ABSTRACT
A set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized using solid phase peptide synthesis and chemical ligation technique. Selective chemical ligation was achieved as a result of hydrazone formation in the course of interaction between NLS hydrazide and GnRH analog modified by pyruvic acid. The efficiency of synthesized peptide carriers was demonstrated in experiments with human cancer cells transfected by reporter luciferase and beta-galactosidase genes or suicide HSV-1 thymidine kinase gene. It was shown that selectivity of action on cancer cells can be achieved as a result of peptide/DNA complex penetration through the cell membrane by GnRH receptor-mediated endocytosis pathway.
Subject(s)
Gene Transfer Techniques , Gonadotropin-Releasing Hormone , Nuclear Localization Signals , Antigens, Viral, Tumor/chemistry , Antigens, Viral, Tumor/pharmacology , Cell Membrane/chemistry , Cell Membrane/metabolism , DNA/chemistry , DNA/pharmacology , Gonadotropin-Releasing Hormone/analogs & derivatives , Gonadotropin-Releasing Hormone/chemical synthesis , Gonadotropin-Releasing Hormone/chemistry , Gonadotropin-Releasing Hormone/pharmacology , Hep G2 Cells , Humans , Nuclear Localization Signals/chemistry , Nuclear Localization Signals/pharmacology , Simian virus 40/chemistryABSTRACT
To improve the efficiency of anticancer drugs due to their delivery to intracellular targets a set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized. NLS was attached to the parent molecule via ε-amino group of D-Lysine in position 1 or 6 of peptide sequence using orthogonal protection strategy. The biological activity studies revealed that incorporation of NLS moiety significantly increases cytotoxic activity of palmitoyl-containing GnRH analogues in vitro. The influence of tested peptides on tumor cells does not accompanied by the destruction of cell membrane, as confirmed in experiments with normal fibroblasts, used as a control.
Subject(s)
Antigens, Viral, Tumor/chemistry , Cell Nucleus/metabolism , Drug Carriers/chemistry , Gonadotropin-Releasing Hormone/analogs & derivatives , Nuclear Localization Signals/chemistry , Simian virus 40/metabolism , Amino Acid Sequence , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Carriers/chemical synthesis , Drug Carriers/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gonadotropin-Releasing Hormone/chemical synthesis , Gonadotropin-Releasing Hormone/chemistry , Gonadotropin-Releasing Hormone/pharmacology , Humans , Molecular Sequence Data , Nuclear Localization Signals/genetics , Protein Binding , Receptors, LHRH/metabolism , Simian virus 40/chemistryABSTRACT
N-Amidinoproline, a hybrid structure modeling key features of the Arg-Pro sequence, was synthesized. The activation of carboxyl group of free N-amidinoproline was found to result in the formation of a cyclic side product, whose structure was confirmed by ESI MS, 1H NMR, and 13C NMR spectra. The preparation of N-(mesitylenesulfonylamidino)-L-proline using the mesitylenesulfonyl derivative of 2-methylisourea was demonstrated to be accompanied by partial racemization. The target product was synthesized by modification of N-amidinoproline by mesitylenesulfonyl chloride. The possibility of using N-amidinoproline in the N-terminal modification of a peptide chain was shown by the example of synthesis of an analogue of the 95-98 fragment of fibrinogen alpha chain.
Subject(s)
Peptides/chemical synthesis , Proline/analogs & derivatives , Dipeptides/chemical synthesis , Dipeptides/chemistry , Fibrinogen/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Peptides/chemistry , Proline/chemistry , Protein Structure, Secondary , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
Luliberin analogues modified at the N-terminus were synthesized to search for drugs exerting a cytotoxic effect on cells of hormone-dependent tumors. A synthetic scheme effective in the preparation of analogues containing fatty acid residues was proposed. The cytotoxic effect of the peptides was studied on a number of cell lines of human tumors in vitro. The dependence of the antitumor effect on the length of peptide chain, amino acid sequence, and structure of the N-terminal group was demonstrated. Modification with palmitic acid was found to result in highly active compounds in the case of analogues containing more than ten aa, whereas modifications with lauric, caproic, or trimethylacetic acid led to compounds with significantly lower activities. Analogues of luliberin containing a palmitic acid residue and effectively inhibiting the growth of tumor cells in vitro were synthesized.
