A high-throughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis

Abstract

Tuberculosis is a disease of global importance for which novel drugs are urgently required. We developed a whole-cell phenotypic screen which can be used to identify inhibitors of Mycobacterium tuberculosis growth. We used recombinant strains of virulent M. tuberculosis which express far-red fluorescent reporters and used fluorescence to monitor growth in vitro. We optimized our high throughput assays using both 96-well and 384-well plates; both formats gave assays which met stringent reproducibility and robustness tests. We screened a compound set of 1105 chemically diverse compounds previously shown to be active against M. tuberculosis and identified primary hits which showed ≥ 90% growth inhibition. We ranked hits and identified three chemical classes of interest – the phenoxyalkylbenzamidazoles, the benzothiophene 1–1 dioxides, and the piperidinamines. These new compound classes may serve as starting points for the development of new series of inhibitors that prevent the growth of M. tuberculosis. This assay can be used for further screening, or could easily be adapted to other strains of M. tuberculosis.

8‐Hydroxyquinolines are bactericidal against Mycobacterium tuberculosis

“…We, and others, identified these compounds in phenotypic screening as hits (Ananthan et al, 2009;Ollinger et al, 2018). We, and others, identified these compounds in phenotypic screening as hits (Ananthan et al, 2009;Ollinger et al, 2018).…”

“…The HQ series has good activity against M. tuberculosis in vitro. We, and others, identified these compounds in phenotypic screening as hits (Ananthan et al, 2009;Ollinger et al, 2018). We wanted to explore the SAR for this series to determine the scope for further development.…”

Mycobacterial drug discovery

“…1A), [22][23][24][25] but alternative methods such as monitoring cell growth through the heterologous expression of a fluorescent marker can be an alternative approach. 26,27 The screens can be modified to mimic the different physiological states of Mtb during infection, such as aerobic and anaerobic conditions, 28,29 starvation, 30 in addition to the requirement of different carbon sources and nutrients 31,32 and in Mtb-infected macrophages. 33,34 The phenotypic-based hits discovered from these screens already circumvent permeability issues and can be further assessed for additional criteria including cytotoxicity, ultimately providing compounds with good selectivity and specificity.…”

Novel Trifluoromethyl Pyrimidinone Compounds With Activity Against Mycobacterium tuberculosis

“…We have previously developed high-throughput screening using fluorescent strains of M. tuberculosis, which enabled us to screen compound libraries (Ollinger et al, 2018). We identified several series with anti-tubercular activity; among these is the trifluoromethyl pyrimidinone series, which has potent activity in vitro.…”