Identification of Host-Targeted Small Molecules That Restrict Intracellular Mycobacterium tuberculosis Growth

Abstract

Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.

Autophagy in the Fight Against Tuberculosis

“…Ridaifen-B, a tamoxifen derivative, was also shown to be an inducer of autophagy, but through a currently unknown Beclin-1-independent mechanism (Nagahara et al, 2013). Gefitinib, an epidermal growth factor receptor (EGFR) inhibitor used to treat breast and other types of cancers, has also been shown to induce autophagy and to be effective in M. tuberculosis killing in a mouse model (Stanley et al, 2014). Nortriptyline and fluoxetine, currently used to treat depression, were also reported to promote autophagy and enhance mycobacteria clearance (Sundaramurthy et al, 2013;Stanley et al, 2014).…”

“…Gefitinib, an epidermal growth factor receptor (EGFR) inhibitor used to treat breast and other types of cancers, has also been shown to induce autophagy and to be effective in M. tuberculosis killing in a mouse model (Stanley et al, 2014). Nortriptyline and fluoxetine, currently used to treat depression, were also reported to promote autophagy and enhance mycobacteria clearance (Sundaramurthy et al, 2013;Stanley et al, 2014). While the mechanisms behind autophagy enhancement by these two drugs are currently unknown, the fluoxetine effect is related to increased TNF-a secretion (Stanley et al, 2014).…”

“…Nortriptyline and fluoxetine, currently used to treat depression, were also reported to promote autophagy and enhance mycobacteria clearance (Sundaramurthy et al, 2013;Stanley et al, 2014). While the mechanisms behind autophagy enhancement by these two drugs are currently unknown, the fluoxetine effect is related to increased TNF-a secretion (Stanley et al, 2014). Since all these FDA-approved drugs have been reported as autophagy inducers, they have the potential to be used as complementary treatment(s) to current TB therapies.…”

Towards host-directed therapies for tuberculosis

“…4 This should include new anti-M.tb agents that are regulators of cellular processes such as growth and proliferation, glucose metabolism, apoptosis, and autophagy and restrict M.tb growth in macrophages include kinase modulators, G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, and anti-inflammatory drugs. 5 Closer collaborations between funders, drug developers, basic science, translational clinical research and clinical trials researchers are required to identify other candidates for HDTs from existing drugs and newer adjunct therapies. Increased donor funding into development and evaluation of novel therapeutic strategies using a range of HDTs are urgently required.…”

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

“…paratuberculosis interactions with host immune cells. Through examination of interaction outcomes, these assays can provide information relevant to the assessment of vaccine efficacy (5-7), pathogenesis (8), and antimicrobial activity (9). However, if in vitro infection assays are to be used as rapid screening tools, it is essential that they function in a high-throughput capacity for large studies and that the time taken to obtain results does not become rate limiting.…”