טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKovachi Tchelet
SubjectStructure-Activity Relationships of Acyl-Lysine Oligomers
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Amram Mor
Full Thesis textFull thesis text - English Version


Abstract

Oligomers of acylated lysines (OAKs) designed to mimic natural antimicrobial peptides were shown to exert potent antimicrobial properties in vitro and in vivo. OAKs can be organized as tandem repeats of acyl-lysyl (an) or lysyl-acyl-lysyl (bn) subunits, with n=4, 8 or 12 carbon acyls. a8-OAKs were selective for gram negative bacteria whereas a12-OAKs displayed a broader activity spectrum but were often hemolytic owing to self assembly in solution. This suggests that the use of intermediate acyl might unravel improved OAKs. Two series (a10 and b10-OAKs) were synthesized, purified (>95%) and assessed for biophysical properties  as to allow selection of the most promising OAK, which was further characterized, including in mechanistic and synergy with conventional antibiotics terms. The b10-OAKs generally displayed improved antibacterial properties which correlated with reduced tendency for self assembly. Namely, while showing low hemolytic activity (LC50>100µM), lauryl-lysyl-3b10 (C12K-3b10) inhibited growth of most of 51 bacteria tested in-vitro (MIC50=6.2 µM) by an essentially bacteriostatic mode of action as observed with four representative bacteria, at 3 or 6 multiples of the MIC value. While in Staphylococcus aureus the bacteriostsic effect was accompanied by depolarization of the cytoplasmic membrane, in Escherichia coli it did not, suggesting membrane related mode of action against Staphylococcus aureus, but different mechanism against Escherichia coli. Since the mode of action against Escherichia coli does not seems to be membrane related, bacterial DNA binding was tested and the results suggested that the OAK does not affect the bacteria through DNA binding mechanism.

C12K-3b10 resembles previously characterized OAKs with respect to activities against gram negative bacteria and red blood cells, but is also active against gram positive bacteria. The data suggest moreover that the OAK might act by distinct mechanisms on distinct bacteria. The effect of C12K-3b10 at sub-MIC concentrations combined with conventional antibiotics against both Staphylococcus aureus and Escherichia coli varied from full synergy through re-sensitization up to no effect at all, depending on various factors such as bacterial species and antibiotics used. This suggests that depending on the bacterial envelop, the OAK enables increased antibiotic penetrability and thus may help overcome some resistance mechanisms against them. Overall, these properties suggest that C12K-3β10 might represent a potential candidate for therapeutic development.