טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRotem Shahar
SubjectEssential Biophysical Requirements for Potency and
Selectivity of Antimicrobial Peptides
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Amram Mor
Full Thesis textFull thesis text - English Version


Abstract

Antimicrobial peptides (AMPs) are widely regarded as a potential source of future antibiotics owing to a remarkable set of advantages ranging from molecular simplicity to low-resistance swift-kill of a broad range of microbial cells. The externally localized site of action and receptor-independent mechanism of peptide-based antimicrobials is considered to be the key element in preventing drug resistance. However, this mechanism is also largely responsible for unselective activity over a wide range of cell types. Thus, a major challenge toward their safe use is the ability to endow AMPs with increased specificity.

My research focused on fine-tuning AMPs physicochemical attributes for improved antimicrobial properties, in terms of potency and selectivity, and in terms of the mechanism of action. First I assessed the extent to which physicochemical properties can be exploited to promote discriminative activity, by manipulating the N-terminal sequence of the 13-mer dermaseptin derivative, K4-S4(1-13). Inhibitory activity against 16 strains of bacteria showed that by manipulating its hydrophobicity and charge, K4-S4(1-13) became predominantly active against either Gram-positive or Gram-negative bacteria.

Next I tested the possibility that AMPs could be derived from random genomic sequences. Using two phage lysins (D3 and ΦKZ) as a model, I selected and produced two putative peptides believed to possess antimicrobial properties based on their physicochemical characteristics. The data support this hypothesis in that the peptides and various analogs displayed antibacterial activity and are believed to act by a mechanism of action resembling that of conventional AMPs when judged by both structural and functional criteria.

However, as drug candidates peptides represent less than ideal formula, namely because of poor bioavailability, potential immunogenicity, optional toxicity and high production costs. To address these issues, we designed novel peptide-mimetics termed oligo-acyl-lysyl (OAK).

To understand the attributes that govern activity of this novel antimicrobial system we compared functional and mechanistic properties of two analogous OAKs: C12K-7α8 and C12K-5α8. The data provide strong evidence for multiple similarities that included tissue stability, low hemolysis, large-spectrum antibacterial activity in-vitro and ability to prevent E. coli-induced mortality in-vivo. Despite these similarities however, C12K-7α8 mode of action involved membrane(s) disruption unlike the C12K-5α8 that acted predominantly through inhibition of DNA functions.

Collectively, the data support the view that physicochemical properties can govern AMPs selectivity and potency and further suggest that, relatively minor differences in the sequence of AMPs are responsible for selecting one mechanism over another.