טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentGoldberg Keren
SubjectMolecular Basis for OAK Synergy with Antibiotics
Targeting Gram-Negative Bacteria
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Amram Mor
Full Thesis textFull thesis text - English Version


Abstract

Bacterial multidrug resistance to conventional antibiotics has become a distressing widespread phenomenon. Therefore, vast effort is being put into developing new drugs, in order to overcome this problem. Host defense peptides (HDPs) represent attractive candidates for drug development due to their ability to target a broad spectrum of pathogens while significantly challenging development of resistance mechanisms. However, HDPs present various drawbacks, including toxicity, bioavailability and high costs production. To overcome these disadvantages, peptide-mimetic compounds have been designed, based on HDPs’s essential biophysical characteristics.

Oligomers of acylated lysines (OAKs) are synthetic mimics of HDPs, composed of tandem repeats of amide linked fatty acids and lysines. Due to their structural simplicity, pharmacokinetic stability and relative low production cost, OAKs represent potential antibacterial agents. The OAK C12(ω7)K-β12 preferentially targets gram-positive bacteria by a bacteriostatic mode of action, whilst practically inactive against gram-negative bacteria. Also, C12(ω7)K-β12 has demonstrated synergistic mode of action with several kinds of antibiotics against Gram-positive bacteria, and, more interestingly, this OAK was suggested to synergies with some antibiotics against Gram-negative bacteria as well. This work was therefore designed to verify/investigate this possibility.

We show that potent antibiotic action can be provoked in-vitro and in-vivo, by a treatment combining two antibacterial compounds that are essentially inactive against Gram-negative bacteria. At sub-minimal inhibitory concentrations, C12(ω7)K-β12 showed the ability to permeabilize the outer membrane of Gram-negative bacteria to large molecules such as nitrocefin. Also, C12(ω7)K-β12 has rapidly achieved partial membrane depolarization, hence depriving bacteria of the proton motive force required for active efflux. Consequently, bacteria became significantly sensitive to intracellular targeting antibiotics such as rifampicin and erythromycin, thereby suggesting a potentially useful approach for expanding the antibiotics sensitivity spectrum of MDR Gram-negative bacteria. Also, the ability to affect disease course systemically was compared for mono- and combination therapy, using the mouse thigh-infection model. Preliminary results showed that combined treatment of C12(ω7)K-β12 and erythromycin was more effective than each treatment alone, unlike the combined treatment with rifampicin.

In attempt to improve activity of the OAK-rifampin combined treatment in-vivo, we initiated the assessment of a novel lipid-OAK encapsulation system, designed to facilitate the simultaneous delivery of this OAK and rifampicin. Our results so far, show the correlation between lipid compositions, efficient drug encapsulation and stability in whole blood.

Collectively, the data suggest a potentially useful approach to expand the currently decreasing antibiotic armamentarium for therapeutic treatment of GNB.