University of Oulu

Wazir, S., Maksimainen, M. M., Alanen, H. I., Galera-Prat, A., & Lehtiö, L. (2021). Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases. SLAS DISCOVERY: Advancing the Science of Drug Discovery, 26(1), 67–76.

Activity-based screening assay for mono-ADP-Ribosylhydrolases

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Author: Wazir, Sarah1; Maksimainen, Mirko M.1; Alanen, Heli I.1;
Organizations: 1Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu,University of Oulu, Oulu, Finland
Format: article
Version: accepted version
Access: open
Online Access: PDF Full Text (PDF, 1.3 MB)
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Language: English
Published: SAGE Publications, 2021
Publish Date: 2020-12-08


ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD⁺, anomer of β-NAD⁺, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD⁺ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases

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Series: SLAS discovery
ISSN: 2472-5552
ISSN-E: 2472-5560
ISSN-L: 2472-5552
Volume: 26
Issue: 1
Pages: 67 - 76
DOI: 10.1177/2472555220928911
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the Academy of Finland (grant nos. 287063 and 294085) and by the Sigrid Jusélius Foundation.
Academy of Finland Grant Number: 287063
Detailed Information: 287063 (Academy of Finland Funding decision)
294085 (Academy of Finland Funding decision)
Copyright information: © 2020 Society for Laboratory Automation and Screening. Reprinted by permission of SAGE Publications. Reuse is restricted to non-commercial and no derivative uses.