Suppression of interferon signaling via small-molecule modulation of TFAM
- Astellas Pharmaceuticals Inc, Oncology Research, Cancer Biology, United States
- Astellas Pharmaceuticals Inc, Oncology Medicinal Chemistry, United States
- Division of Rheumatology and Clinical Immunology, University of Pittsburgh, United States
- Aging Institute, University of Pittsburgh/UPMC, United States
- Department of Medicine, Division of Cardiology, University of Pittsburgh, United States
- Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, United States
- Generian Pharmaceuticals, United States
- Vascular Medicine Institute, University of Pittsburgh, United States
Figures
Figure 1
Development of a high-throughput screen for the identification of potent small-molecule activators to TFAM.
(A) Diagram of primary screen and secondary assays (CETSA, stabilizing TFAM to increase mtDNA copy number, prevent escape and inhibit activation of STING pathway). (B) Results from CETSA screen. (C) Hit validation in mtDNA copy number assay—compounds 1, 2, and 15 evaluated at increasing concentrations in HeLa cells. (D) Compound 2 evaluated at increasing concentrations across multiple cell lines. Figures are representatives of at least 2 independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=4 biological replicates. Source data for this figure are available in Figure 1—source data 1 (raw data and analysis).
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Figure 1—source data 1
Raw data and analysis results to generate the graphs shown in Figure 1.
- https://cdn.elifesciences.org/articles/108742/elife-108742-fig1-data1-v1.xlsx
Figure 2 with 2 supplements
Compounds 2, 3, and 15 repress ISG signaling mediated by mtDNA damage and the cGAS/STING pathway.
(A) Flowchart of the CXCL-10 experiment. (B) Compounds 2, 3, and 15 repressed ISG signaling in CXCL-10 production. THP-1 cells were pre-treated with individual compounds 48 h prior to 100 ng/mL TNFa stimulation for 48 h. CXCL-10 levels were measured. (C) Flowchart of the ISRE experiment. (D) Compounds 2, 3, and 15 repressed ISG signaling in IRF3 production. THP-1-Dual cells were pre-treated with individual compounds 24 h prior to 100 ng/mL TNFa stimulation for 48 h. The inhibition of type I IFN response was monitored via ISRE reporter activation. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in Figure 2—source data 1 (raw data and analysis).
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Figure 2—source data 1
Raw data and analysis results to generate the graphs shown in Figure 2 .
- https://cdn.elifesciences.org/articles/108742/elife-108742-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
TNFa stimulates upregulation of CXCL10 protein levels.
(A) CXCL10 protein levels from THP-1 cells stimulated with increasing concentrations of TNFa for 24 h and 48 h. (B) Pre-treatment of cells with H151 for 48 h inhibited CXCL10 secretion in a dose-dependent fashion. THP-1 cells were pre-treated with H151 for 48 h prior to TNFa (100 ng/mL) stimulation for 48 h followed by measurement of CXCL-10. (C) Pre-treatment of cells with VBIT-4 for 48 h inhibited CXCL10 secretion in a dose-dependent fashion. THP-1 cells were pre-treated with VBIT-4 for 48 h prior to TNFa (100 ng/mL) stimulation for 48 h followed by measurement of CXCL-10. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in Figure 2—source data 1 (raw data and analysis).
Figure 2—figure supplement 2
Assessment of compound assay interference.
(A) ISRE reporter activation of THP-1 dual cells stimulated with 100 ng/mL TNFa for 48 h. Compounds were added to the cells 5 min before the assay detection. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in Figure 2—source data 1 (raw data and analysis).
Figure 3
Hit expansion – compound 11 identified as a TFAM activator with sub-micromolar potency.
(A) Chemical structures of hit compounds and related analogs. (B) TFAM modulators were profiled in the ISRE assay and display dose-dependent suppression of ISG signaling. THP-1-Dual cells were pre-treated with individual compounds 24 h prior to 100 ng/mL TNFa stimulation for 48 h. ISRE reporter activation was measured. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in Figure 3—source data 1 (raw data and analysis).
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Figure 3—source data 1
Raw data and analysis results to generate the graphs shown in Figure 3.
- https://cdn.elifesciences.org/articles/108742/elife-108742-fig3-data1-v1.xlsx
Figure 4
Mechanism of action.
(A) Compounds 2, 3, and 15 do not repress cGAMP induced ISG signaling. THP-1-Dual cells were pre-treated with individual compounds 48 h prior to 10 ug/mL cGAMP stimulation for 24 h. ISRE reporter activation was measured. (B) Compounds 2, 3, and 11 impart a dose-dependent increase in TFAM protein levels. Immunoblot analysis of TFAM from T47D cells treated with indicated compounds for 5 days. (C) Compounds exhibit minimal impact on TFAM mRNA levels. (D) Downregulation of TFAM attenuates the function of compounds 2 and 3 in repression of ISG signaling. THP-1-Dual cells were treated with individual compounds 24 h after siRNA transfection. After incubation for 24 h, THP-1-Dual cells were stimulated with 100 ng/mL TNFa for another 48 h. ISRE reporter activation was measured and normalized to protein concentration. (E) Compound 3 inhibits mtDNA cytosolic leakage. THP-1 cells were pre-treated with individual compounds 72 h prior to 100 ng/mL TNFa stimulation for 48 h. Cytosolic mtDNA was extracted and quantified using a qPCR assay. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in Figure 4—source data 1 (original uncropped blots) and Figure 4—source data 2 (annotated uncropped blots), and Figure 4—source data 3 (raw data and analysis).
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Figure 4—source data 1
Original uncropped western blot images for Figure 4.
- https://cdn.elifesciences.org/articles/108742/elife-108742-fig4-data1-v1.pdf
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Figure 4—source data 2
Annotated uncropped western blot images for Figure 4, with treatment conditions and protein identities indicated.
- https://cdn.elifesciences.org/articles/108742/elife-108742-fig4-data2-v1.zip
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Figure 4—source data 3
Raw data and analysis results to generate the graphs shown in Figure 4.
- https://cdn.elifesciences.org/articles/108742/elife-108742-fig4-data3-v1.xlsx
Figure 5
Impact of compound 2 in representative cellular disease models.
(A) Compound 2 dose-dependently increases ATP levels in MELAS 80% cybrid cells after 48 h treatment. Cells were treated with increasing concentrations of Compound 2 or DMSO (vehicle control) for 48 h. ATP levels were measured using the CellTiter-Glo luminescent assay (Promega). Data are presented as mean ± SEM from n=8 biological replicates. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test; *p<0.05; **p<0.01; ***p<0.005; ***p<0.001 compared to DMSO control. (B) Western blot analysis Compound 2 dose-dependently decreases fibrotic markers (Fibronectin and α-SMA) of SSc patient fibroblasts after 48 h treatment. Actin is shown as a loading control. (C) Compound 2 increases the suppressing capacity of Tregs against effector T cells in a dose-dependent fashion. ITreg cells from four donors stained with Celltrace violet (CTV) are cocultured with anti-CD3/CD28 stimulated autologous CFSE-labeled responder cells (Tresp) for 4 days followed by staining for viability and CD4 and analysis on flow cytometer for dilution of CFSE. The percentage suppression of Tresp cells was calculated for each group with different Treg:Tresp ratio. Source data for Figure 5 are provided in Figure 5—source data 1 (original uncropped blots) and Figure 5—source data 2 (annotated uncropped blots).
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Figure 5—source data 1
Original uncropped western blot images for Figure 5.
- https://cdn.elifesciences.org/articles/108742/elife-108742-fig5-data1-v1.zip
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Figure 5—source data 2
Annotated uncropped western blot images for Figure 5, with treatment conditions and protein identities indicated.
- https://cdn.elifesciences.org/articles/108742/elife-108742-fig5-data2-v1.zip
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Suppression of interferon signaling via small-molecule modulation of TFAM
eLife 14:RP108742.
https://doi.org/10.7554/eLife.108742.2
