A High Throughput Luminescent Assay for Glycogen Synthase Kinase-3<i>β</i> Inhibitors

Baki, Andrea; Bielik, Attila; Molnár, László; Szendrei, Gyorgyi I.; Keserü, György M.

doi:10.1089/adt.2006.029

Cited by 112 publications

(112 citation statements)

References 18 publications

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“…Enzyme assays to determine GSK-3b activity were carried out as described by Baki et al 23 Measurement of AchE activity was adapted from the colorimetric assay described by Ellman et al 24 for a microplate test system. Reverse transcriptase activity was assayed using a colorimetric reverse transcriptase enzyme-linked immunosorbent assay kit (Cat.…”

Section: Biological Activitymentioning

confidence: 99%

Gombapyrones, new α-pyrone metabolites produced by Streptomyces griseoruber Acta 3662

et al. 2009

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Gombapyrones A-D, new members of the a-pyrone family of secondary metabolites, were produced by Streptomyces griseoruber Acta 3662, which was isolated from bamboo tree rhizosphere. The strain was characterized by its morphological and chemotaxonomical features and by 16S rDNA sequencing as S. griseobuber. The gombapyrone structures were determined by mass spectrometry and by NMR experiments, and were found to have an inhibitory activity against protein tyrosine phosphatase 1B and glycogen synthase kinase 3b. The Journal Antibiotics ( Keywords: fermentation; isolation; a-pyrone; protein-tyrosine phosphatase inhibitor; Streptomyces; structure elucidation INTRODUCTION In our screening program to detect novel secondary metabolites from actinomycetes for pharmaceutical applications (http://www. actapha rm.org) freshly isolated strains from selected European and Malaysian ecosystems were included. The strains were grown as submerged cultures in different complex media, extracts were prepared from mycelia and from culture filtrates at various fermentation times and were screened by HPLC-diode array analysis to determine their chemical diversity. Evaluation of chromatograms was performed using our in-house developed HPLC-UV-Vis database, which contains 933 entries, most of them being antibiotics. 2 Strain Acta 3662, which was isolated from soil collected from the rhizosphere of bamboo trees in the tropical rainforest of the University of Malaya Field Station at Gombak, Selangor, Malaysia, was of interest because of the presence of a family of metabolites in the mycelium extract having nearly congruent UV-Vis spectra, which were unlike those of all reference compounds of the database. This study describes the taxonomy of the producing strain, the fermentation, isolation, structural elucidation and biological activity of the new family of metabolites from strain Acta 3662, which were named gombapyrones, composed from the collection site Gombak and the a-pyrone scaffold of their structures, which are shown in Figure 1.

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Section: Biological Activitymentioning

confidence: 99%

Gombapyrones, new α-pyrone metabolites produced by Streptomyces griseoruber Acta 3662

et al. 2009

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“…Overall, these results indicate that 6-gingerol exhibited protective effects on apoptosis induced by A␤1-42 in cultured PC12 cells by reducing oxidative stress and inflammatory responses, suppressing the activation of GSK-3␤ and enhancing the activation of Akt, thereby exerting neuroprotective effects. Kim DS et al [28] have isolated four shogaols from the roots of Zingiber officinale that protect IMR32 human neuroblastoma and normal human umbilical vein endothelial cells from beta-amyloid (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) insult at EC 50 = 4.5-81 M. In their further studies, they have synthesized ten shogaols to evaluate the importance of the side-chain length in protecting cells from amyloid betaA(1-42) insult using PC12 rat pheochromocytoma and IMR-32 human neuroblastoma cells [29]. Taking into account this in vitro studies Moon M et al [30] has shown 6-shogaol was capable to attenuate neuroinflammation and cognitive deficits in animal models of dementia in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Kinase-Glo assays were performed in assay buffer using black 96-well plates as described previously by Baki et al [25] In a typical assay, 10 l (10 M) of test compound (dissolved in dimethyl sulfoxide [DMSO] at 1 mM concentration and diluted in advance in assay buffer to the desired concentration) and 10 l (20 ng) of enzyme were added to each well followed by 20 l of assay buffer containing 25 M substrate and 1 M ATP. The final DMSO concentration in the reaction mixture did not exceed 1%.…”

Section: Gsk-3β Activitymentioning

confidence: 99%

GSK-3beta inhibitory effects of 6-gingerol and 6-shogaol help to the recovery of SHSY-5Y cells after amyloid beta1–42 oligomer or aggregate toxicity

Yerer¹,

Tiryaki²,

Demirpolat³

2017

JCB

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Abstract.BACKGROUND: GSK-3␤, has been shown to regulate APP cleavage resulting in the increased production of A␤ and the inhibition of the GSK-3␤ reduced the amyloid beta induced neurotoxicity in several studies. OBJECTIVE: This study was designed to investigate the GSK-3␤ inhibitory effects of 6-gingerol and 6-shogaol the major components of Zingiber officinale could be able to recover the SHSY-5Y cells from Amyloid beta 1-42 oligomer and aggregate toxicity. METHODS: SHSY-5Y (ATCC; CRL-2266) cells were maintained in Dulbecco's modified eagle medium (DMEM, Gibco) containing 10% fetal calf serum, 100 U/mL penicillin, 50 g/mL streptomycin, at 37 • C and 5% CO2 in a humidified incubator. Cells were transferred to sterile 96-well e-plates at 5 × 10 4 cells per well and followed real time for 78 hours via Xcelligence system. Ferulic acid was used as a positive control as a GSK-3␤ inhibitor at 4 M dose and 6-gingerol and 6-shogaol were applied to the cells at 0.01 M, 0.1 M, 1 M, 10 M, 100 M doses/50 L with or without ␤-Amyloid 1-42 oligomers or aggregates. GSK-3␤ activity was determined by Kinase-Glo assay. RESULTS: Both the amyloid beta aggregates and the oligomers had cytotoxic effect on SHSY-5Y cells followed by the real time cell analyzer system. 6-gingerol and 6 shogaol inhibited the GSK-3␤ enzyme up to % 20 levels in a dose dependent manner until 1 M. However, parallel with the reduction of the inhibition for 6-shogaol over 1 M, showed a toxicity itself on SHSY-5Y cells, whereas 6-gingerol did not show any cytotoxic effect at any doses applied. CONCLUSIONS: 6-gingerol and 6-shogaol increased the cell viability followed in a real time manner after around 24 hours than the amyloid beta aggregate or oligomer toxicity as much as the ferulic acid could. Furthermore these effects supported the GSK-3␤ inhibitory effects of both compounds at low doses up to 1 M. These results reveal that 6-gingerol and 6-shogaol the major components of Zingiber officinale help to recovery from amyloid beta toxicity which might further be investigated by clinical trials.

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“…The antimicrobial assays were performed as described by Helaly et al 23 To determine the cytotoxicity of compound 1, the sensitivity of the cell line NIH-3T3 was evaluated by monitoring the metabolic activity using the CellTiter-Blue À Cell Viability Assay (Promega, Mannheim, Germany). The cultivation of the cell line and the bioassay were performed as described by Schneemann et al 24 GSK-3b inhibition with compound 1 was determined in an in vitro assay adapted from a luminescent assay described by Baki et al 25 …”

Section: Producing Organism and Its Classificationmentioning

confidence: 99%

Benzoxacystol, a benzoxazine-type enzyme inhibitor from the deep-sea strain Streptomyces sp. NTK 935

et al. 2011

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Benzoxacystol, a new 1,4-benzoxazine-type metabolite, was produced by strain NTK 935, a marine member of the Streptomyces griseus 16S rRNA clade, isolated from deep-sea sediment collected from the Canary Basin. The structure of benzoxacystol was determined by mass spectrometry, NMR experiments and X-ray analysis. The compound showed an inhibitory activity against the enzyme glycogen synthase kinase 3b and a weak antiproliferative activity against mouse fibroblast cells. Keywords: 1,4-benzoxazin-3-one; glycogen synthase kinase 3b; marine Streptomyces INTRODUCTION Actinomycetes from marine sediments collected at various sites in the Atlantic and Pacific Oceans were screened by HPLC-diode array analysis for the production of novel secondary metabolites. Strain NTK 935 was isolated from the same deep-sea sediment core as Streptomyces sp. NTK 937, which we reported recently to produce caboxamycin, a novel antibiotic with a benzoxazole scaffold. 2 Extracts of strain NTK 935 contained a metabolite having an unusual UV-vis spectrum, which was not identified by means of our in-house developed HPLC database. 3 HPLC-ESI-MS analysis revealed a molecular mass of 380, which was-together with UV-vis data-not in accordance with other natural products available in the DNP database. 4 Therefore, strain NTK 935 was selected for fermentation studies, isolation and characterization of the unusual metabolite; this resulted in the identification of the novel 1,4-benzoxazine structure shown in Figure 1.This study describes the taxonomy of the producing strain, fermentation and isolation, structural elucidation and biological activity of the new benzoxazine-type metabolite, which was named benzoxacystol.

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A High Throughput Luminescent Assay for Glycogen Synthase Kinase-3β Inhibitors

Cited by 112 publications

References 18 publications

Gombapyrones, new α-pyrone metabolites produced by Streptomyces griseoruber Acta 3662

Gombapyrones, new α-pyrone metabolites produced by Streptomyces griseoruber Acta 3662

GSK-3beta inhibitory effects of 6-gingerol and 6-shogaol help to the recovery of SHSY-5Y cells after amyloid beta1–42 oligomer or aggregate toxicity

Benzoxacystol, a benzoxazine-type enzyme inhibitor from the deep-sea strain Streptomyces sp. NTK 935

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