Branched aminoglycosides: biochemical studies and antibacterial activity of neomycin B derivatives

Abstract-The C5 00 -OH group in neomycin B was glycosylated with a variety of mono-and di-saccharides to probe the effect of introduction of additional binding elements on antibacterial activity and interaction with the aminoglycosides modifying enzyme APH(3 0 )-IIIa. The designed structures show antibacterial activity superior to that of neomycin B against pathogenic and resistant strains, while in parallel they demonstrate poor substrate activity with APH(3 0 )-IIIa

Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model

Thirty-five percent of patients with Rett syndrome carry nonsense mutations in the MECP2 gene. We have recently shown in transfected HeLa cells that readthrough of nonsense mutations in the MECP2 gene can be achieved by treatment with gentamicin and geneticin. This study was performed to test if readthrough can also be achieved in cells endogenously expressing mutant MeCP2 and to evaluate potentially more effective readthrough compounds. A mouse model was generated carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the new mouse model were treated with gentamicin and the novel aminoglycosides NB30, NB54, and NB84. The localization of the readthrough product was tested by immunofluorescence. Readthrough of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells. As expected, the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, while NB30 was less effective. Readthrough of nonsense mutations can be achieved not only in transfected HeLa cells but also in fibroblasts of the newly generated Mecp2R168X mouse model. NB54 and NB84 were more effective than gentamicin and are therefore promising candidates for readthrough therapy in Rett syndrome patients

Ex Vivo Treatment with a Novel Synthetic Aminoglycoside NB54 in Primary Fibroblasts from Rett Syndrome Patients Suppresses MECP2 Nonsense Mutations

BACKGROUND: Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2) comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT). Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro. RESULTS: We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X) common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%), R270X (27%) and R294X (18%). In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF). CONCLUSION: Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT

Novel cellular model system and therapeutic molecules for the development of a read-through approach for CF caused by stop codon mutations of the CFTR gene-Progetto n°: 02/2010 FFC Finanziato da: Fondazione per la Ricerca sulla Fibrosi Cistica-Bando 2010 Durata: 12 mesi (dal 01/09/2010 al 31/08/2011)

Progetto n°: 02/2010 FFC Finanziato da: Fondazione per la Ricerca sulla Fibrosi Cistica-Bando 2010 Durata: 12 mesi (dal 01/09/2010 al 31/08/2011)- Nonsense mutations promote premature translational termination and are the leading cause of approximately 30% of inherited diseases, including cystic fibrosis. In the last few years, it has been demonstrated that drugs (like aminoglycoside antibiotics) can be designed and produced to suppress premature translation termination, inducing a ribosomal read-through of premature, but not normal termination codons. Moreover the treatments with aminoglycosides may provide a mean of restoring CFTR function in patients with stop mutations and have introduced new hopes for the development of a pharmacologic approach to the cure of CF. The rationale supporting this project is to optimize the read-through molecules leading to restoration of CFTR production in cystic fibrosis caused by non-sense stop codon mutations. The objectives of this project are: development of in vitro cellular model systems and biochemical screening systems; synthesis of novel drugs exhibiting read-through effects on CFTR mRNA carrying stop codon mutations; development of experimental strategies to reduce NMD of the CFTR mRNA and biological assays on treated experimental model systems, testing the activity of the read-through drugs on CFTR function of chloride transport

Aminoglycosides Affect Intracellular Salmonella enterica Serovars Typhimurium and Virchow▿

The high antibacterial activity and selectivity of aminoglycosides and their low activity against intracellular bacteria associated with eukaryotic cells make them the antibiotics of choice for the elimination of extracellular bacteria during intracellular studies. Given the evidence that aminoglycosides can penetrate the eukaryotic cell membrane, the goal of this study was to examine the influence of aminoglycosides on macrophage-associated Salmonella. Herein, we show that gentamicin, kanamycin, and tobramycin at concentrations between 15 to 150 μg ml−1 do not kill intracellular Salmonella but have other effects on the bacterial physiology. By using Salmonella enterica serovars Typhimurium and Virchow harboring luciferase reporter plasmid, we observed that the light produced by intracellular Salmonella declined immediately upon exposure to aminoglycosides, indicating that the bacteria were under stress. The extent of this effect was dependent on the macrophage host, on the identity of the aminoglycoside and its concentration, on the exposure time, and on the Salmonella serovar. Salmonella associated with Nramp1-negative macrophages, in which the phagosomal pH is higher, were more susceptible to aminoglycosides than Salmonella associated with Nramp1-expressing macrophages. These results verify that aminoglycosides affect intracellular bacteria and that the extent of this effect is dependent on the acidity level within the phagosome, suggesting that for the study of intracellular bacteria, the aminoglycoside concentration should be limited to two to five times the MIC for the bacterial strain studied. This precaution should guarantee the complete execution of extracellular bacteria with minimal effects on the intracellular bacteria and the host cells

Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells

Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity