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Small-Molecule Screening Assay for Mono-ADP-Ribosyltransferases
Teemu Haikarainen1, Sudarshan Murthy1, Mirko M Maksimainen1
1Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, Oulu, Finland.
Methods in Molecular Biology (Clifton, N.J.)
|August 12, 2018
Summary
Researchers developed a new assay to screen for inhibitors of mono-ADP-ribosyltransferases (ARTs). This cost-effective method uses the natural substrate NAD+ to detect enzyme activity, aiding in cancer drug discovery.
Area of Science:
- Biochemistry and Molecular Biology
- Enzymology
- Drug Discovery
Background:
- Mono-ADP-ribosyltransferases (ARTs), part of the PARP/ARTD family, transfer a single ADP-ribose unit to proteins.
- These enzymes are crucial for cellular processes like DNA repair and transcription.
- ARTs are implicated in cancer, making them targets for novel therapeutic strategies.
Purpose of the Study:
- To develop and validate a novel activity-based screening assay for mono-ADP-ribosyltransferases.
- To provide a robust and cost-effective method for identifying small-molecule inhibitors of ART enzymes.
- To enable screening for both mono- and poly-ADP-ribosyltransferases.
Main Methods:
- An activity-based assay was designed utilizing the natural substrate NAD+.
- The assay chemically converts unreacted NAD+ into a measurable fluorophore.
- Enzyme activity is quantified by measuring the relative concentration of the generated fluorophore post-reaction.
Main Results:
- The developed assay is homogenous, robust, and cost-effective.
- The assay successfully measures the activity of mono-ADP-ribosyltransferases.
- The assay is also applicable for screening poly-ADP-ribosyltransferases.
Conclusions:
- A novel, versatile activity-based screening assay for ART enzymes has been established.
- This assay facilitates the discovery and development of small-molecule inhibitors for cancer therapy.
- The assay's applicability to both mono- and poly-ARTs enhances its utility in drug discovery.
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