Identification of Small Molecule Inhibitors against Mycobacteria in Activated Macrophages

Abstract

Mycobacterial pathogens are intrinsically resistant to many available antibiotics, making treatment extremely challenging, especially in immunocompromised individuals and patients with underlying and chronic lung conditions. Even with lengthy therapy and the use of a combination of antibiotics, clinical success for non-tuberculous mycobacteria (NTM) is achieved in fewer than half of the cases. The need for novel antibiotics that are effective against NTM is urgent. To identify such new compounds, a whole cell high-throughput screen (HTS) was performed in this study. Compounds from the Chembridge DIVERSet library were tested for their ability to inhibit intracellular survival of M. avium subsp. hominissuis (MAH) expressing dtTomato protein, using fluorescence as a readout. Fifty-eight compounds were identified to significantly inhibit fluorescent readings of MAH. In subsequent assays, it was found that treatment of MAH-infected THP-1 macrophages with 27 of 58 hit compounds led to a significant reduction in intracellular viable bacteria, while 19 compounds decreased M. abscessus subsp. abscessus (Mab) survival rates within phagocytic cells. In addition, the hit compounds were tested in M. tuberculosis H37Ra (Mtb) and 14 compounds were found to exhibit activity in activated THP-1 cells. While the majority of compounds displayed inhibitory activity against both replicating (extracellular) and non-replicating (intracellular) forms of bacteria, a set of compounds appeared to be effective exclusively against intracellular bacteria. The efficacy of these compounds was examined in combination with current antibiotics and survival of both NTM and Mtb were evaluated within phagocytic cells. In time-kill dynamic studies, it was found that co-treatment promoted increased bacterial clearance when compared with the antibiotic or compound group alone. This study describes promising anti-NTM and anti-Mtb compounds with potential novel mechanisms of action that target intracellular bacteria in activated macrophages.