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Fine-tuning of macrophage activation using synthetic rocaglate derivatives.

Bhattacharya B, Chatterjee S, Devine WG, Kobzik L, Beeler AB, Porco JA, Kramnik I - Sci Rep (2016)

Bottom Line: Several active compounds belonged to the flavagline (rocaglate) family.These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage.These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary Center, Department of Medicine, Boston University School of Medicine, National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, Boston, MA, 02118, USA.

ABSTRACT
Drug-resistant bacteria represent a significant global threat. Given the dearth of new antibiotics, host-directed therapies (HDTs) are especially desirable. As IFN-gamma (IFNγ) plays a central role in host resistance to intracellular bacteria, including Mycobacterium tuberculosis, we searched for small molecules to augment the IFNγ response in macrophages. Using an interferon-inducible nuclear protein Ipr1 as a biomarker of macrophage activation, we performed a high-throughput screen and identified molecules that synergized with low concentration of IFNγ. Several active compounds belonged to the flavagline (rocaglate) family. In primary macrophages a subset of rocaglates 1) synergized with low concentrations of IFNγ in stimulating expression of a subset of IFN-inducible genes, including a key regulator of the IFNγ network, Irf1; 2) suppressed the expression of inducible nitric oxide synthase and type I IFN and 3) induced autophagy. These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage. These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

No MeSH data available.


Related in: MedlinePlus

CMLD candidate compound identification and validation.(a) Dose response in J774-21 GFP-Ipr1 reporter cell line. J7-21 cells were treated with different doses of C9433 (0.010, 0.033, 0.1, 0.33, 1, 3.3 μM) in the presence of 0.2 U/mL IFNγ for 24 hrs, followed by addition of 1 μg/mL dox for 24 hrs. GFP-Ipr1 expression was measured using automated cytometry. Results are representative of at least two independent experiments performed in triplicates. (b) Toxicity of compounds in primary BMDM. BMDM were treated with compounds C9433, C5557 and C8808 at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (c) Dose-dependent translation inhibition by rocaglates. 293TR-FLuc cells were treated with rocaglates C9433, C5557 and C8808 (0.033, 0.1, 0.33, 1, 3.3 μM) and luciferase activity was measured after 18 hrs. Two independent experiments were performed in triplicates. (d) Comparative effect of 1 μM rocaglates C9433, C5557 and C8808 on gene expression. BMDM were treated with 1 μM compound for 24 hr and the mRNA expression of Irf1, Igtp and Irgm1, Irf5, Gadd45b and Ptgs2 was measured by real-time PCR. (e) Effect of C9433 on IFNγ-inducible gene expression. BMDM was treated with different doses of C9433 (0.33, 1, 3.3 μM) in presence and absence of 0.2U IFNγ for 24 hrs and mRNA expression of Irf7, Irf1 and Ido1 was measured by real-time PCR. Cells treated with 20 U/mL IFNγ served as a positive control of gene expression. PCR data are representative of at least two independent experiments.
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f3: CMLD candidate compound identification and validation.(a) Dose response in J774-21 GFP-Ipr1 reporter cell line. J7-21 cells were treated with different doses of C9433 (0.010, 0.033, 0.1, 0.33, 1, 3.3 μM) in the presence of 0.2 U/mL IFNγ for 24 hrs, followed by addition of 1 μg/mL dox for 24 hrs. GFP-Ipr1 expression was measured using automated cytometry. Results are representative of at least two independent experiments performed in triplicates. (b) Toxicity of compounds in primary BMDM. BMDM were treated with compounds C9433, C5557 and C8808 at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (c) Dose-dependent translation inhibition by rocaglates. 293TR-FLuc cells were treated with rocaglates C9433, C5557 and C8808 (0.033, 0.1, 0.33, 1, 3.3 μM) and luciferase activity was measured after 18 hrs. Two independent experiments were performed in triplicates. (d) Comparative effect of 1 μM rocaglates C9433, C5557 and C8808 on gene expression. BMDM were treated with 1 μM compound for 24 hr and the mRNA expression of Irf1, Igtp and Irgm1, Irf5, Gadd45b and Ptgs2 was measured by real-time PCR. (e) Effect of C9433 on IFNγ-inducible gene expression. BMDM was treated with different doses of C9433 (0.33, 1, 3.3 μM) in presence and absence of 0.2U IFNγ for 24 hrs and mRNA expression of Irf7, Irf1 and Ido1 was measured by real-time PCR. Cells treated with 20 U/mL IFNγ served as a positive control of gene expression. PCR data are representative of at least two independent experiments.

Mentions: To identify novel IFNγ-synergistic compounds, we screened a chemical library from the Boston University Center for Molecular Discovery (BU-CMD, www.bu.edu/cmd) which is freely available to biological collaborators. The library consisted of 3840 compounds and is comprised of novel chemotypes rich in structural diversity. Testing the BU-CMD library led to the identification of 30 initial hits; ten of the hits were subsequently confirmed after re-testing in triplicates using quality-controlled compounds from frozen stocks. These candidate compounds (CCs) were subjected to in-depth analyses to exclude assay artifacts and to assess IFNγ-dependence of their activity. First, we tested the candidate compounds in J774 cells that do not express GFP-Ipr1, resulting in exclusion of two DNA intercalating fluorophores with non-specific nuclear fluorescence. Next we tested CCs in J7-21 cells in the presence of IFNγ but in the absence of doxycycline to exclude false positive compounds that would be able to substitute for Dox - none were found. Ultimately, three BU-CMD compounds were selected based on their activity and specificity. All of the top compounds [CMLD005557 (C5557), CMLD008808 (C8808), and CMLD009433 (C9433)] were structurally related derivatives of the natural product rocaglamide A (rocaglates) (see below). Compounds were subjected to quality control (QC) analysis and their activity was confirmed using our initial screening assay. We measured dose-response effects on GFP-Ipr1 expression using the J7-21 clone-based assay of all three CCs in a concentration range 0.03–3.3 μM. The specific activity of C9433 was the highest and dose-dependent with an IC50 of 160 nM (Fig. 3a). Toxicity of C5557 and C8808 was higher in comparison to C9433 in J7-21 cells (not shown), but in primary macrophages all three CCs exhibited low toxicity (Fig. 3b). Because rocaglates are known to inhibit protein translation, we compared the effects of C9433, C8808 and C5557 effects on protein biosynthesis using a reporter cell line 293TR-Fluc expressing firefly luciferase under the control of a constitutive promoter22. Within the range of concentrations used in our assays, C9433 and C5557 displayed similar translation inhibition activities (IC50 values 53 nM and 45 nM respectively) while C8808 was less potent (IC50 value 100 nM), suggesting that specific activities of the CCs in our assay did not strictly parallel their translational inhibition activities (Fig. 3c).


Fine-tuning of macrophage activation using synthetic rocaglate derivatives.

Bhattacharya B, Chatterjee S, Devine WG, Kobzik L, Beeler AB, Porco JA, Kramnik I - Sci Rep (2016)

CMLD candidate compound identification and validation.(a) Dose response in J774-21 GFP-Ipr1 reporter cell line. J7-21 cells were treated with different doses of C9433 (0.010, 0.033, 0.1, 0.33, 1, 3.3 μM) in the presence of 0.2 U/mL IFNγ for 24 hrs, followed by addition of 1 μg/mL dox for 24 hrs. GFP-Ipr1 expression was measured using automated cytometry. Results are representative of at least two independent experiments performed in triplicates. (b) Toxicity of compounds in primary BMDM. BMDM were treated with compounds C9433, C5557 and C8808 at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (c) Dose-dependent translation inhibition by rocaglates. 293TR-FLuc cells were treated with rocaglates C9433, C5557 and C8808 (0.033, 0.1, 0.33, 1, 3.3 μM) and luciferase activity was measured after 18 hrs. Two independent experiments were performed in triplicates. (d) Comparative effect of 1 μM rocaglates C9433, C5557 and C8808 on gene expression. BMDM were treated with 1 μM compound for 24 hr and the mRNA expression of Irf1, Igtp and Irgm1, Irf5, Gadd45b and Ptgs2 was measured by real-time PCR. (e) Effect of C9433 on IFNγ-inducible gene expression. BMDM was treated with different doses of C9433 (0.33, 1, 3.3 μM) in presence and absence of 0.2U IFNγ for 24 hrs and mRNA expression of Irf7, Irf1 and Ido1 was measured by real-time PCR. Cells treated with 20 U/mL IFNγ served as a positive control of gene expression. PCR data are representative of at least two independent experiments.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834551&req=5

f3: CMLD candidate compound identification and validation.(a) Dose response in J774-21 GFP-Ipr1 reporter cell line. J7-21 cells were treated with different doses of C9433 (0.010, 0.033, 0.1, 0.33, 1, 3.3 μM) in the presence of 0.2 U/mL IFNγ for 24 hrs, followed by addition of 1 μg/mL dox for 24 hrs. GFP-Ipr1 expression was measured using automated cytometry. Results are representative of at least two independent experiments performed in triplicates. (b) Toxicity of compounds in primary BMDM. BMDM were treated with compounds C9433, C5557 and C8808 at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. (c) Dose-dependent translation inhibition by rocaglates. 293TR-FLuc cells were treated with rocaglates C9433, C5557 and C8808 (0.033, 0.1, 0.33, 1, 3.3 μM) and luciferase activity was measured after 18 hrs. Two independent experiments were performed in triplicates. (d) Comparative effect of 1 μM rocaglates C9433, C5557 and C8808 on gene expression. BMDM were treated with 1 μM compound for 24 hr and the mRNA expression of Irf1, Igtp and Irgm1, Irf5, Gadd45b and Ptgs2 was measured by real-time PCR. (e) Effect of C9433 on IFNγ-inducible gene expression. BMDM was treated with different doses of C9433 (0.33, 1, 3.3 μM) in presence and absence of 0.2U IFNγ for 24 hrs and mRNA expression of Irf7, Irf1 and Ido1 was measured by real-time PCR. Cells treated with 20 U/mL IFNγ served as a positive control of gene expression. PCR data are representative of at least two independent experiments.
Mentions: To identify novel IFNγ-synergistic compounds, we screened a chemical library from the Boston University Center for Molecular Discovery (BU-CMD, www.bu.edu/cmd) which is freely available to biological collaborators. The library consisted of 3840 compounds and is comprised of novel chemotypes rich in structural diversity. Testing the BU-CMD library led to the identification of 30 initial hits; ten of the hits were subsequently confirmed after re-testing in triplicates using quality-controlled compounds from frozen stocks. These candidate compounds (CCs) were subjected to in-depth analyses to exclude assay artifacts and to assess IFNγ-dependence of their activity. First, we tested the candidate compounds in J774 cells that do not express GFP-Ipr1, resulting in exclusion of two DNA intercalating fluorophores with non-specific nuclear fluorescence. Next we tested CCs in J7-21 cells in the presence of IFNγ but in the absence of doxycycline to exclude false positive compounds that would be able to substitute for Dox - none were found. Ultimately, three BU-CMD compounds were selected based on their activity and specificity. All of the top compounds [CMLD005557 (C5557), CMLD008808 (C8808), and CMLD009433 (C9433)] were structurally related derivatives of the natural product rocaglamide A (rocaglates) (see below). Compounds were subjected to quality control (QC) analysis and their activity was confirmed using our initial screening assay. We measured dose-response effects on GFP-Ipr1 expression using the J7-21 clone-based assay of all three CCs in a concentration range 0.03–3.3 μM. The specific activity of C9433 was the highest and dose-dependent with an IC50 of 160 nM (Fig. 3a). Toxicity of C5557 and C8808 was higher in comparison to C9433 in J7-21 cells (not shown), but in primary macrophages all three CCs exhibited low toxicity (Fig. 3b). Because rocaglates are known to inhibit protein translation, we compared the effects of C9433, C8808 and C5557 effects on protein biosynthesis using a reporter cell line 293TR-Fluc expressing firefly luciferase under the control of a constitutive promoter22. Within the range of concentrations used in our assays, C9433 and C5557 displayed similar translation inhibition activities (IC50 values 53 nM and 45 nM respectively) while C8808 was less potent (IC50 value 100 nM), suggesting that specific activities of the CCs in our assay did not strictly parallel their translational inhibition activities (Fig. 3c).

Bottom Line: Several active compounds belonged to the flavagline (rocaglate) family.These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage.These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary Center, Department of Medicine, Boston University School of Medicine, National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, Boston, MA, 02118, USA.

ABSTRACT
Drug-resistant bacteria represent a significant global threat. Given the dearth of new antibiotics, host-directed therapies (HDTs) are especially desirable. As IFN-gamma (IFNγ) plays a central role in host resistance to intracellular bacteria, including Mycobacterium tuberculosis, we searched for small molecules to augment the IFNγ response in macrophages. Using an interferon-inducible nuclear protein Ipr1 as a biomarker of macrophage activation, we performed a high-throughput screen and identified molecules that synergized with low concentration of IFNγ. Several active compounds belonged to the flavagline (rocaglate) family. In primary macrophages a subset of rocaglates 1) synergized with low concentrations of IFNγ in stimulating expression of a subset of IFN-inducible genes, including a key regulator of the IFNγ network, Irf1; 2) suppressed the expression of inducible nitric oxide synthase and type I IFN and 3) induced autophagy. These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage. These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

No MeSH data available.


Related in: MedlinePlus