טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShapira Katya
SubjectEvaluation of Nonsense Mutation Suppression Efficiency of
Novel Aminoglycoside Derivatives
DepartmentDepartment of Biotechnology
Supervisor Professor Timor Baasov
Full Thesis textFull thesis text - English Version


Abstract

A large number of human genetic diseases result from nonsense mutations that cause premature termination of the synthesis of proteins encoded by mutant genes. Currently, hundreds of such nonsense mutations are known, and several where shown to account for certain cases of fatal diseases, including cystic fibrosis, Duchenne muscular dystrophy, Hurler syndrome,  and more. For many of these diseases there is presently no effective treatment and the only treatment used is symptomatic. Recent studies have shown that some aminoglycoside antibiotics have the ability to impose the mammalian ribosome to read past a false-stop signal and generate full-length functional proteins. However, high toxicity of those drugs in humans limits their therapeutic use. To date, no efforts have been made to optimize aminoglycosides activity as stop codon readthrough inducers.

The main objective of the proposed research is to develop novel aminoglycoside variants with efficient termination suppression activity and with reduced toxicity, for the treatment of genetic disorders resulting from premature stop mutations.

Towards these ends, series of new derivatives of paromomycin were designed, synthesized and their ability to readthrough stop codon mutations was examined in both in vitro and ex vivo mammalian systems. First-generation lead, NB30, exhibited the most significant increase in readthrough activity and significantly reduced cytotoxicity relative to the scaffold Paromamine. Since the discovery of NB30, numerous new aminoglycoside based structures were rationally design and synthesized by optimizing the Paromamine and NB30. Continuing the structure-activity-toxicity relationship studies, lead us to the discovery of a series of new mono and disaccharide scaffolds: compounds NB148 and NB146, that perform better than paromamine. In parralel to the research towards finding new scaffolds, we succeeded to generate a new series of trisaccharide structures: NB124, NB125, NB127, and NB128. NB127 demonstrated low toxicity as well as high level of readthrough activity, than those of previous generation leads. Furthermore, NB127 outreach the activity of G418, the most active but highly toxic natural aminoglycoside until now. The compound NB127 was selected as the 5th generation lead. We also showed that our new lead compound demonstrats greater selectivity to the eukaryotic versus prokaryotic ribosome, contrary the currently known aminoglycoside antibiotics.

Therefore, this study provides a new direction for the development of novel aminoglycoside-based structures by means of optimizing drug-induced suppression efficacy and toxicity; further progress in this direction may offer promise for the treatment of many genetic diseases caused by nonsense mutations.