A Simple Method to Study ADP-Ribosylation Reversal: From Function to Drug Discovery.

ADP-ribosylation is an ancient modification of proteins, nucleic acids, and other biomolecules found in all kingdoms of life as well as in certain viruses. The regulation of fundamental (patho)physiological processes by ADP-ribosylation, including the cellular stress response, inflammation, and immune response to bacterial and viral pathogens, has created a strong interest into the study of modification establishment and removal to explore novel therapeutic approaches. Beyond ADP-ribosylation in humans, direct targeting of factors that alter host ADP-ribosylation signaling (e.g., viral macrodomains) or utilize ADP-ribosylation to manipulate host cell behavior (e.g., bacterial toxins) were shown to reduce virulence and disease severity. However, the realization of these therapeutic potentials is thus far hampered by the unavailability of simple, high-throughput methods to study the modification "writers" and "erasers" and screen for novel inhibitors.Here, we describe a scalable method for the measurement of (ADP-ribosyl)hydrolase activity. The assay relies on the conversion of ADP-ribose released from a modified substrate by the (ADP-ribosyl)hydrolase under investigation into AMP by the phosphodiesterase NudT5 into bioluminescence via a commercially available detection assay. Moreover, this method can be utilized to study the role of nudix- or ENPP-type phosphodiesterases in ADP-ribosylation processing and may also be adapted to investigate the activity of (ADP-ribosyl)transferases. Overall, this method is applicable for both basic biochemical characterization and screening of large drug libraries; hence, it is highly adaptable to diverse project needs.