Structure, Function, and Inhibition of Adenylosuccinate Lyase (ADSL) from Mycobacterium tuberculosis

Adenylosuccinate lyase (ADSL), encoded by the purB gene, is an essential enzyme in the purine biosynthesis pathway of Mycobacterium tuberculosis (Mtb), making it a promising target for antimicrobial drug development. Here, we report the expression, purification, kinetic characterization, high-throughput screening (HTS), and structural analysis of Mtb ADSL. We developed a highly sensitive and scalable bioluminescent assay using a PPDK-luciferase coupling system to quantify ADSL enzymatic activity via AMP detection. This assay enabled reliable kinetic analysis and successful pilot HTS of a small-molecule library, identifying bithionol and tetraiodothyroacetic acid (Tetrac) as inhibitors of Mtb ADSL. Inhibitory activity was confirmed using an orthogonal fluorescence polarization (FP) assay and further validated using the AMP-Glo luminescence assay. Specificity was evaluated using human ADSL (huADSL) to confirm that the compounds selectively inhibited Mtb ADSL while sparing the human enzyme. Thermal shift and gel-based protein stability assays demonstrated direct binding of bithionol and Tetrac to Mtb ADSL. Furthermore, bithionol and Tetrac displayed antibacterial activity against M. tuberculosis strains H37Ra and H37Rv, with moderate to low cytotoxicity toward human cells. Supplementation with exogenous AMP restored the growth of M. tuberculosis H37Ra inhibited by bithionol and Tetrac, confirming that both compounds act through on-target engagement of Mtb ADSL. The phagocytosis assay demonstrated that the compounds retained intracellular efficacy against M. tuberculosis. Finally, we determined the crystal structures of Mtb ADSL in two apo forms at high resolution (1.78 Å and 2.1 Å), revealing conserved tetrameric architecture with distinct active-site features that differentiate Mtb from human ADSL. Modeling suggested that both compounds bind to an allosteric site adjacent to the active site. These findings provide a framework for structure-guided development of selective ADSL inhibitors as potential antitubercular agents.