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Published on: August 17, 2019
Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases
Sarah Wazir1, Mirko M Maksimainen1, Heli I Alanen1
1Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Oulu, Finland.
This study introduces a new assay for measuring the activity of mono-ADP-ribosylhydrolases, enzymes that remove ADP-ribose from proteins. The assay uses α-NAD+, a compound similar to ADP-ribose, as a substrate. After the enzyme acts on α-NAD+, the remaining amount is measured using fluorescence. The method was tested on MacroD2 and successfully identified compounds that inhibit its activity. However, these compounds were found to destabilize proteins, so they are not suitable for further development. The assay is a valuable tool for drug discovery and can be adapted for other hydrolases.
Area of Science:
- Enzymology within biochemistry
- Drug discovery in pharmacology
- Post-translational modification research in molecular biology
Background:
Post-translational modifications like ADP-ribosylation regulate essential cellular functions. Enzymes such as ADP-ribosyltransferases and hydrolases control these modifications. Human macrodomain proteins, including MacroD1, MacroD2, and ARH3, are known to interact with ADP-ribose. Their activities include reading or removing ADP-ribosylation. These proteins are linked to diseases like cancer, making them potential drug targets. However, robust biochemical tools for studying hydrolases remain limited. This gap motivated the development of new screening methods. Prior research has shown the importance of macrodomains in cellular regulation. No prior work had resolved the need for a high-throughput assay for mono-ADP-ribosylhydrolases.
Purpose Of The Study:
The study aimed to develop an activity-based screening assay for mono-ADP-ribosylhydrolases. These enzymes are crucial for reversing ADP-ribosylation, but few tools exist to study them. The researchers focused on MacroD2 and ARH3, which are involved in ADP-ribose hydrolysis. The goal was to create a reliable assay for identifying inhibitors. The assay needed to be sensitive and adaptable for high-throughput screening. The researchers also wanted to validate the assay’s effectiveness. They tested it with known substrates and inhibitors. The study sought to demonstrate the assay’s potential for drug discovery.
Main Methods:
The researchers designed an assay using α-NAD+, an anomer of β-NAD+. This compound mimics protein-linked ADP-ribose and is accepted as a substrate by MacroD1, MacroD2, and ARH3. The assay measures α-NAD+ remaining after hydrolysis. A fluorescent compound is generated from the remaining α-NAD+ on a microtiter plate. The method was optimized specifically for MacroD2. A proof-of-concept compound screening was performed. Three compounds were identified as hits with micromolar potency. The compounds were further analyzed for mechanism of action.
Main Results:
The assay successfully detected MacroD2 activity using α-NAD+. The fluorescent conversion provided a measurable signal. The assay was optimized for sensitivity and reproducibility. A screening campaign identified three compounds with micromolar potency. Further analysis revealed these compounds destabilized proteins. This mechanism excluded them from further study. The assay demonstrated usability for MacroD2. The researchers also tested the assay on other hydrolases. The method proved adaptable for additional targets. The results suggest the assay could be used in drug discovery.
Conclusions:
The study demonstrated a functional assay for mono-ADP-ribosylhydrolases. The assay uses α-NAD+ as a substrate and detects hydrolysis through fluorescence. It was validated for MacroD2 and showed adaptability for other enzymes. The screening identified compounds with micromolar potency. However, these compounds acted as protein destabilizers. This mechanism limited their further use. The assay remains a valuable tool for inhibitor discovery. The authors suggest it could be applied to other hydrolases. The findings support the potential of macrodomains as drug targets. The study provides a foundation for future biochemical investigations.
Frequently Asked Questions
The assay uses α-NAD+, an anomer of β-NAD+, as a substrate for hydrolases like MacroD2. The remaining α-NAD+ is converted into a fluorescent compound to measure enzyme activity.
α-NAD+ resembles protein-linked ADP-ribose and is accepted by MacroD1, MacroD2, and ARH3. This makes it a suitable substrate for detecting hydrolase activity.
Three compounds were identified as hits with micromolar potency. However, they were found to destabilize proteins, which limited their further use.
The assay measures the amount of α-NAD+ remaining after hydrolysis by converting it to a fluorescent compound on a microtiter plate.
MacroD2 is a human macrodomain protein involved in ADP-ribose hydrolysis. The assay was optimized for MacroD2 and demonstrated its usability as a screening tool.
The assay can be used to screen for inhibitors of mono-ADP-ribosylhydrolases. It provides a functional tool for studying macrodomains linked to diseases like cancer.
