ELUCIDATION OF THE MECHANISM OF EFFECTIVE MEMBRANE PERMEATION OF BUFORIN 2 AND ITS APPLICATION TO VECTOR FOR INTRACELLULAR DRUG DELIVERY

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MORE THAN 500 ANTIMICROBIAL PEPTIDES HAVE BEEN DISCOVERED FROM VARIOUS ANIMALS INCLUDING HUMAN AND PLANTS. THESE PEPTIDES ARE NOW RECOGNIZED TO PLAY AN IMPORTANT ROLE IN INNATE IMMUNITY. MANY ANTIMICROBIAL PEPTIDES, LIKE MAGAININ 2 ISOLATED FROM XENOPUS LAEVIS, TARGET BACTERIAL CELL MEMBRANES, EXERTING CYTOTOXICITY BY PERMEABILIZING THEM. IN CONTRAST, BUFORIN 2 ISOLATED FROM BUFO BUFO GARGARIZANS EFFECTIVELY CROSS MEMBRANES WITHOUT PERMEABILIZATION, KILLING BACTERIA BY BINDING DNA AND RNA.THE AIMS OF THIS STUDY ARE 1)TO ELUCIDATE THE MECHANISM OF THE EFFECTIVE TRANSLOCATION OF BUFORIN 2 AND 2)TO APPLY THIS UNIQUE PROPERTY TO INTRACELLULAR DRUG DELIVERY.1)MECHANISM OF TRANSLOCATION : BUFORIN 2 TRANSLOCATES ACROSS LIPID BILAYERS BY A MECHANISM ESSENTIALLY THE SAME AS THAT OF MAGAININ 2, I.E. VIA THE FORMATION OF A TRANSIENT PEPTIDE-LIPID SUPRAMOLECULAR COMPLEX PORE. THE SHORT AMPHIPATHIC HELIX (RESIDUES 5-21) FORMED BY THE PRESENCE OF PROLINE11 CONTAINS MANY POSITIVE CHARGES. THEREFORE, THE ELECTROSTATIC REPULSION EXTREMELY DESTABILIZES THE PORE, MINIMIZING MEMBRANE PERMEABILIZATION.2)INTRACELLULAR DRUG DELIVERY : BUFORIN 2 DERIVATIVES WITH THE TEXAS RED DYE AS A DRUG MODEL AT THE N- OR C-TERMINUS WERE SYNTHESIZED, AND THEIR INTERACTIONS WITH HUMAN CELL LINES (HELA AND TM12) WERE INVESTIGATED. THESE PEPTIDES TEMPERATURE-INDEPENDENTLY PENETRATED CELLS EVEN IN THE PRESENCE OF A METABOLIC INHIBITOR, FURTHERMORE, THE TOXICITIES OF THESE PEPTIDES WERE VERY LOW. THUS, BUFORIN 2 IS A PROMISING CANDIDATE OF A VECTOR FOR INTRACELLULAR DRUG DELIVERY.FINALLY, THE AIMS OF THIS STUDY COULD BE ACHIEVED BY THE AID OF THIS GRANT.