G418

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Timor Baasov - One of the best experts on this subject based on the ideXlab platform.

  • Increased Selectivity toward Cytoplasmic versus Mitochondrial Ribosome Confers Improved Efficiency of Synthetic Aminoglycosides in Fixing Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense Mutations
    2016
    Co-Authors: Jeyakumar Kandasamy, Dana Atia-glikin, Eli Shulman, Katya Shapira, Michal Shavit, Valery Belakhov, Timor Baasov
    Abstract:

    Compelling evidence is now available that gentamicin and Geneticin (G418) can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits the strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity toward cytoplasmic ribosome correlates with the increased activity and that the decreased specificity toward mitochondrial ribosome confers the lowered cytotoxicity

  • increased selectivity toward cytoplasmic versus mitochondrial ribosome confers improved efficiency of synthetic aminoglycosides in fixing damaged genes a strategy for treatment of genetic diseases caused by nonsense mutations
    Journal of Medicinal Chemistry, 2012
    Co-Authors: Jeyakumar Kandasamy, Eli Shulman, Katya Shapira, Michal Shavit, Valery Belakhov, Dana Atiaglikin, Timor Baasov
    Abstract:

    Compelling evidence is now available that gentamicin and Geneticin (G418) can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits the strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity toward cytoplasmic ribosome correlates with the increased activity and that the decreased specificity toward mitochondrial ribosom...

Jeyakumar Kandasamy - One of the best experts on this subject based on the ideXlab platform.

  • Increased Selectivity toward Cytoplasmic versus Mitochondrial Ribosome Confers Improved Efficiency of Synthetic Aminoglycosides in Fixing Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense Mutations
    2016
    Co-Authors: Jeyakumar Kandasamy, Dana Atia-glikin, Eli Shulman, Katya Shapira, Michal Shavit, Valery Belakhov, Timor Baasov
    Abstract:

    Compelling evidence is now available that gentamicin and Geneticin (G418) can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits the strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity toward cytoplasmic ribosome correlates with the increased activity and that the decreased specificity toward mitochondrial ribosome confers the lowered cytotoxicity

  • increased selectivity toward cytoplasmic versus mitochondrial ribosome confers improved efficiency of synthetic aminoglycosides in fixing damaged genes a strategy for treatment of genetic diseases caused by nonsense mutations
    Journal of Medicinal Chemistry, 2012
    Co-Authors: Jeyakumar Kandasamy, Eli Shulman, Katya Shapira, Michal Shavit, Valery Belakhov, Dana Atiaglikin, Timor Baasov
    Abstract:

    Compelling evidence is now available that gentamicin and Geneticin (G418) can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits the strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity toward cytoplasmic ribosome correlates with the increased activity and that the decreased specificity toward mitochondrial ribosom...

Youngchoon Moon - One of the best experts on this subject based on the ideXlab platform.

John H. Andorfer - One of the best experts on this subject based on the ideXlab platform.

  • A Novel G418 Conjugate Results In Targeted Selection of Genetically Protected Hepatocytes without Bystander Toxicity
    Bioconjugate chemistry, 2007
    Co-Authors: Martina Volarevic, Robert Smolić, John H. Andorfer
    Abstract:

    G418, an aminoglycoside neomycin analogue, is an antimicrobial agent that interferes with protein synthesis and has been used extensively for selection of mammalian cell lines that possess neomycin resistance (NR). It is potent and nonspecific in its effects that occur through tight binding to ribosomal elements. Because of the potent intracellular effect, we wondered whether G418 could be used to select a specific cell type based on receptor-mediated endocytosis. The objective of this study was to target G418 specifically to liver cells via asialoglycoprotein receptors (AsGR) which are known to be highly selective for these cells. A novel G418 conjugate was synthesized chemically by coupling G418 to a galactose-terminating carrier protein, asialoorosomucoid (AsOR), in a molar ratio of 5:1. AsOR–G418 conjugates inhibited viability of AsGR (+) cells by 84.3%, while inhibition in AsGR (–) cells was only by 19%. In AsGR (+) cells, stably transfected with a NR gene, the conjugate decreased viability by less t...

  • Receptor- Mediated Targeting of Toxin Results in Hepatotoxicity Without Collateral Damage in Rats
    2007
    Co-Authors: Martina Volarevic, John H. Andorfer, Robert Smolic, Catherine H. Wu, George Y. Wu
    Abstract:

    We recently developed a rat model with a chimeric human liver. However, that model contained relatively few human hepatocytes. To increase the ratio of human cells, a selection strategy was devised to eliminate host hepatocytes by transfection of a neomycin resistance gene into human hepatocytes and use of G418 for selection.However, G418 has extrahepatic toxicity in mammals. Aim: Our objective was to target G418 specifically to liver via asialoglycoprotein receptors (AsGR). Methods: A novel G418 conjugate was created by chemically coupling G418 to a galactose-terminal asialoglycoprotein, asialoorosomucoid (AsOR). Groups of normal rats were injected IV with radiolabeled conjugate and organ distribution determined. Groups of 7 days old SD rats (n=4-7) were also injected with AsOR-G418 (100 mg/kg, iv), free G418, AsOR or PBS. Serum samples were collected for alanine aminotransferase (ALT), an assay for liver damage ; and creatinine release, an assay for kidney toxicity. Six days after treatment, animals were sacrificed and liver and kidneys collected for total RNA isolation, fixed and stained with hematoxylin/eosin and periodic acid-Schiff(PAS). Results: Fifteen min after IV injection, 82% of labeled conjugate was found in liver. This uptake was the same as for AsOR alone suggesting that G418 conjugate recognition in animals was similar to AsOR alone. In rats treated with 100mg/kg free G418, the survival rate was 50% at 48 h, and 0% at 72 h. In contrast, in groups that received identical G418 as a conjugate, 100mg/kg IV, the 48 h survival rate was 100%, the same as in AsOR and PBS controls. However, ALT levels in conjugate-treated rats doubled to a mean of 224+/- 3 U/L, by 48 h, compared to no change in AsOR, G418 and PBS treated groups. Creatinine levels in conjugate-treated rats were the same as AsOR, and PBS control groups, while in the G418-treated group, levels increased to 3.2 mg/dl by 48 h. In kidney sections of G418-treated rats, PAS- positive brush border disappeared, not found in conjugate-treated group. In contrast, liver sections of conjugate-treated rats showed mild liver injury, not present in AsOR, G418 and PBS control treated groups. Conclusion: G418 in the form of a conjugate retains toxicity that can be targeted specifically to liver cells through AsGR highly expressed on those cells. Conjugate-mediated hepatotoxicity did not result in damage to kidney or other tissues. Targeted selection may be useful in the manipulation of populations of various cell types in laboratory animals.

Leonard Petrucelli - One of the best experts on this subject based on the ideXlab platform.

  • Premature termination codon readthrough upregulates progranulin expression and improves lysosomal function in preclinical models of GRN deficiency
    Molecular Neurodegeneration, 2020
    Co-Authors: Jonathan Frew, Alireza Baradaran-heravi, Aruna D. Balgi, Xiujuan Wu, Steve Arns, Fahimeh S. Shidmoossavee, James B. Jaquith, Karen R. Jansen-west, Francis C. Lynn, Leonard Petrucelli
    Abstract:

    Background Frontotemporal lobar degeneration (FTLD) is a devastating and progressive disorder, and a common cause of early onset dementia. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene ( GRN ) is an important cause of FTLD (FTLD- GRN ), and nearly a quarter of these genetic cases are due to a nonsense mutation. Premature termination codons (PTC) can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. Restoring endogenous PGRN through PTC readthrough has not previously been explored as a therapeutic intervention in FTLD. Methods We studied whether the aminoglycoside G418 could increase PGRN expression in HEK293 and human induced pluripotent stem cell (hiPSC)-derived neurons bearing the heterozygous S116X, R418X, and R493X pathogenic GRN nonsense mutations. We further tested a novel substituted phthalimide PTC readthrough enhancer in combination with G418 in our cellular models. We next generated a homozygous R493X knock-in hiPSC isogenic line (R493X^−/− KI), assessing whether combination treatment in hiPSC-derived neurons and astrocytes could increase PGRN and ameliorate lysosomal dysfunction relevant to FTLD- GRN . To provide in vivo proof-of-concept of our approach, we measured brain PGRN after intracerebroventricular administration of G418 in mice expressing the V5-tagged GRN nonsense mutation R493X. Results The R418X and R493X mutant GRN cell lines responded to PTC readthrough with G418, and treatments increased PGRN levels in R493X^−/− KI hiPSC-derived neurons and astrocytes. Combining G418 with a PTC readthrough enhancer increased PGRN levels over G418 treatment alone in vitro. PGRN deficiency has been shown to impair lysosomal function, and the mature form of the lysosomal protease cathepsin D is overexpressed in R493X^−/− KI neurons. Increasing PGRN through G418-mediated PTC readthrough normalized this abnormal lysosomal phenotype in R493X^−/− KI neuronal cultures. A single intracerebroventricular injection of G418 induced GRN PTC readthrough in 6-week-old AAV- GRN -R493X-V5 mice. Conclusions Taken together, our findings suggest that PTC readthrough may be a potential therapeutic strategy for FTLD caused by GRN nonsense mutations.