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In order to identify anti-tubercular agents with a novel scaffold, commercial libraries of small organic compounds were screened against a fluorescent strain of Mycobacterium tuberculosis H37Rv, using a dual phenotypic assay. Compounds were assessed against bacteria replicating in broth medium, as well as inside macrophages, and thienothiazolocarboxamide (TTCA) scaffold was identified as hit in both assays, with submicromolar inhibitory concentrations. Derivatives of TTCA were further synthesized and evaluated for their inhibitory effects on M. tuberculosis H37Rv. In the present study we report the structure–activity relationship of these TTCA derivatives. Compounds 28, 32 and 42 displayed good anti-tubercular activities, as well as favorable ADME and PK properties. Compound 42 exhibited excellent oral bioavailability in mice with high distribution to lungs, within 1 h. It was found to be efficacious in a dose dependent manner in a murine model of M. tuberculosis infection. Hence, compound 42 is now under evaluation as a potential lead candidate for treatment of tuberculosis.

Lung alveolar macrophages (AMs) are in the first line of immune defense against respiratory pathogens and play key roles in the pathogenesis of Mycobacterium tuberculosis (Mtb) in humans. Nevertheless, AMs are available only in limited amounts for in vitro studies, which hamper the detailed molecular understanding of host-Mtb interactions in these macrophages. The recent establishment of the self-renewing and primary Max Planck Institute (MPI) cells, functionally very close to lung AMs, opens unique opportunities for in vitro studies of host-pathogen interactions in respiratory diseases. Here, we investigated the suitability of MPI cells as a host cell system for Mtb infection. Bacterial, cellular, and innate immune features of MPI cells infected with Mtb were characterized. Live bacteria were readily internalized and efficiently replicated in MPI cells, similarly to primary murine macrophages and other cell lines. MPI cells were also suitable for the determination of anti-tuberculosis (TB) drug activity. The primary innate immune response of MPI cells to live Mtb showed significantly higher and earlier induction of the pro-inflammatory cytokines TNFα, interleukin 6 (IL-6), IL-1α, and IL-1β, as compared to stimulation with heat-killed (HK) bacteria. MPI cells previously showed a lack of induction of the anti-inflammatory cytokine IL-10 to a wide range of stimuli, including HK Mtb. By contrast, we show here that live Mtb is able to induce significant amounts of IL-10 in MPI cells. Autophagy experiments using light chain 3B immunostaining, as well as LysoTracker labeling of acidic vacuoles, demonstrated that MPI cells efficiently control killed Mtb by elimination through phagolysosomes. MPI cells were also able to accumulate lipid droplets in their cytoplasm following exposure to lipoproteins. Collectively, this study establishes the MPI cells as a relevant, versatile host cell model for TB research, allowing a deeper understanding of AMs functions in this pathology.

Mycobacterium chelonae (Mch) is an atypical rapidly growing mycobacterium (RGM) that belongs to the M. chelonae complex, which can cause a variety of human infections. During this type of mycobacterial infection, macrophage-derived chemokines play an important role in the mediation of intracellular communication and immune surveillance by which they orchestrate cellular immunity. However, the intracellular signaling pathways involved in the macrophage-induced chemokine production during Mch infections remain unknown. Thus, the present study aimed to determine the molecular mechanisms by which Mch activates the gene expressions of chemokine (C-C motif) ligand 2 (CCL2) and CCL5 in murine bone marrow-derived macrophages (BMDMs) and in vivo mouse model. Toll-like receptor 2 (TLR2)-deficient mice showed increased bacterial burden in spleen and lung and decreased protein expression of CCL2 and CCL5 in serum. Additionally, Mch infection triggered the mRNA and protein expression of CCL2 and CCL5 in BMDMs via TLR2 and myeloid differentiation primary response gene 88 (MyD88) signaling and that it rapidly activated nuclear factor (NF)-κB signaling, which is required for the Mch-induced expressions of CCL2 and CCL5 in BMDMs. Moreover, while the innate receptor Dectin-1 was only partly involved in the Mch-induced expression of the CCL2 and CCL5 chemokines in BMDMs, the generation of intracellular reactive oxygen species (ROS) was an important contributor to these processes. Taken together, the present data indicate that the TLR2, MyD88, and NF-κB pathways, Dectin-1 signaling, and intracellular ROS generation contribute to the Mch-mediated expression of chemokine genes in BMDMs.

New and improved treatments for tuberculosis (TB) are urgently needed. Recently, it has been demonstrated that verapamil, an efflux inhibitor, can reduce bacterial drug tolerance caused by efflux pump activity when administered in combination with available antituberculosis agents. The aim of this study was to evaluate the effectiveness of verapamil in combination with the antituberculosis drug candidate Q203, which has recently been developed and is currently under clinical trials as a potential antituberculosis agent. We evaluated changes in Q203 activity in the presence and absence of verapamil in vitro using the resazurin microplate assay and ex vivo using a microscopy-based phenotypic assay for the quantification of intracellular replicating mycobacteria. Verapamil increased the potency of Q203 against Mycobacterium tuberculosis both in vitro and ex vivo , indicating that efflux pumps are associated with the activity of Q203. Other efflux pump inhibitors also displayed an increase in Q203 potency, strengthening this hypothesis. Therefore, the combination of verapamil and Q203 may be a promising combinatorial strategy for anti-TB treatment to accelerate the elimination of M. tuberculosis .