DOI: 10.5061/dryad.0rxwdbs5h
title: Glycogen synthase kinase 3 inhibition controls Mycobacterium tuberculosis Infection

description: 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.
description: Lysates were prepared
 from<strong> </strong>parental or GSK3β knockout THP-1 cells
 (0.5 x 10<sup>6</sup> cells/well) that were differentiated
 overnight and then infected with WT Mtb or Δ<em>ptpA</em>
 Mtb<em> </em>and<em> </em>treated with or without
 10 µM P-4423632. Lysates were subjected to Kinexus Kinex™ KAM-2000
 antibody microarray analyses as described (Yue, L., <em>et al</em> 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
 (<em>i.e.,</em> the average of 2 normalized net signal values
 of each antibody on the microarray). See the README file for processing
 details.
description: Microsoft Excel CAT-PETR (av-gay-ubc.shinyapps.io/CAT-PETR/): Flanagan K, Pelech S, Av-Gay Y, Dao Duc K. CAT PETR: A graphical user interface for differential analysis of phosphorylation data. Statistical Applications in Genetics and Molecular Biology. 2023 Aug 21;22(1). doi: 10.1515/sagmb-2023-0017.
description: # Title: Glycogen Synthase Kinase 3 inhibition controls *Mycobacterium
 tuberculosis* Infection [Access this dataset on
 Dryad](https://datadryad.org/stash/dataset/doi:10.5061/dryad.0rxwdbs5h)
 Data showing modulation of macrophage phosphorylation upon infection and
 treatment with P-4423632 using antibody microarray analysis. ##
 Description of the data and file structure The phosphorylation status of
 THP-1 cells were analysed from seven experimental conditions: UP: Parental
 THP-1 cells, Uninfected, Untreated IP: Parental THP-1 cells, Mtb-infected,
 Untreated UPD1: Parental THP-1 cells, Uninfected, P-4423632-treated IPD1:
 Parental THP-1 cells, Mtb-infected, P-4423632-treated UO: GSK3B KO THP-1
 cells, Uninfected, Untreated IO: GSK3B KO THP-1 cells, Mtb-infected,
 Untreated IPK01: Parental THP-1 cells, ptpA KO Mtb-infected, Untreated
 #### Excel Data Excel files include descriptions in the Read Me First tab
 Excel File S1 compares UO and UPD1 to the UP control Excel File S2
 compares IO, IPD1 and IPK01 to the IP control Excel File S3 compares IPD1
 to UPD1 Calculations: Standard error and percent standard deviation of 2
 separate measurements of globally normalized signal intensity values for
 each different antibody on the microarray were calculated. Data were
 determined as percent change from selected controls (% CFC). A positive
 value corresponds to an increase in signal intensity in response to the
 treatment, with a value of 100% corresponding to a 2-fold increment in
 signal intensity. A negative CFC value indicates the degree of reduction
 in signal intensity from that of the control. #### Raw Txt Files Text
 files R1 - R7 are the raw Kinexus data files for the seven conditions
 tested. These files can be directly uploaded into
 [CAT-PETR](https://av-gay-ubc.shinyapps.io/CAT-PETR/) for plotting Volcano
 Plots, Scatter Plots, and Heatmaps in Figure 4A-D. CAT-PETR settings:
 Phospho-specific comparisions using Log Transform normalization, Cyber-T
 t-test, and Benjamini Hochberg correction were used to plot the data. ####
 PDF File KinexusReport.pdf gives a detailed description of the methods,
 results and analysis of the antibody microarray.