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  3. Screening assay to monitor mono-ADP-ribosylhydrolase activity of viral macrodomains in cells

Screening assay to monitor mono-ADP-ribosylhydrolase activity of viral macrodomains in cells

  • Commun Biol. 2026 Mar 12;9(1):386. doi: 10.1038/s42003-026-09832-3.
Sarah Knapp 1 2,  Verena Weber 3 4,  Maud Verheirstraeten 1,  Ani Sabcheva 1 5,  Tanner Wright 6,  Lea Herkens 1 7,  Lukas Hauser 1,  Lea Hirschen 1,  Alexandra Golzmann 1,  Barbara Lippok 1,  Sarah Krieg 1,  Dana Ferraris 8,  Stefan Knapp 9 10 11,  Andreas G Ladurner 6,  Giulia Rossetti 3 12 13,  Bernhard Lüscher 1,  Patricia Korn 14 15
Affiliations

Affiliations

  • 1 Institute of Biochemistry and Molecular Biology, Medical School of the RWTH Aachen University, Aachen, Germany.
  • 2 Institute of Pharmacology, Medical School of the RWTH Aachen University, Aachen, Germany.
  • 3 Institute of Neuroscience and Medicine (INM-9)/Institute for Advanced Simulation (IAS-5), Forschungszentrum Jülich GmbH, Jülich, Germany.
  • 4 Faculty of Mathematics, Computer Science and Natural Sciences, RWTH Aachen, Aachen, Germany.
  • 5 AVT-Biochemical Engineering, RWTH Aachen University, Aachen, Germany.
  • 6 Biomedical Center (BMC), Department of Physiological Chemistry, Faculty of Medicine, LMU Munich, Planegg-Martinsried, Germany.
  • 7 Institute of Pathology, RWTH Aachen University, Aachen, Germany.
  • 8 McDaniel College Department of Chemistry, Westminster, MD, USA.
  • 9 Institut für Pharmazeutische Chemie, Goethe-University Frankfurt, Biozentrum, Frankfurt am Main, Germany.
  • 10 Structural Genomics Consortium, Buchmann Institute for Life Sciences, Goethe-University Frankfurt, Frankfurt am Main, Germany.
  • 11 German Cancer Consortium (DKTK)/German Cancer Research Center (DKFZ), DKTK site Frankfurt-Mainz, Heidelberg, Germany.
  • 12 Jülich Supercomputing Centre (JSC), Forschungszentrum Jülich GmbH, Jülich, Germany.
  • 13 Department of Neurology, University Hospital Aachen, RWTH Aachen University, Aachen, Germany.
  • 14 Institute of Biochemistry and Molecular Biology, Medical School of the RWTH Aachen University, Aachen, Germany. [email protected].
  • 15 Department of Cardiac Surgery, Medical Faculty, RWTH Aachen University, Aachen, Germany. [email protected].
PMID: 41820535 DOI: 10.1038/s42003-026-09832-3
Abstract

Mono-ADP-ribosylation, a modification of both proteins and nucleic acids, is implicated in innate immunity. Intracellularly, this modification is catalyzed by PARP Enzymes, some induced in response to interferons. Mono-ADP-ribosylation is reversed by hydrolases including proteins with macrodomains, which are conserved across all kingdoms of life. Macrodomains encoded by certain positive-sense single-stranded RNA viruses, such as Chikungunya virus and SARS-CoV-2, antagonize host MARylation to enhance viral replication and suppress the immune response. While macrodomain hydrolase activity is essential for CHIKV replication, in SARS-CoV-2 it predominantly contributes to immune evasion, underscoring viral macrodomains as potential Antiviral drug targets. Efforts to develop macrodomain inhibitors include computational modeling, crystallography-based methods, and in vitro assays. However, tools to study macrodomain activity directly in cells remain rare. Here, we established a cell-based assay using PARP15 isoform 1, which we found forms nuclear foci dependent on its ADP-ribosyltransferase activity. Enzymatically active macrodomains dissolve these foci, enabling hydrolase activity monitoring in living cells. Using stable cell lines, this system allows the screening of macrodomain inhibitors while simultaneously addressing cell permeability, toxicity, and physiological relevance. Adaptable to various macrodomains, our platform offers a versatile tool to study macrodomain function in living cells, analyzing mutants, and advancing drug discovery efforts.

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Inhibitors & Agonists
  • Cat. No.
    Product Name
    Description
    Target
    Research Area
  • HY-148566
    99.86%, PARP10 Inhibitor
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