High-Content Screening of Eukaryotic Kinase Inhibitors Identify CHK2 Inhibitor Activity Against Mycobacterium tuberculosis

Abstract

A screen of a eukaryotic kinase inhibitor library in an established intracellular infection model identified a set of drug candidates enabling intracellular killing of Mycobacterium tuberculosis (M.tb). Screen validity was confirmed internally by a Z = 0.5 and externally by detecting previously reported host-targeting anti-M.tb compounds. Inhibitors of the CHK kinase family, specifically checkpoint kinase 2 (CHK2), showed the highest inhibition and lowest toxicity of all kinase families. The screen identified and validated DDUG, a CHK2 inhibitor, as a novel bactericidal anti-M.tb compound. CHK2 inhibition by RNAi phenocopied the intracellular inhibitory effect of DDUG. DDUG was active intracellularly against M.tb, but not other mycobacteria. DDUG also had extracellular activity against 4 of 12 bacteria tested, including M.tb. Combined, these observations suggest DDUG acts in tandem against both host and pathogen. Importantly, DDUG's validation highlights the screening and analysis methodology developed for this screen, which identified novel host-directed anti-M.tb compounds.

A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice

“…nuclei staining) and number of virus-infected cells (based on dsRNA and nucleocapsid staining) was performed using the CellInsight CX7 HCS platform (Thermo Fisher), as previously described 55,56 .…”

Advances in host‐based screening for compounds with intracellular anti‐mycobacterial activity

“…Most of the screens performed to identify compounds acting inside host system have used mycobacteria expressing a fluorescent protein, with reduction in the fluorescence being the major readout. Green fluorescent protein (GFP) expressing mycobacteria are the most common (Christophe et al, 2009;Han et al, 2019;Koo et al, 2008;Pethe et al, 2013;Stanley et al, 2014;Sundaramurthy et al, 2013), although screens have been performed with mycobacteria expressing red fluorescent protein (RFP) (Richter et al, 2019), mCherry (Huang, Kushner, et al, 2018;VanderVen et al, 2015) or tdTomato (Shapira et al, 2020) (Figure 2C). In all the cases, the fluorescent protein is expressed under a strong promoter.…”

“…Unlike the murine cell lines, THP1 is a monocyte cell line that requires an additional activation step for differentiation to macrophages. Shapira et al screened 400 compounds(Shapira et al, 2020) in Mtb-infected THP1 cells. The intracellular bacteria were measured using tomato RFP signal by high content imaging using CellInsight CX5 High Content platform and Thermo Fisher Scientific TM HCS StudioTM Cell Analysis Software.…”

From infection niche to therapeutic target: the intracellular lifestyle of Mycobacterium tuberculosis

“…Drugs first developed to control cancer growth have similarly been shown useful from screening for potential antimycobacterial agents [ 154 ]. A recent study by our group has revealed through the screening of kinase inhibitors on intracellular Mtb, that the CHK2 pathway is of importance to Mtb infection dynamics [ 155 ]. We reported the CHK2 inhibition by kinase inhibitor DDUG to be both strongly toxic to intracellular Mtb, and show low host-cell loss; a combination not seen in many campaigns against intracellular bacteria.…”