Rapid Screening and Validation of Intracellularly Active Antimycobacterial Compounds with Efficacy in Cellular and Murine Tuberculosis Models

The discovery of effective intracellular antimycobacterial agents is limited by the slow growth of Mycobacterium tuberculosis (Mtb) and a lack of scalable models that effectively replicate host-pathogen interactions. To address this, we established a robust ex vivo infection model using human monocyte-derived THP-1 macrophages and the fast-growing pathogenic surrogate Mycobacterium marinum (Mmar) which recapitulates key features of Mtb intracellular infection, including macrophage colonization and innate immune activation. This model supports medium-throughput screening workflows for assessing antibiotic-mediated control of intracellular mycobacteria. Using this platform, we screened FDA-approved antipsychotics and identified the phenothiazines- trifluoperazine (TFP) and fluphenazine (FFP), as potent adjuncts to frontline anti-tuberculosis therapy (ATT). Combinations of either phenothiazine with frontline TB drugs enhanced intracellular bacterial clearance more effectively than the previously reported antidepressant sertraline, initiating significant control within 12 hours of treatment in macrophages. In murine models of TB infection, TFP and FFP further potentiated bacterial clearance and tissue recovery when used in combination with standard anti-TB therapy (ATT). Together, our results validate the THP1–Mmar infection model as a rapid, robust, and scalable platform for identifying intracellular antimycobacterial agents and host-directed therapeutics, offering a valuable tool to accelerate TB drug discovery and development.