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Updated: Feb 20, 2026
Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
Published on: October 23, 2019
Development of an Inhibitor Screening Assay for Mono-ADP-Ribosyl Hydrolyzing Macrodomains Using AlphaScreen
Teemu Haikarainen1, Mirko M Maksimainen1, Ezeogo Obaji1
11 Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland.
Abstract:
Protein mono-ADP-ribosylation is a posttranslational modification involved in the regulation of several cellular signaling pathways. Cellular ADP-ribosylation is regulated by ADP-ribose hydrolases via a hydrolysis of the protein-linked ADP-ribose. Most of the ADP-ribose hydrolases share a macrodomain fold. Macrodomains have been linked to several diseases, such as cancer, but their cellular roles are mostly unknown. Currently, there are no inhibitors available targeting the mono-ADP-ribose hydrolyzing macrodomains. We have developed a robust AlphaScreen assay for the screening of inhibitors against macrodomains having mono-ADP-ribose hydrolysis activity. We utilized this assay for validatory screening against human MacroD1 and identified five compounds inhibiting the macrodomain. Dose-response measurements and an orthogonal assay further validated four of these compounds as MacroD1 inhibitors. The developed assay is homogenous, easy to execute, and suitable for the screening of large compound libraries. The assay principle can also be adapted for other ADP-ribose hydrolyzing macrodomains, which can utilize a biotin-mono-ADP-ribosylated protein as a substrate.
