Integrating computational and biological approaches for the discovery of putative MmpL3 inhibitors against Mycobacterium tuberculosis

The rapid emergence of drug-resistant Mycobacterium tuberculosis strains has progressively undermined the efficacy of existing anti-tuberculosis therapies, underscoring the urgent need for new therapeutic agents. MmpL3, an essential transporter involved in mycobacterial cell wall biosynthesis, represents a promising drug target. In this study, an integrated virtual screening strategy that combined pharmacophore-based and structure-based approaches was applied to screen the ChemDiv database, followed by biological evaluation, to identify potential MmpL3 inhibitors. Six compounds (F784-0295, Y205-0192, F624-3971, C200-4969, Y508-1233, and 8018 − 0330) showed measurable antimycobacterial activity. Among them, the previously unreported pyrazolo[1,5-a]pyrimidine derivative Y508-1233 exhibited potent activity against both H37Rv and multidrug-resistant Mycobacterium tuberculosis strains, with an MIC₉₀ of 37.3 µM for each strain. ADMET predictions indicated generally acceptable pharmacokinetics, though somewhat lipophilic with limited solubility. Molecular dynamics simulations revealed that Y508-1233 remains stably bound within the central channel of the MmpL3 transmembrane domain, primarily through nonpolar interactions with helices TM4 and TM10, accompanied by a persistent hydrogen bond between its carboxamide nitrogen and Asp640. Structural analysis identified an unoccupied subpocket delineated by Leu243 and Ile244, providing a structural basis for further molecular optimization. Furthermore, τ-random acceleration molecular dynamics simulations predicted that Y508-1233 may exhibit a relatively favorable residence time. These findings indicate that Y508-1233 is a putative MmpL3-targeting candidate with promising activity against drug-resistant tuberculosis, warranting further optimization and experimental validation.