Hongmin Li

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  Abstract Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis, hemorrhagic fever, and microcephaly, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to Flavivirus replication, such as the viral protease. During viral replication, the Flavivirus genome is translated as a single-polyprotein precursor, which must be cleaved into individual proteins by host proteases and a complex of the viral protease, NS3, and its cofactor, NS2B. As this cleavage is an obligate step of the viral life cycle, the Flavivirus protease is an attractive target for antiviral drug development. In this chapter, we will survey the flaviviral protease and recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from Flavivirus protease crystal structures.

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