A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin ß chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites.

Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability (1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect, (2) they are stable in human serum at least for 24 h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in the structure of oxime bond-linked daunorubicin-GnRH-III bioconjugates is not required for their antitumor activity. Moreover, the antitumor activity is influenced by the structure of the metabolites (daunorubicin-amino acid derivatives) and their DNA-binding properties.

Effect of conjugation with polypeptide carrier on the enzymatic degradation of Herpes simplex virus glycoprotein D derived epitope peptide.

Two conjugates with epitope peptide (278)LLEDPVGTVA (287) derived from glycoprotein D (gD-1) of Herpes simplex virus (HSV) were synthesized for analysis of the effect of conjugation on protection against enzymatic degradation. In this design, the turn-forming epitope core (281)DPVG (284) was positioned in the central part of the peptide and elongated by three amino acids from the native sequence at both termini. Conjugation was achieved by the introduction of amide bond or thioether linkage between the C-terminal of the HSV peptide and the side chain of four lysine residues of the oligotuftsin derivative used as carrier molecule. We compared the proteolytic stability of the conjugates in diluted human sera as well as in rat liver lysosomal preparation. The data obtained in lysosomal preparation at two pH values (pH 3.5 and 5.0) show that the type of covalent bond between the carrier and the epitope peptide had no significant effect, as compared to the stability of the free, unconjugated peptide. Based on the identification of degradation fragments by mass spectrometry we found marked differences in the lengths and amounts of oligopeptides obtained. In contrast, in 10% and 50% human serum the conjugation provided full protection against enzymatic hydrolysis over 96 h, while the free peptide was decomposed quickly.

Characterization of a trypsin-like serine protease of activated B cells mediating the cleavage of surface proteins.

Activated B cells may cleave their surface receptors due to the proteolytic activity on the cell membrane or in its vicinity. We attempted to isolate and characterize the protease(s) responsible for this cleavage. Zymograms prepared from the supernatant and the plasma membrane fraction of activated human B cells and BL41/95 cell line exhibited a 85-90 kDa doublet band with protease activity, while that of resting B cells did not. Soybean trypsin inhibitor (STI), Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and EDTA treatment abolished the activity of this protease. The excess of Zn(2+) ions in EDTA did not restore the enzymatic activity, while it was completely recovered in the presence of Ca(2+). We affinity-purified a 85-90 kDa protease from the supernatant of BL41/95 cells using STI coupled to Sepharose 4B beads, and measured its kinetic parameters. For the arginyl substrate K(M) was 358+/-59 microM and for the lysyl substrate 582+/-103 microM. TLCK and benzamidine inhibited the protease at micromolar, while STI at nanomolar concentrations. Both the inhibition profile and the substrate specificity suggest that it is a trypsin-like serine protease. We assume that the 85-90 kDa serine protease expressed on and secreted by activated B cells and BL41/95 cell line is responsible for the cleavage of various membrane proteins, including Fcgamma receptors; thus it may play a crucial role in regulating B cell's function.

The effect of cyclization on the enzymatic degradation of herpes simplex virus glycoprotein D derived epitope peptide.

One linear and three cyclic peptides corresponding to the 278-287 ((278)LLEDPVGTVA(287)) sequence of glycoprotein D (gD-1) of herpes simplex virus were synthesized for the analysis of the effect of cyclization on protection against enzymatic degradation. In this design, the turn-forming motif ((281)DPVG(284)) was positioned in the central part of the peptide and elongated by three amino acids at both termini. Cyclopeptide formation was achieved by the introduction of a peptide bond, a disulfide bridge or a thioether link. The stability of these peptides was compared in human serum and also in rat lysosomal preparations. The data obtained in 10% and 50% human serum show that all three types of cyclization enhanced the stability, but at different levels. Complete stability was only achieved by the introduction of a thioether link, while the presence of a disulfide or peptide bond resulted in improved, but partial resistance against hydrolytic decomposition. In lysosomal preparations the presence of cyclic primary structure provided full protection against enzymatic hydrolysis. Taken together, these findings indicate that by appropriate structural modification it is feasible to construct a synthetic antigen with high stability against enzymatic degradation in complex biological fluids. Further studies are in progress to identify enzymes responsible for degradation in diluted human sera as well as in the lysosomal preparations and to gain more detailed information on the mechanism of action.

Partial D-amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide.

The stability of an immunogen against enzymatic degradation is considered an important factor for the design of synthetic vaccines. For our studies, we have selected an epitope from the tandem-repeat unit of the high-molecular-weight MUC2 mucin glycoprotein, which can be underglycosylated in case of colon cancer. In this study, we prepared a MUC2 peptide containing the PTGTQ epitope of a MUC2 protein backbone-specific mAb 996 and its derivatives. In these peptides, the N- and C-terminal flanking regions were systematically substituted by up to three d-amino acids. Peptides prepared by solid-phase synthesis were tested for their mAb 996 binding in competitive ELISA experiments, and their stability was studied in serum and lysosomal preparation. Our data show that the epitope function of peptide (15)TPTPTGTQTPT(25) is retained even in the presence of two d-amino acid residues at its N-terminal flanking region and up to three at its C-terminal flanking region (tpTPTGTQtpt). Also, this partly d peptide shows high resistance against proteolytic degradation in diluted human serum and in lysosomal preparation. These findings suggest that, by appropriate combination of structural modifications (namely, d-amino acid substitution) in the flanks of an Ab epitope, it is feasible to construct a synthetic antigen with preserved recognition properties and high stability against enzymatic degradation. Peptides tPTPTGTQTpt and tpTPTGTQTpt derived from this study can be used for immunization experiments and as potential components of synthetic vaccines for tumor therapy.

The ubiquitin-proteasome system in Creutzfeldt-Jakob and Alzheimer disease: intracellular redistribution of components correlates with neuronal vulnerability.

Creutzfeldt-Jakob (CJD) and Alzheimer disease (AD) are accompanied by selective neuronal loss in the brain. We examined the regional and subcellular immunolocalization of ubiquitin, proteasomal subunits, and the heat-shock protein Hsp72 in control, CJD, and AD cases. In control and non-affected areas of disease cases, 20S proteasomes, 19S regulatory subunits, S6a, S6b, and S10b exhibit mainly cytoplasmic, whereas S4 and S7 show predominantly nuclear localization. The intensity of immunostaining for ubiquitin, proteasomal subunits, and Hsp72 varies in different anatomical regions both in disease and control brains. Areas with weaker immunolabeling correspond to affected areas in CJD and AD. In disease cases, antibodies for 20S, S4, S6b, S7, and ubiquitin intensely immunolabel neuronal nuclei of vulnerable cells in affected areas. Our results suggest that the ubiquitin-proteasome system takes part in the pathogenesis of neurodegeneration. Ubiquitin, Hsp72, and proteasomal ATPases possibly play a role in protecting certain neuronal populations in CJD and AD.