Confocal Fluorescence Detection Expanded to UV Excitation: The First Continuous Fluorimetric Assay of Human Steroid Sulfatase in Nanoliter Volume
| Autor: | Nicole-Claudia Meisner, Peter Nussbaumer, Andreas Neubauer, Melitta Bilban, Stefan Jäger, Andreas Billich, Manfred Auer |
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| Rok vydání: | 2004 |
| Předmět: |
Chemical Phenomena
Photochemistry Ultraviolet Rays Confocal Drug Evaluation Preclinical CHO Cells law.invention Absorbance chemistry.chemical_compound Dehydroepiandrosterone sulfate Coumarins law Cricetinae Drug Discovery Steroid sulfatase Animals Humans Nanotechnology Fluorometry Enzyme Inhibitors chemistry.chemical_classification Microscopy Confocal Chromatography Chemistry Physical Chinese hamster ovary cell Fluorescence Kinetics Enzyme chemistry Recombinant DNA Molecular Medicine Female Indicators and Reagents Spectrophotometry Ultraviolet Steryl-Sulfatase Algorithms |
| Zdroj: | ASSAY and Drug Development Technologies. 2:21-30 |
| ISSN: | 1557-8127 1540-658X |
| DOI: | 10.1089/154065804322966289 |
| Popis: | Steroid sulfatase is an enzyme that currently enjoys considerable interest as a potential drug target in the treatment of estrogen- and androgen-dependent diseases, in particular breast cancer. We have purified human steroid sulfatase to apparent homogeneity from recombinant Chinese hamster ovary cells, and we established an assay with a new fluorogenic substrate, 3,4-benzocoumarin-7-O-sulfate (1). Substrate 1 features a K(m) value of 22.5 microM, which is close to the value for the natural substrate dehydroepiandrosterone sulfate (26 microM) and much lower than the K(m) values of other synthetic substrates (276-736 microM). Importantly, the cleavage of substrate 1 can be monitored continuously during the enzymatic cleavage, since a change in fluorescence intensity is detectable at the pH where the enzyme is active; in contrast, all other synthetic substrates described so far require alkalization to reveal a measurable absorbance or fluorescence signal. The adaptation of the assay to the 96-well format allows continuous monitoring of multiple wells in a microplate fluorescence reader. Applications of the assay for the determination of IC(50) and K(i) values of novel steroid sulfatase inhibitors are presented. Most importantly the assay was transferred to the nanoscale format (1-microl assay volume) in 2080-well plates with confocal fluorescence detection. This miniaturization will permit screening with a minimum throughput of 20000 compounds per day. The system presented demonstrates that the confocal detection platform used for nanoscreening can be successfully adapted to assays for which conventional ultraviolet dyes like coumarins are necessary. This strongly broadens the application range of confocal readers in drug screening. |
| Databáze: | OpenAIRE |
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