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.
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Pázmány P. stny. 1/A, 1117, Budapest, Hungary.
Erika
E
Orbán
Gábor
G
Mezo
Pascal
P
Schlage
Gabriella
G
Csík
Zarko
Z
Kulić
Philipp
P
Ansorge
Erzsébet
E
Fellinger
Heiko Michael
HM
Möller
Marilena
M
Manea
D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D002401:Cathepsin B / Q000737:chemistry; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D004247:DNA / Q000737:chemistry*; D003630:Daunorubicin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D005453:Fluorescence; D007987:Gonadotropin-Releasing Hormone / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D015394:Molecular Structure; D010091:Oximes / Q000493:pharmacokinetics / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D044967:Serum / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D010091:Oximes; D010446:Peptide Fragments; C448022:gonadotropin-releasing hormone-III; D007987:Gonadotropin-Releasing Hormone; D004247:DNA; D002401:Cathepsin B; D011761:Pyrrolidonecarboxylic Acid; D003630:Daunorubicin