A FRET-Based High-Throughput Screening Assay for the Discovery of <i>Mycobacterium tuberculosis</i> DNA 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 Mycobacterium tuberculosis . 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 of M. tuberculosis DarG and its bacterial orthologues, including Thermus aquaticus DarG and SCO6735 in Streptomyces coelicolor . Notably, pranlukast did not inhibit human macrodomains, indicating strong selectivity for bacterial targets. Since pranlukast has previously been reported to reduce M. tuberculosis burden, further investigation into its action mechanism in this context would be valuable.