New screening test finds drug that blocks a tuberculosis survival switch

Tuberculosis remains a global health threat in part because the bacterium Mycobacterium tuberculosis can enter states that help it survive stress and resist treatment. Researchers are looking for new ways to tip the balance against the bacterium by targeting its own regulatory systems rather than the usual antibiotic targets. One such system is DarTG2, a conserved toxin-antitoxin ADP-ribosylation pair found in many pathogenic bacteria including Mycobacterium tuberculosis. In this system the toxin, DNA ADP-ribosyltransferase DarT2, mono-ADP-ribosylates a short single-stranded DNA sequence, a change that can push the bacterium toward dormancy. The paired antitoxin, DNA ADP-ribosylglycohydrolase DarG, reverses that modification and restores bacterial growth. Because blocking DarG could keep the toxin’s modification in place and disrupt bacterial recovery, DarG-selective inhibitors have been proposed as a novel strategy to fight drug-resistant tuberculosis. However, prior to this work there were no reported small molecule inhibitors of DarG and creating a practical assay to screen thousands of compounds for DarG activity presented a major technical challenge. In response, the team led by corresponding author L. Lehtiö set out to build a reliable, high-throughput way to find DarG inhibitors.

To search for DarG inhibitors the researchers designed and optimized a fluorescence resonance energy transfer (FRET)-based high-throughput screening assay tailored to DarG’s DNA ADP-ribosylhydrolase activity. They produced a FRET pair that combined the M. tuberculosis DarG macrodomain with a poly-ADP-ribosylated peptide, each fused to compatible fluorophores so that hydrolysis of the ADP-ribose modification would change the fluorescent signal. This simple and robust readout let them screen chemical libraries efficiently for compounds that block DarG activity. Using this assay to screen a target-focused phenotypic library, the team identified pranlukast as a hit. Follow-up testing showed that pranlukast selectively inhibited the DNA ADP-ribosylhydrolase activity of DarG from Mycobacterium tuberculosis and its orthologues, while not affecting human mono-ADP-ribosyl binders and erasers. The study therefore both produced a workable screening tool and a selective chemical probe, addressing the earlier lack of small molecule DarG inhibitors.

The work has two clear implications. First, the new FRET-based high-throughput screening assay gives researchers a practical method to discover and optimize DarG inhibitors, opening a path toward a previously unexplored class of anti-tuberculosis agents. Second, the identification of pranlukast as a selective inhibitor of DarG activity is noteworthy because pranlukast has already been reported to reduce Mycobacterium tuberculosis burden, suggesting it or related compounds might be repurposed or used as starting points for drug development. Importantly, the reported selectivity against bacterial DarG and lack of activity on human mono-ADP-ribosyl binders and erasers suggests a possible therapeutic window, although the authors note that further investigation into pranlukast’s mechanism of action in this context would be valuable. Overall, the study led by L. Lehtiö provides both a tool and a candidate that could accelerate efforts to exploit bacterial ADP-ribosylation systems against drug-resistant tuberculosis, while underscoring the need for follow-up studies to translate these early findings into clinical advances.