Discovery and Mechanism of Action of a Novel Desloratadine Derivative Inhibiting Mycobacterium tuberculosis 3-Dehydroquinate Synthase.

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), remains a leading cause of mortality from infectious disease worldwide. The continued emergence of multidrug-resistant strains highlights the urgent need for new therapeutic agents with novel mechanisms of action. Here, we report the discovery and comprehensive characterization of DL25, a synthetic desloratadine-derived small molecule exhibiting potent antimycobacterial activity (minimum inhibitory concentration (MIC) 2-4 μg/mL against multiple mycobacterial species). Using a supra-inhibitory DL25 selection strategy, we isolated and characterized spontaneous resistant mutants. Whole-genome sequencing identified recurrent mutations in aroB, which encodes 3-dehydroquinate synthase (DHQS), an essential enzyme of the shikimate pathway. Consistent with target involvement, aroB overexpression reduced susceptibility to DL25, while DL25 decreased DHQS-associated biochemical activity in vitro with an apparent IC50 of approximately 5.0 μg/mL. In addition, DL25 exhibited concentration- and time-dependent bactericidal activity and showed partial synergy with several established antitubercular agents. Structural modeling, molecular docking, and 100 ns molecular dynamics simulations supported stable binding of DL25 within the DHQS active site and identified interactions with residues implicated in substrate recognition and catalysis. Collectively, the genetic, biochemical, and computational findings support DHQS as a biologically relevant target associated with DL25 activity and establish DL25 as a promising lead-like scaffold for the development of future shikimate pathway-directed antimycobacterial agents. More broadly, this study demonstrates the value of integrating genetic, biochemical, and computational approaches for mechanism-guided antituberculosis drug discovery.