Related Experiment Video
Updated: May 6, 2026
06:07
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
2.5K
A new, sensitive ecto-5'-nucleotidase assay for compound screening.
Marianne Freundlieb1, Herbert Zimmermann2, Christa E Müller1
1PharmaCenter Bonn, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical Institute, Pharmaceutical ChemistryI, University of Bonn, D-53121 Bonn, Germany.
Analytical Biochemistry
|October 23, 2013
Summary
A new radiometric assay detects ecto-5'-nucleotidase (eN) activity with high sensitivity. This assay is crucial for developing new cancer and inflammatory disease treatments by targeting eN.
Area of Science:
- Biochemistry
- Enzymology
- Pharmacology
Background:
- Ecto-5'-nucleotidase (eN) is a membrane enzyme implicated in cancer progression.
- Elevated eN expression in cancer cells promotes proliferation, angiogenesis, metastasis, and immunosuppression.
- eN is a potential therapeutic target for cancer and inflammatory diseases.
Purpose of the Study:
- To develop and optimize a sensitive radiometric assay for quantifying eN activity.
- To validate the assay's suitability for high-throughput screening (HTS).
- To compare the assay's sensitivity with existing methods.
Main Methods:
- A radiometric assay utilizing [³H]adenosine-5'-monophosphate (AMP) as a substrate.
- Separation of the product [³H]adenosine from the substrate [³H]AMP via lanthanum chloride precipitation and filtration.
- Optimization of reaction conditions for reproducible quantitative determination.
Main Results:
- The assay demonstrated high sensitivity with a mean Z' factor of 0.73, indicating suitability for HTS.
- The limit of detection was at least 30-fold lower compared to common colorimetric assays.
- The developed radiometric assay outperformed existing malachite green, capillary electrophoresis, and luciferase-based assays.
Conclusions:
- A highly sensitive and validated radiometric assay for eN activity has been established.
- This assay is superior to current methods and suitable for HTS.
- The assay will facilitate the discovery of novel therapeutic agents targeting eN for cancer and inflammatory diseases.
