Ανάπτυξη ανιμικροβιακών πεπτιδίων: σχεδιασμός, σύνθεση, διαμορφωτικές και βιολογικές μελέτες

Abstract

The excessive use of antibiotics, which has led to multi-resistant bacteria, makes it necessary to develop new therapeutic agents. Antimicrobial peptides (AMPs) are used as the first defensive barrier of the organism against microbial infection. They possess a broad spectrum of antimicrobial activity against Gram negative and Gram positive bacteria, as well as against fungi and protozoa. Most of antimicrobial peptides have positive charge, in order to interact electrostatically with the negatively charged bacterial membranes and can adopt amphipathic helical conformations, which facilitates the disruption of the bacterial membranes. Elements desirable for therapeutics include antimicrobial activity, as well as low toxicity and proteolytic stability. In the present study, cationic helical peptides of type Y-(Aib-Arg-Aib-X)n-NH2, where n = 2 ή 3, Υ = Leu ή Phe θαη Y= H- ή Ac- ή Ahx, were synthesized by the conventional stepwise Fmoc/tBu solid-phase method on a Rink-Amide AM resin. The peptides were purified by RP-HPLC and identified by ESI-MS. Conformational studies of the peptides were evaluated by CD spectroscopy and indicated high helical conformations. The above peptides were tested for their antimicrobial activity against Gram negative bacteria, E. coli DH5a, P. aeruginosa PAO and Gram positive bacteria, M. smegmatis mc2155 and B. subtilis DELTA, as well as against fungi C. parapsilosis. The peptides with the best activity against bacteria were tested for their hemolytic activity, whereas proteolytic stability assays were performed. The peptides Ζ-(Aib-Arg-Aib-Leu)3-NH2, Ahx-(Aib-Arg-Aib-Leu)3-NH2, Ζ-(Aib- Arg-Aib-Phe)3-NH2 and Ahx-(Aib-Arg-Aib-Leu)2-NH2 exhibits the best antimicrobial activities (lowest MIC) against Gram negative bacterial strain E. coli DH5a. They are not toxic at this concentration and retain their activity for a long time after incubation with the proteolytic enzyme trypsin.