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.

GSK3β has been proposed to have an essential role in Coronaviridae infections. Screening of a targeted library of GSK3β inhibitors against both SARS-CoV-2 and HCoV-229E to identify broad-spectrum anti-Coronaviridae inhibitors resulted in the identification of a high proportion of active compounds with low toxicity to host cells. A selected lead compound, T-1686568, showed low micromolar, dose-dependent activity against SARS-CoV-2 and HCoV-229E. T-1686568 showed efficacy in viral-infected cultured cells and primary 2D organoids. T-1686568 also inhibited SARS-CoV-2 variants of concern Delta and Omicron. Importantly, while inhibition by T-1686568 resulted in the overall reduction of viral load and protein translation, GSK3β inhibition resulted in cellular accumulation of the nucleocapsid protein relative to the spike protein. Following identification of potential phosphorylation sites of Coronaviridae nucleocapsid, protein kinase substrate profiling assays combined with Western blotting analysis of nine host kinases showed that the SARS-CoV-2 nucleocapsid could be phosphorylated by GSK3β and PKCa. GSK3β phosphorylated SARS-CoV-2 nucleocapsid on the S180/S184, S190/S194 and T198 phospho-sites, following previous priming in the adjacent S188, T198 and S206, respectively. Such inhibition presents a compelling target for broad-spectrum anti-Coronaviridae compound development, and underlies the mechanism of action of GSK3β host-directed therapy against this class of obligate intracellular pathogens.

GSK3β has been proposed to have an essential role in Coronaviridae infection. Screening of a targeted library of GSK3β inhibitors against SARS-CoV-2 and HCoV-229E resulted in identification of high proportion of active compounds with low toxicity to host cells. A select lead compound, T-1686568, showed dose-dependent activity against SARS-CoV-2 transcription, translation and viral particle release in multiple cell lines and primary organoids. A protein kinase substrate profiling assay combined with western blot analysis showed that SARS-CoV-2 nucleocapsid is phosphorylated by GSK3β on S180/S184, S190/S194 and T198 which have already been primed in the adjacent phospho-sites S188, T198 and S206 respectively. Inhibition by T-1686568 resulted in reduction of the S1 Spike protein levels, an accumulation of the Nucleocapsid (N) protein and maintenance of the non-structural (NSP2) level in infected Huh-7.5.1 cells, indicating that N phosphorylation might serve as a critical precursor for processing and release of mature viruses.

Pathogenic mycobacteria gain entry to their hosts by inhalation or ingestion where they adhere to different cell types and are subsequently internalized by professional phagocytic cells, such as macrophages or dendritic cells. Multiple pathogen-associated molecular patterns present on the mycobacterial surface are recognized by and interact with a diverse panel of phagocytic pattern recognition receptors, representing the first step of the infection process. This review summarizes the current knowledge on the numerous host cell receptors and their associated mycobacterial ligands or adhesins. It further discusses the downstream molecular and cellular events resulting from the engagement of the various receptor-mediated pathways, leading to either intracellular survival of mycobacteria or to activation of host immune defenses. The content presented herein on adhesins and host receptors may serve as a resource for those developing novel therapeutic approaches, e.g., in the design of anti-adhesin molecules to prevent bacterial attachment and infection. The collection of mycobacterial surface molecules highlighted in this review may also provide potential new therapeutic targets, diagnostic markers, or vaccine candidates to combat these notoriously challenging and persistent pathogens.