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)be nzoic 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 (phi0) of the coloured peptides were also determined because phi0 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. The effect of chromophore (EPAB) and/or penta-lysine solubilizing tag on the biological activity was also studied. Potencies of the bovine neurotensin 8-13 fragment and its different coloured and penta-lysine derivatives were compared in isolated longitudinal muscle strips of guinea pig ileum. It was shown that the hexapeptide with penta-lysine tag had almost the same activity as the 8-13 fragment itself. The activity of the EPAB-derivative was found to be rather low. However, the presence of the solubilizing tag in the coloured hexapeptide compensated the negative effect of the chromophore.

Binary synthesis of multicomponent peptide mixtures by the portioning-mixing technique.

By introducing a new operation (non-coupling), our portioning-mixing method has become suitable for preparing binary peptide libraries. We demonstrate that all the expected components of a simple library are present in the mixture. The number of components in such libraries, the molar ratio of peptides as well as the possibilities of screening are discussed.

General method for rapid synthesis of multicomponent peptide mixtures.

A method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences. The main point of modification is that before every coupling cycle the resin is divided into equal parts and each portion is coupled with a different amino acid. Then the portion are mixed and before the next coupling cycle the resin is again distributed into equal portions. The method is illustrated by the synthesis of a mixture of 27 tetrapeptides and that of 180 pentapeptides.

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%.

Synthesis of gamma-L-glutamyl-taurine.

Glu(Tau), a bioactive substance previously isolated from the protein free aqueous extract of bovine parathyroid powder, has been synthesized. The intermediate derivative Z-Glu(Tau)-OBzl was prepared in three different ways from Z-Glu-OBzl and (1) cystamine by using the mixed anhydride method followed by oxidation, (2) Tau by the active ester procedure via Z-Glu-(ONp)-OBzl, (3) Tau applying mixed anhydride coupling. The protecting groups were removed by hydrogenolysis.