A molecular toolbox for ADP-ribosyl binding proteins.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Proteins interacting with ADP-ribosyl groups are implicated in disease pathways and viral infections.
• These proteins represent significant targets for therapeutic intervention.
• Existing assays may have limitations in detecting diverse ADP-ribosyl interactions.

Purpose of the Study:

• To develop a robust and accessible assay for detecting interactions with mono- and poly-ADP-ribosyl groups.
• To create tools for high-throughput screening of inhibitors targeting ADP-ribosylation.
• To facilitate drug discovery efforts related to ADP-ribosylation.

Main Methods:

• Utilized a C-terminal G protein alpha subunit peptide (GAP) tag for site-specific ADP-ribosylation.
• Fused GAP-tagged proteins and ADP-ribosyl binders to fluorescent proteins for FRET analysis.
• Employed nanoluciferase fusion for blot-based detection of ADP-ribosylated proteins.
• Demonstrated high-throughput screening using SARS-CoV-2 nsp3 macrodomain.

Main Results:

• Successfully generated robust FRET partners to confirm interactions with 22 known ADP-ribosyl binders.
• Identified suramin as a moderate-affinity, non-specific inhibitor of the SARS-CoV-2 nsp3 macrodomain.
• Developed tools producible in *Escherichia coli* for broad research application.

Conclusions:

• The developed assay is versatile for studying both hydrolyzing and non-hydrolyzing ADP-ribosyl binders.
• This technology enhances ADP-ribosylation research and accelerates the discovery of novel therapeutic agents.
• The tools provide a foundation for screening inhibitors and understanding disease mechanisms involving ADP-ribosylation.