A FRET-Based High-Throughput Screening Assay for the Discovery ofDNA ADP-Ribosylglycohydrolase DarG Inhibitors.

DarTG2 is a conserved toxin-antitoxin ADP-ribosylation system that regulates bacterial survival and the antiphage response found in many pathogenic bacteria, including. While DNA ADP-ribosyltransferase (DarT2) toxin mono-ADP-ribosylates a single-stranded DNA sequence motif and potentially induces bacterial dormancy, DNA ADP-ribosylglycohydrolase (DarG) antitoxin, containing a highly conserved macrodomain, reverses the modification and restores bacterial growth. Therefore, developing DarG-selective inhibitors may represent a promising strategy to combat tuberculosis. However, no small-molecule inhibitors targeting DarG have been identified. Here, we developed and optimized a simple and robust fluorescence resonance energy transfer (FRET)-based binding assay to identify small-molecule inhibitors targeting the DarG macrodomain. The assay utilized fluorescent fusion proteins to detect the interaction between the DarG macrodomain and an ADP-ribosylated peptide. Screening the target-focused phenotypic library using this method led to the identification of pranlukast, which selectively inhibited the DNA ADP-ribosylhydrolase activity ofDarG and its bacterial orthologues, includingDarG and SCO6735 inNotably, pranlukast did not inhibit human macrodomains, indicating strong selectivity for bacterial targets. Since pranlukast has previously been reported to reduceburden, further investigation into its action mechanism in this context would be valuable.