Synthesis of peptide analogues of the natural human peptide Cathelicidin LL-37 and study of their antimicrobial and anticancer activity (Master thesis)

Μιχαήλ, Βασίλειος

Cationic antimicrobial peptides (AMPs) are being produced in nature by all living organisms as part of their natural immunity system against invading pathogens and therefore, they are characterized as natural antibiotics. They have relatively low mass (<10 kDa), their sequences range between 10 to 50 amino acids, they carry a positive charge and the oxidation state is at least +2 (usually 4, 5, 6), because of the existence of the basic amino acids arginine, lysine and histidine. They consist more than 50% of hydrophobic amino acids and can be folded into a tertiary structure, amphipathic conformation, when interacting with the cell membrane of bacteria. The motivation of researchers is based on the range of the applications offered by these peptides, as they can act as effective antimicrobial agents either by themselves or with other antibiotics and other antimicrobial peptides, leading to rapid death of the pathogen and promoting angiogenesis and wound healing. Furthermore, AMPs can’t cause microbial resistance because they usually act by disrupting the cell membrane. Studies showed that AMPs have antimicrobial activity against a broad spectrum of pathogens, including gram-negative and gram-positive bacteria, fungi, viruses, parasites and protozoa. Some of the peptides have anticancer activity. The antimicrobial cationic peptides must meet certain requirements in order to be used as therapeutic agents. Thus, besides high antimicrobial activity, they should have low to none toxicity against human erythrocytes as well as proteolytic stability. In this work we synthesized six cationic peptide analogues of natural human antimicrobial peptide Cathelicidin LL-37. The sequences of synthesized peptide analogues are the follows. The first sequence corresponds to the central volute 17-29 of natural peptide Cathelicidin LL-37. . Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-NH2 .Leu-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-NH2 . Ala-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-NH2 . Phe-Lys-Arg-Ile-Val-Gln-Lys-Ile-Lys-Asp-Phe-Leu-Arg-NH2 . Ac-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Leu-Asp-Phe-Leu-Arg-NH2 . Ac-Phe-Lys( )-Arg-Ile-Val-Gln-Arg-Ile-Leu-Asp-Phe-Leu-Arg-NH2 The peptides were synthesized by the stepwise solid phase synthesis procedure by Merrifield, according to Fmoc-tBu method. The purification of the peptides was carried out using RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) and particularly they were purified by semi-preparative and preparative RP-HPLC. The purity of the peptides has been tested by analytical RP-HPLC, while the identification of their structure was confirmed by ESI-MS (Electrospray Ionization Mass Spectroscopy). The conformational characteristics of three peptide analogues were evaluated by circular dichroism spectroscopy (CD). All of the synthesized peptides were tested for their antimicrobial activity against three gram-negative bacteria, two gram-positive bacteria and a fungus. Experiments were carried out in order to test the cytotoxic activity of the peptides against the cell line A549 using the MTT method. A549 cells are epithelial lung cancer cells, which were isolated from Caucasian male, aged 58 years old. Also, the peptides were tested for the toxicity against human erythrocytes. The experiments for the synthesis, the purification and the identification of the peptides were performed in the Laboratory of Peptide chemistry of Chemistry Department. The study of antimicrobial activity and hemolytic stability took place in the Laboratory of Biochemistry of Chemistry Department. The study of cytotoxic activity against the cancer cells A549 was performed in the Unit of Technical Infrastructures and Methods of Characterization and Testing of Bioactive Substances. The experiments for the conformational characteristics of the peptides using circular dichroism spectroscopy were performed in the Laboratory of Biological Chemistry of Medical Department.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείας
Subject classification: Πεπτίδια
Keywords: Αντιμικροβιακά πεπτίδια,Αντικαρκινικά πεπτίδια,Antimicrobial peptides,Anticancer peptides
Appears in Collections:Διατριβές Μεταπτυχιακής Έρευνας (Masters)

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