טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNudelman Igor
SubjectRedesign of Aminoglycosides for Treatment of Human
Genetic Diseases Caused by Premature Stop
Mutations
DepartmentDepartment of Chemistry
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,  Rett syndrome, Hemophilia 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 significantly reduced cytotoxicity in comparison to gentamicin and paromomycin, and promoted dose-dependent suppression of nonsense mutations of the PCDH15 gene, one of the underlying causes of type 1 Usher syndrome, its suppression potency was significantly lower relative to that of gentamicin and paromomycin. In attempts to further improve the suppression efficiency and reduce the toxicity of NB30, we have developed NB54 as a second-generation lead structure. NB54 exhibited significantly reduced cell, cochlear and acute toxicities, and has substantially higher stop-codon readthrough potency in both in vitro and ex vivo studies, than those of gentamicin and paromomycin. Continuing systematic fine-tuning of the developed leads, we designed and synthesized third-generation lead structures NB74, and NB84. They differ from the previous leads (NB30 and NB54) by the presence of the (R)-6'-methyl group on the glucosamine ring and show significantly reduced cell toxicity while in parallel exhibit substantially higher stop-codon readthrough potency in both in vitro and ex vivo studies, than those of gentamicin. 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.