiCl4/NaI. Reduction of the methionine sulfoxide residue in these model peptides yielded the desired product as the major component (yield ca. 70%) when a reaction time of four minutes was used. Methionine sulfoxide appears to be the most readily reducible species by low valent titanium. The competing side reactions observed were disulfide bond reduction by low valent titanium and/or tryptophan oxidation by the I2 generated by reduction of the TiCl4 with NaI. These side reactions became a serious problem when longer reaction times were used. The levels of contaminants generated by these side reactions were observed to increase with time, reducing the yield of the desired product.

To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

A complete series of N- and C-blocked, monodispersed homo-oligopeptides from the sterically hindered (R)-isovaline residue to the hexamer level was synthesized step by step by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuteriochloroform solution by Fourier transform infrared absorption and 1H nuclear magnetic resonance. In addition, the molecular structures of tripeptide, tetrapeptide and pentapeptide were assessed in the crystal state by x-ray diffraction. The results obtained confirm the conclusions from previous studies, namely that beta-bends and 3(10)-helices are preferentially adopted by isovaline-rich peptides, a clear indication that this C alpha, alpha-disubstituted glycine tends to induce folded structures much more extensively than its unmethylated parent compound alpha-aminobutyric acid. Furthermore, in this work we were able to demonstrate unambiguously for the first time that the relationship between isovaline chirality and helix screw sense is the same as that promoted by C alpha-monosubstituted glycines [(R)-amino acids give left-handed helical structures]. A comparison is also made with the conclusions extracted from published work on homo-oligopeptides from other C alpha-methylated amino acids.

N-(2,2,5,7,8-Pentamethylchroman-6-sulfonyl)-N'-3- (N-9-fluorenylmethoxycarbonyl-glycinyl)propylguanidine (1) was prepared and utilized as an arginine surrogate (Narg) building block compatible with solid-phase synthesis according to the Fmoc methodology. Narg is potentially useful in the assembly of combinatorial compound libraries or in the preparation of modified peptides. The applicability of this building block was demonstrated by its incorporation into an analogue of Thr-Arg-Ser-Ala-Trp, a pentapeptide for which inhibition of osteoclastic bone resorption was claimed. The modified pentapeptide showed an increased proteolytic stability when compared to the original inhibitor.

A technique of generating libraries in which every compound is displayed only once and all possible combinations are prepared with absolute certainty is presented. The method is based on the stepwise division of a continuous carrier in each step of randomization. Polypropylene membrane and cotton thread were evaluated as potential supports for this approach to library construction. Cotton thread accommodates 400 nmol of test compound per cm, but its mechanical properties limit the dimension of libraries. An alternative combinatorial library of "restructurable toothbrushes" is suggested.

A cyclic peptide template combinatorial library in a positional scanning format, composed of three positional libraries, was synthesized using solid-phase chemistry and four orthogonal protecting groups (Fmoc, Boc, Dde, OAll). The cyclic peptide template is composed of three lysine residues and one glutamic acid residue. The chemical diversity was introduced by acylating the epsilon-amino groups of the lysine residues using 10 carboxylic acids in addition to the 20 proteinogenic amino acids. The library components have been shown to be stable towards proteolytic degradation. Compounds with chymotrypsin inhibitory activity were identified through the screening of this library.

Peptides able to adopt an environment-dependent secondary structure represent attractive models for enzyme mimics. An approach to design new synthetic enzyme-like peptides involves the use of binding-step based mimics. The polypeptide melittin, based on its intrinsic ability to bind to phospholipids, was selected as a potential binding-step based catalytic mimic. Synthetic melittin was found to exhibit weak but reproducible phospholipase-like activity. The rate enhancement of the hydrolysis of phospholipids, although far from that of the natural enzyme, was encouraging as a first step in the development of new catalytic peptides.

Pseudopeptide analogues of the C-terminal hexapeptide of histone H3 (-Ile-Arg-Gly-Glu-Arg-Ala-OH) were obtained by systematically replacing, in each analogue, one peptide bond at a time by a reduced peptide bond psi (CH2-NH). The resulting analogues were then examined, in ELISA and in the BIAcore system, for their ability to bind polyclonal and monoclonal antibodies generated against the parent natural peptide and the protein. The comparative results show that reduced bond pseudopeptide analogues can mimic the parent peptide. These results present the first unequivocal example for the potent applicability of reduced peptide bond pseudopeptides in the immunological field.