Glycogen synthase kinase 3 inhibition controls Mycobacterium tuberculosis infection

Summary 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.

The table shows the MIC50 and MIC25 values obtained from the dose-dependency assays testing six selected GSK3 inhibitors against intracellular M. tuberculosis H37Rv (WT) and M. tuberculosis ΔptpA.The MIC50 and MIC25 values were determined by non-linear regression with the omission of up to two outliers.The MIC25s were not determined (ND) for compounds 2 and 6, as the values were outside the range of the observed x values.

Figure S1 :
Figure S1: Number of hits per kinase target, related to Figure 1.Of the 313 unique compounds tested in the PKIS/UNC library (Fig 1A), approximately one third (103) displayed at least 20% reduction of intracellular growth of Mtb with an acceptable toxicity in THP-1 cells (viability > 70%).Stacked bars represent the number of active compounds grouped by compound chemotype targeting the same host kinase.Hatched sections represent chemotypes that inhibit more than one kinase (shown in brackets).
Figure S2: GSK3 inhibitors has limited effect on growth of M. tuberculosis in broth, related to Figure 1 and Star Methods Broth Activity analysis.A qualitative analysis of Takeda's GSK3 inhibitor library against Mtb cultured in broth.The images show a resazurin assay of 96 well-plates of M. tuberculosis treated with the GSK3 inhibitors or rifampicin (positive control) all at 20 µM concentration.Panel A shows Mtb treated with GSK3 inhibitors #1-59.Panel B shows Mtb treated with GSK3 inhibitors #60-88.Each inhibitor was tested in duplicate.The conversion of resazurin (blue) to resorufin (pink) indicates the presence of live bacteria.

Figure S3 :
Figure S3: in vitro inhibition of GSK3β by selected inhibitors, related to Star Methods: Determination of IC50 of GSK3 inhibitors.GSK3β activity was measured using the ADP-Glo TM Kinase assay and normalized to the negative control (100% kinase activity).Curve was fit using non-linear regression analysis calculated with GraphPad Prism software.

Figure
Figure S4: Dose-dependent activity of GSK3β inhibitor P-4423632 on intracellular bacteria.Related to Star Methods describing C. Jejuni and S. enterica infections.A.Caco-2 cells were infected with C. jejuni (MOI 1:500) and treated with two-fold serial dilutions of P-4423632 for 24h.Percent inhibition was calculated by subtracting CFU values as a percentage of the untreated DMSO control (plotted at 0.1 µM) from 100%.Data represents three biological experiments performed in duplicate ± SEM, N = 6.Curve was fit using non-linear regression analysis of log (inhibitor) vs. response (variable slope) calculated with GraphPad Prism 10.B. Dose dependent inhibition of Salmonella enterica serovar Typhimurium in THP-1 cells (MOI 10:1).Data from three independent experiments.Non-linear regression curve plotted following constraining the range from 0-100% using GraphPad Prism 10.

Figure S5 :
Figure S5: Images from HCS of Mtb-infected hMDMs treated with P-4423632, related to Figure 3A.Representative images taken at 72h post-treatment with twofold serial dilutions of P-4423632.Images were captured at 40x magnification using the Phoenix Opera HCS system and are a composite of two fluorescent channels: blue = DAPI-stained nuclei of hMDMs, red = Mtb expressing E2-Crimson.Images represent one of 25 fields per well, four wells per compound concentration, of three biological replicates used to calculate the dose-response curve in Fig 3 A.

FigureFigure S7 :
Figure S6: P-4423632 inhibition of Mtb-infected hMDM cells, related to Figure 3A.hMDMs from three independent donors were infected with Mtb expressing GFP and were monitored over the course of 328 h using the Sartorius IncuCyte S3 HCS platform with automated analysis of live cell imaging every 8h A. Mtb growth represented by GFP fluorescence area, B. Confluence area of hMDMs cells, C. hMDMs cell viability (dead count) represented by RFP spot counts of DRAQ7 dye.Infected hMDMs were treated with two-fold serial dilutions of P-4423632 from 20 µM to 0.078 µM (highest four and lowest concentrations shown), as well as 0.1% DMSO vehicle control (0 µM) and 1 µg/mL of Rifampicin and Isoniazid (R+I) positive control.Box and whisker plots represent the median, quartile, and outlier (points) data from three blood donors performed in duplicate (N = 6).D. Variability among hMDM donors.Data from A at 0 µM (DMSO control) were plotted as a line graph by donor ± SEM which demonstrated complete control of Mtb by one of the donors and large variability of Mtb control among the donors over the time course of the experiment.N = 2.

Figure S8 :
Figure S8:Venn diagram showing the involvement of 16 highly modulated proteins in GSK3, phagocytosis, and apoptosis related pathways, related to Figure 4. GSK3b related pathways are defined as any KEGG pathway which involves GSK3b.Disease specific pathways such as cancer pathways were filtered out.Phagocytosis and apoptosis related pathways were provided directly by KEGG.The 16 proteins were chosen from the 25 proteins with the highest difference in log fold change between infection and infection + treatment as shown in Fig. 4 C. Circle color shows the pathway group: GSK3b = blue, phagocytosis = green, apoptosis = red.

Table S2 . Disk assay examining P-4423632 in vitro activity against a variety of gram negative, gram positive and mycobacterial strains related to Figure 1
. R denotes resistance (no bacterial clearing) up to 25 μg of P-4423632 as the maximum concentration tested unless otherwise indicated; Gen: Gentamycin control; BDQ: Bedaquiline control.