Ferenc Sebestyén

Chemical Synthesis of Peptide Libraries

Abstract The efficiency of the “portioning-mixing” method is demonstrated by the synthesis of more than one million hexapeptides in 5 days. An example is described to illustrate the possibility of completion of partial peptide libraries. Our method is also shown to be suitable to carry out binary peptide synthesis. In addition, a new screening method is outlined based on specific interaction of peptides with living cells.

Characterization of chemotactic ability of peptides containing N‐formyl‐methionyl residues in Tetrahymena fMLP as a targeting ligand

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C‐terminal f‐Met and N‐terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f‐MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f‐Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis‐activating processes.

INDUCTION OF APOPTOSIS BY A SHORT-CHAIN NEUROPEPTIDE ANALOG IN SMALL CELL LUNG CANCER

Small cell lung cancer (SCLC) cells express a variety of neuropeptides which act as autocrine growth factors. Although several neuropeptide analogs have been reported to antagonize SCLC proliferation, the development of these compounds has been limited by their low potency and the cytostatic nature of their effects. In the present study we evaluated the cytotoxic activity of four short-chain substance P analogs (NY3460, NY3238[-pHOPA], NY3238[Phe1], NY3238[Lys5]) against a panel of five SCLC cell lines. NY3460 was the most potent compound in all five SCLC cell lines (IC50 = 2.8-3.7 microM) as assessed by a MTT growth inhibitory assay. NY3238[Phe1] was also relatively active in all cell lines (IC50 = 3.5-11.2 microM), while NY3238[Lys5] and NY3238[-pHOPA] were substantially less active. NY3460 was the only agent to induce an increase in the percentage of cells with subdiploid DNA content suggestive of apoptosis by flow cytometric DNA content analysis. The induction of apoptosis was confirmed by fluorescent microscopy in NCI-H69, NCI-H82, NCI-H446, and NCI-H510 cells after exposure to 5.0 microM NY3460 for 48 h. These findings suggest that NY3460 is a relatively potent cytotoxic inhibitor of SCLC growth, and that short-chain neuropeptide analogs deserve further evaluation as anti-SCLC agents.

Coloured peptides: synthesis, properties and use in preparation of peptide sub-library kits.

Several methods were developed for the solid‐phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4‐(4‐(N‐ethyl‐N‐(3‐(tert‐butyloxycarbonyl)aminopropyl)amino)phenylazo)benzoic acid (Boc‐EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C‐termini. N‐terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe‐Ala‐Val‐Leu‐Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta‐lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP‐HPLC hydrophobicity indexes (φ0) of the coloured peptides were also determined because φ0 values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning‐mixing method. Each component of this library contained the red azo dye (EPAB) and the penta‐lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub‐libraries obtained after cleavage were readily soluble in water, giving intense red solutions.

An improved method for isolation of the C-terminal fragment of proteins

An efficient and easily realizable method for the isolation of the C-terminal fragment is described. Proteins are esterified by methanolic HCl and subsequently digested with pepsin. The peptide mixture is submitted to paper electrophoresis in pH 2.1 buffer. The identification of the C-terminal peptide is performed by preparing a guide peptide map, using pH 5.5 buffer in the second dimension. The C-terminal fragment appears as an on-diagonal spot. It can be isolated by a pH 5.5 run of the corresponding band from the first (pH 2.1) electrophoretogram. Since the C-terminal peptide is the fastest moving component, there is no need for its further purification. The expected yield is about 40%.

Fluorescent derivatives of bovine neurotensin 8-13 fragment

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.

Chemotaxis induced by SXWS tetrapeptides in Tetrahymena-overlapping chemotactic effects of SXWS sequences and their identical amino acids

The chemotactic potential of SXWS peptides and the components of the extracellular domain of cytokine receptors were investigated in Tetrahymena as a functional index of substitution with different amino acids in the position ‘X’ of the tetrapeptide. Data obtained demonstrate that position X plays a special determining role in the ligand, SEWS and STWS possess extremely strong chemoattractant ability, and aromatic amino acids result in chemorepellent ligands. Diverse effects of structurally related molecules, for example, SNWS–SDWS, demonstrate a highly sensitive discrimination potential in the applied model system. Physicochemical characteristics (hydropathy, residue size, and solvent‐exposed area) of the amino acids were correlated with the chemotactic activity. Data obtained by computer‐assisted conformation analysis of SXWS peptides and the highly overlapping chemotactic effects of the investigated SXWS peptides as well as the presence of the amino acids in the ‘X’ position indicate that member ‘X’ of the SXWS sequence performs a special role in interactions with the chemotaxis receptors in the membrane. Copyright

Analysis and Characterization of Combinatorial Mixtures of Mucin-2 Antigen Peptides

An epitope motif, TX1TX2T, of mucin-2 glycoprotein was identified by means of a monoclonal antibody, Mab 994 raised against a synthetic mucin-derived 13-mer pep-tide-conjugate [1,2]. For determination of the epitope sequence recognised with highest affinity by the antibody, a combinatorial approach was applied. We have prepared 19 sub-libraries (TATX2T, TDTX2T, TYTX2T) with portioning-mixing technique [3] using Boc/Bzl chemistry. Each library contained one pre-selected amino acid in position X1 and all proteinogenic amino acids, except Cys, in position X2 [1]. Antibody binding of sub-libraries was most profound when Gin was at the X1 position. 19 individual peptides corresponding to the TQTX2T sub-library were synthesised and tested. Binding data showed that apart from the native TQTPT peptide other compounds (e.g., TQTAT) were also recognised [4]. For the analytical characterization of the TQTX2T sub-library MALDI-TOF-MS and ultrahigh-resolution ESI-FTICR-MS as well as amino acid analysis were applied (cysteine was omitted for practical reasons). Shorter libraries corresponding to X2T and TX2T were also produced for amino acid analysis. To evaluate the resolution power of these MS methods we have prepared and tested “artificial” peptide mixtures of different complexity by mixing 6, 10, or 19 individual peptides corresponding to the TQTX2T. Thus equimolar mixtures of penta-peptides were analysed and compared with the combinatorial mixture.