Σύνθεση πεπτιδικών αναλόγων του φυσικού ανθρώπινου πεπτιδίου Cathelicidin LL-37 και μελέτη της αντιμικροβιακής και αντικαρκινικής τους δράσης
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Ημερομηνία
Συγγραφείς
Μιχαήλ, Βασίλειος
Τίτλος Εφημερίδας
Περιοδικό ISSN
Τίτλος τόμου
Εκδότης
Πανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείας
Περίληψη
Τύπος
Είδος δημοσίευσης σε συνέδριο
Είδος περιοδικού
Είδος εκπαιδευτικού υλικού
Όνομα συνεδρίου
Όνομα περιοδικού
Όνομα βιβλίου
Σειρά βιβλίου
Έκδοση βιβλίου
Συμπληρωματικός/δευτερεύων τίτλος
Περιγραφή
Τα πεπτίδια συντέθηκαν με τη μέθοδο πεπτιδικής σύνθεσης σε στερεή φάση κατά Merrifield, σύμφωνα με την Fmoc-tBu στρατηγική. Ο καθαρισμός των πεπτιδίων πραγματοποιήθηκε με την τεχνική της υγρής χρωματογραφίας υψηλής απόδοσης ανάστροφης φάσης, RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) και πιο συγκεκριμένα με την ημιπαρασκευαστική και παρασκευαστική RP-HPLC. Ο έλεγχος της καθαρότητάς τους πραγματοποιήθηκε με αναλυτική RP-HPLC, ενώ η ταυτοποίηση της δομής τους με φασματοσκοπία μάζας ιονισμού με ηλεκτροψεκασμό ESI-MS (Electrospray Ionization Mass Spectroscopy). Η διαμόρφωση των τριών πεπτιδικών αναλόγων μελετήθηκε με φασματοσκοπία κυκλικού διχρωϊσμού (CD). Όλα τα πεπτίδια μελετήθηκαν ως προς την αντιμικροβιακή τους δράση απέναντι σε τρία gram-αρνητικά, δύο gram-θετικά βακτήρια και σε ένα μύκητα. Πραγματοποιήθηκαν πειράματα ελέγχου της κυτταροτοξικής δράσης των πεπτιδίων με τη μέθοδο ΜΤΤ απέναντι στην κυτταρική σειρά Α549, που είναι επιθηλιακά καρκινικά κύτταρα από πνεύμονα ανδρός Καυκάσιας φυλής, ηλικίας 58 ετών. Επίσης, τα πεπτίδια μελετήθηκαν ως προς την τοξικότητά τους εναντίον των ανθρώπινων ερυθροκυττάρων. Η σύνθεση, ο καθαρισμός και η ταυτοποίηση των πεπτιδίων πραγματοποιήθηκαν στο Εργαστήριο Πεπτιδοχημείας του Τμήματος Χημείας. Η μελέτη της αντιμικροβιακής δράσης και της αιμολυτικής σταθερότητας πραγματοποιήθηκαν στο Εργαστήριο Βιοχημείας του Τμήματος Χημείας. Η μελέτη της κυτταροτοξικής δράσης εναντίον των καρκινικών κυττάρων Α549 πραγματοποιήθηκε στη Μονάδα Τεχνικών Υποδομών και Μεθόδων Χαρακτηρισμού και Ελέγχου Βιοδραστικότητας Ουσιών. Η διαμορφωτική μελέτη με φασματοσκοπία κυκλικού διχρωϊσμού (CD) πραγματοποιήθηκε στο Εργαστήριο Βιολογικής Χημείας του Τμήματος Ιατρικής.
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.
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.
Περιγραφή
Λέξεις-κλειδιά
Αντιμικροβιακά πεπτίδια, Αντικαρκινικά πεπτίδια, Antimicrobial peptides, Anticancer peptides
Θεματική κατηγορία
Πεπτίδια
Παραπομπή
Σύνδεσμος
Γλώσσα
el
Εκδίδον τμήμα/τομέας
Πανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείας
Όνομα επιβλέποντος
Πάνου-Πομώνη, Ευγενία
Εξεταστική επιτροπή
Πάνου-Πομώνη, Ευγενία
Λέκκα, Μαρία-Ελένη
Κούκκου, Άννα-Ειρήνη
Λέκκα, Μαρία-Ελένη
Κούκκου, Άννα-Ειρήνη
Γενική Περιγραφή / Σχόλια
Ίδρυμα και Σχολή/Τμήμα του υποβάλλοντος
Πανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείας
Πίνακας περιεχομένων
Χορηγός
Βιβλιογραφική αναφορά
Βιβλιογραφία : σ. 53, 88, 149, 168
Ονόματα συντελεστών
Αριθμός σελίδων
269 σ.
Λεπτομέρειες μαθήματος
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Άδεια Creative Commons
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