Homogeneous Time-Resolved Fluorescence-Based Assay to Screen ADP-Ribosyl Hydrolase Inhibitors

ADP ribosylation (ADPr) is a crucial post-translational modification that plays a vital role in DNA damage repair. Catalyzed by ADP ribose polymerases using NAD⁺ as a substrate, ADPr activates DNA repair pathways rapidly, thereby maintaining genomic integrity. The involvement of ADP ribose hydrolases in this process is significant, as they hydrolyze PAR chains, facilitating the release of ADPr-modified proteins from DNA or other proteins, which is essential for subsequent DNA repair steps. This protocol outlines a high-throughput screening method for identifying inhibitors of ADP ribose hydrolases, utilizing His-Tb-conjugated and ADPr-modified His-ADP ribose polymerase as the signal donor, and GST-d2-conjugated GST-XRCC1 as the signal receptor. The detection of time-resolved fluorescence signals enables efficient evaluation of compounds with potential therapeutic activity against cancer. Key features • A high-throughput screening method is provided for efficiently identifying inhibitors of ADP ribose hydrolases. • The method employs homogeneous time-resolved fluorescence (HTRF) for sensitive detection of inhibitory effects on ADP ribose hydrolases, enabling high-throughput screening without complex separation steps. • The research aims to accelerate the development of novel cancer therapies targeting ADP ribose hydrolases to improve treatment outcomes.