|M.Sc Student||Firzon Vera|
|Subject||Synthesis and Evaluation of Aminoglycosides Derivatives|
as Prodrugs for Treatment of Genetic Diseases
|Department||Department of Chemistry||Supervisor||Professor Timor Baasov|
|Full Thesis text|
There are more than 1800 inherited human diseases caused by nonsense mutations, that is, alterations in the genetic code that prematurely stop the translation process and lead to the production of truncated, nonfunctional proteins. Depending on the disorder, nonsense mutations account for 5-70% of cases of genetic diseases, including cystic fibrosis, Duchenne muscular dystrophy, Usher syndrome, Hurler syndrome and more. For many of those diseases there is presently no effective treatment and the widely used treatment is only symptomatic.
In the last several years, numerous in vitro and in vivo experiments including clinical trials have demonstrated the ability of certain types of aminoglycosides to induce mammalian ribosome to readthrough disease-causing nonsense mutations, and partially restore full-length functional proteins. Despite these promising results, severe side-effects of standard aminoglycosides, including high toxicity to mammals, have limited their clinical benefit for suppression therapy. To address these issues, our laboratory has recently developed a series of new derivatives of aminoglycosides that show significantly improved readthrough activity and reduced toxicity.
However, due to their highly charged nature, aminoglycosides exhibit poor permeability into eukaryotic cells, which requires their administration in higher dosages that causes harmful side effects, and limits their use in translational therapy.
In attempt to solve these limitations, we decided to increase the lipophilicity of our developed lead structures (e.g. NB124) by converting them to the corresponding polyester derivatives (e.g. NB124-ester in which all hydroxyls are converted to esters). Initially, for the proof-of-concept we tested this method on the commercial aminoglycoside G418 and synthesized the corresponding ester derivatives G418-Ac, G418-iBut and G418-Bz (Fig. A; Ac-acetate, iBu-isobutirate, Bz-benzoate), which were evaluated in various in vitro and ex vivo nonsense suppression test. The best results were obtained with G418-Bz, when tested in primary cells of mouse embryonic fibroblasts (MEFs) derived from homozygous Idua-W402X mice: the nonsense W402X mutation suppression activity of G418-Bz significantly exceeded that of the parent G418. Encouraged, we then synthesized the similar derivative of NB124, NB124-Bz, which then did undergo similar biological tests, including the suppression of W402X mutation in MEFs. The observed results demonstrated that the activity of NB124-Bz significantly exceeds that of NB124 at all incubation times and concentrations tested.
Unlike NB124, which was developed for treatment of genetic diseases and lacks antibacterial activity, G418 is antibiotic. Therefore, the synthetic G418-Bz derivative, in addition to nonsense suppression tests, was also tested for its antibacterial activity. We found that while G418-Bz was about as active as G418 against both Gram-negative and Gram-positive bacteria, it also maintained activity against series of G418-resistant strains and exhibited significantly reduced toxicity relative to that of G418.
In conclusion, this study provides a new direction in development of aminoglycoside-based pro-drugs to improve their efficiency for suppression therapy. This progress may offer promise for the treatment of many genetic diseases. Additionally, a new direction for improvement of aminoglycosides as antibiotics that are in current clinical use is suggested.