Identification of novel inhibitors of intracellular M. tuberculosis and their mechanisms of action

Abstract

Tuberculosis (TB) is the second leading cause of death from infectious disease worldwide, accounting for 1.5 million deaths per year. The emergence of drug-resistant TB strains further highlights the urgent need for new drugs and new drug targets. TB is caused by the bacterium Mycobacterium tuberculosis, a facultative intracellular pathogen that can survive and replicate inside macrophages. Thus, drug screening in the macrophage mimics the physiological context of the disease, which makes it possible to identify inhibitors of targets/pathways that are involved in the M. tuberculosis adaptation to the intracellular environments and virulence. Our lab has developed and validated a fluorescence-based, live-cell, high-content analysis (HCA) assay to examine drug efficacy against intracellular M. tuberculosis. Here, we describe a high throughput screening where 10,000 diverse small molecules were screened to identify novel inhibitors of M. tuberculosis growth within macrophage-like cells using high content analysis. We selected a number of series for follow-up based on their physicochemical properties and the novelty of the chemotypes and identified five chemical classes of interest. We tested available analogs for each series to complete a limited structure-activity relationship study. A benzene amide ether (BAE) series was identified from the screen. BAE compounds exhibited good activity against intracellular M. tuberculosis as well as low cytotoxicity against the murine RAW 264.7 and human THP-1 cell lines. Compounds had minimal inhibitory activity against M. tuberculosis cultured axenically but did show bactericidal activity against nutrient-starved M. tuberculosis, suggesting that the compounds primarily target the bacteria. Understanding the target and mechanism of action of compounds identified from phenotypic screens is important for downstream drug development to optimize their potency better, identify combination therapies more rationally, and better predict toxic side effects. Therefore, we conducted a series of assays to identify the mechanism of action of the BAE series. An increase in reactive oxygen species (ROS) was observed in M. tuberculosis treated with BAE compounds. In addition, we demonstrated that BAE compounds depleted ATP levels of treated M. tuberculosis culture under both replicating and starved culture.Additionally, BAE compounds increased the oxygen consumption rate. Finally, we examined the transcriptomic changes induced by BAE compound treatment in replicating and nutrient-starved wild-type strains of M. tuberculosis. BAE compound treatment induced changes in energy metabolism under both conditions. Notably, the mechanism of action of one of the BAE compounds was predicted as targeting respiration using the PROSPECT screen. These results suggest that this novel BAE series targets respiration in M. tuberculosis. We also conducted a series of assays to investigate the mode of action of the Phenylthiourea series. We were able to exclude common targets which suggest that the PTU compounds exert antitubercular activity through a distinct or novel target and/or mechanism. Future work will focus on target identification for both series.