Glycogen synthase kinase 3 inhibition controls Mycobacterium tuberculosis Infection

Compounds targeting host control of infectious diseases provide an attractive alternative to antimicrobials. A phenotypic screen of a kinase library identified compounds targeting glycogen synthase kinase 3 as potent inhibitors of Mycobacterium tuberculosis (Mtb) intracellular growth in the human THP-1 cell line and primary human monocytes-derived macrophages (hMDM). CRISPR knockouts and siRNA silencing showed that GSK3 isoforms are needed for the growth of Mtb and that a selected compound, P-4423632 targets GSK3β. GSK3 inhibition was associated with macrophage apoptosis governed by the Mtb secreted protein tyrosine phosphatase A (PtpA). Phospho-proteome analysis of macrophages response to infection revealed a wide array of host signaling and apoptosis pathways controlled by GSK3 and targeted by P-4423632. P-4423632 was additionally found to be active against other intracellular pathogens. Our findings strengthen the notion that targeting host signaling to promote the infected cell's innate antimicrobial capacity is a feasible and attractive host-directed therapy approach.

Methods
Lysates were prepared from parental or GSK3β knockout THP-1 cells (0.5 x 106 cells/well) that were differentiated overnight and then infected with WT Mtb or ΔptpA Mtb and treated with or without 10 µM P-4423632. Lysates were subjected to Kinexus Kinex™ KAM-2000 antibody microarray analyses as described (Yue, L., et al Clinical Proteomics & Bioinformatics. 2017;2(1):1-10). The KAM-2000 microarrays utilized 2059 commercial, pan-specific antibodies for 939 non-redundant human proteins targets that included protein kinases, phosphatases, transcription factors, stress proteins and many other signaling proteins. The KAM-2000 microarray featured 1165 pan-specific and 894 phosphosite-specific commercial antibodies, produced principally by Kinexus Bioinformatics (Vancouver, BC, Canada) as well as from other suppliers following their in-house validation, with each antibody printed in quadruplicate on each Nexterion P slide (Schott AG, Jena, Germany).

Two 16-bit images from each KAM-2000 microarray were then captured using a ScanArray Reader (Perkin-Elmer). Signal quantification was performed with ImaGene 9.0 from BioDiscovery (El Segundo, CA) with predetermined settings for spot segmentation and background correction. The output of the array consisted of the average normalized net signals (i.e., the average of 2 normalized net signal values of each antibody on the microarray).

See the README file for processing details.