Structure, Function, and Inhibition of Adenylosuccinate Lyase (ADSL) from <i>Mycobacterium tuberculosis</i>

<i>Adenylosuccinate lyase</i> (ADSL), encoded by the <i>purB</i> gene, is an essential enzyme in the purine biosynthesis pathway of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), 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 <i>Mtb</i> ADSL. We developed a highly sensitive and scalable bioluminescent assay using a PPDK-luciferase coupling system to quantify ADSL enzymatic activity <i>via</i> 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 <i>Mtb</i> 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 (<i>hu</i>ADSL) to confirm that the compounds selectively inhibited <i>Mtb</i> ADSL while sparing the human enzyme. Thermal shift and gel-based protein stability assays demonstrated direct binding of bithionol and Tetrac to <i>Mtb</i> ADSL. Furthermore, bithionol and Tetrac displayed antibacterial activity against <i>M. tuberculosis</i> strains H37Ra and H37Rv, with moderate to low cytotoxicity toward human cells. Supplementation with exogenous AMP restored the growth of <i>M. tuberculosis</i> H37Ra inhibited by bithionol and Tetrac, confirming that both compounds act through on-target engagement of <i>Mtb</i> ADSL. The phagocytosis assay demonstrated that the compounds retained intracellular efficacy against <i>M. tuberculosis</i>. Finally, we determined the crystal structures of <i>Mtb</i> 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 <i>Mtb</i> 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.