טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMariana Hainrichson
SubjectDesign and Biochemical Characterization of Novel
Aminoglycosides as Potential Drugs: Overcoming
the Bacterial Resistance and Toxicity
DepartmentDepartment of Biotechnology
Supervisor Full Professor Baasov Timor
Full Thesis textFull thesis text - English Version


Abstract

Aminoglycosides are highly potent, broad-spectrum antibiotics that have important clinical applications in the treatment of bacterial infections. Aminoglycosides exert their effect by interfering with translational fidelity. Prolonged clinical and veterinary use of the currently available aminoglycosides has resulted in the rapid spread of antibiotic resistance to this family of antibiotics in pathogenic bacteria. Another limitation in using aminoglycosides is their high toxicity to mammals through kidney and ear-associated illnesses.

The main objective of this research is to design novel aminoglycosides that can resist or inhibit aminoglycoside-modifying enzymes while simultaneously target the ribosomal RNA. The investigations of the mechanisms of aminoglycosides selectivity to bacterial versus eukaryotic cells and their toxicity were in the heart of our research.

Initially we have generated a new class of branched aminoglycosides by linking a variety of sugars at C5”-OH group of neomycin B. Some of the compounds were found to be poorer substrates than the original neomycin B to the APH(3’)-IIIa enzyme. Our new derivatives exhibited similar or better antibacterial activities than that of the parent neomycin B, especially against Pseudomonas  aeruginosa. Therefore, we decided to explore the resistance mechanisms of this bacterium in order to improve the anti-pseudomonal potency of new derivatives. We have cloned, overexpressed and purified for the first time the chromosomally encoded enzyme APH(3’)-IIb from P. aeruginosa. The biochemical characterization of the enzyme revealed that it is highly regiospecific to the C3’-OH position of aminoglycosides. The conclusion derived from this study was that the future design of aminoglycoside derivatives against P. aeruginosa should consider the blocking of C3'-OH position from phosphorylation.

In a parallel research running in our lab, several novel analogs of aminoglycosides have been developed with aim to use them for the treatment of certain human genetic diseases. Series of toxicity tests on NB30 and NB54 have shown that they exhibit significantly reduced toxicity than those of the parent aminoglycoside drug paromomycin. In order to better understand the origin of aminoglycoside's toxicity, we have tested the ability of NB30 and NB54 along with a series of clinical aminoglycoside drugs, to inhibit mitochondrial protein synthesis on the intact mitochondria. We found that unlike clinical aminoglycoside drugs that significantly inhibit mitochondrial protein synthesis, NB30 and NB54 are lack of inhibitory activity. We also demonstrated that the aminogycoside-induced inhibition of mitochondrial protein synthesis promotes the formation of Fe(II) in mitochondria which in turns triggers formation of reactive oxygen species and cell death.