All authors are employed by GlaxoSmithKline. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: HY WZ HP HGF CM SL ZZ VS CL. Performed the experiments: HY HGF EL CM TC TR CL. Analyzed the data: HY HGF EL CM TC TR CL. Contributed reagents/materials/analysis tools: BW HZ SS. Wrote the paper: HY CL. Critically revised the manuscript: JLE GPV.
Chronic inflammation is a major contributing factor in the pathogenesis of many age-associated diseases. One central protein that regulates inflammation is NF-κB, the activity of which is modulated by post-translational modifications as well as by association with co-activator and co-repressor proteins. SIRT1, an NAD+-dependent protein deacetylase, has been shown to suppress NF-κB signaling through deacetylation of the p65 subunit of NF-κB resulting in the reduction of the inflammatory responses mediated by this transcription factor. The role of SIRT1 in the regulation of NF-κB provides the necessary validation for the development of pharmacological strategies for activating SIRT1 as an approach for the development of a new class of anti-inflammatory therapeutics. We report herein the development of a quantitative assay to assess compound effects on acetylated p65 protein in the cell. We demonstrate that small molecule activators of SIRT1 (STACs) enhance deacetylation of cellular p65 protein, which results in the suppression of TNFα-induced NF-κB transcriptional activation and reduction of LPS-stimulated TNFα secretion in a SIRT1-dependent manner. In an acute mouse model of LPS-induced inflammation, the STAC SRTCX1003 decreased the production of the proinflammatory cytokines TNFα and IL-12. Our studies indicate that increasing SIRT1-mediated NF-κB deacetylation using small molecule activating compounds is a novel approach to the development of a new class of therapeutic anti-inflammatory agents.
Inflammation is a physiological response to remove injurious stimuli and initiate the healing process. However, unresolved or sustained low-grade inflammation leads to development of chronic diseases including chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, type 2 diabetes (T2D), cancer, Alzheimer’s disease, cardiovascular, and renal diseases, many of which are associated with aging. Upregulation of inflammatory biomarkers is a characteristic of the aging process
One key protein that regulates inflammatory responses is the transcription factor NF-κB which is held quiescent in the cytoplasm when in complex with IκBα. In response to a proinflammatory stimulus (e.g. lipopolysaccharide (LPS), tumor necrosis factor (TNFα), or interleukin-1 (IL-1)) via Toll-like receptors or cytokine receptors, IκBα is phosphorylated by IKK and subject to ubiquitin-dependent proteasomal degradation, thereby allowing NF-κB to translocate to the nucleus and activate the transcription of a cascade of proinflammatory cytokines and chemokines to induce inflammatory responses
SIRT1 is an NAD+-dependent protein deacetylase that plays important roles in regulating metabolism, inflammation, stress resistance, DNA repair and cell survival through deacetylation of key transcription factors, enzymes and proteins
The pivotal role of SIRT1 in regulating inflammation suggests a new avenue for attenuating inflammation by modulating SIRT1 activity. SIRT1 activity can be regulated by the endogenous activator AROS (active regulator of SIRT1)
Resveratrol (RES) was first identified as a naturally occurring small molecule that biochemically activates SIRT1
We are interested in understanding the mechanism by which STACs regulate inflammation. We therefore developed a quantitative assay to measure the cellular levels of acetylated p65 protein. In this cell-based system we demonstrate that overexpression of SIRT1 attenuates, while knockdown or inhibition of SIRT1 increases p65 acetylation. We also show that compounds from two chemical series of STACs activate SIRT1
Structures of benzimidazole STACs and the core structure of quinolone STACs of this study are shown in
(A) Structures of the benzimidazole STACs. (B) Core structure of the quinolone STACs.
U2OS cells (ATCC, HTB-96) and HEK 293 cells (cell line used for BacMam transduction was derived at GSK from ATCC CRL-1573
BacMam p65 and BacMam p300-HAT viruses were prepared internally according to the standard procedures
Acetylated p65 protein in cell lysates was measured by an AlphaScreen format based assay (Bosse
U2OS cells in six-well plates were pretreated with test compounds for 1 hour followed by doxorubicin treatment at 1 µg/ml for four hours to induce p53 acetylation. For the experiments to evaluate knockdown of SIRT1 on doxorubicin-induced p53 acetylation, SIRT1-siRNA or NT-siRNA was transfected into cells 40 hours prior to compound pretreatment. An ELISA assay was developed and used to measure acetylated p53 protein in cell lysates. After coating an anti-acetylated p53 antibody (Cell Signaling, 2525) onto a 96-well plate at 4°C overnight, the plate was blocked for 2 hours. Cell lysates were then transferred onto the ELISA plate and incubated at RT for 3 hours. Detection of acetylated p53 protein was achieved by using a HRP-labeled anti-p53 antibody (Santa Cruz, sc-126 HRP) followed by exposing the ELISA plate to HRP substrate (BioFX Laboratories, TMBC-0100-01). Absorbance at OD 450 nm was read by a SpectraMax M5 plate reader. The quantity of acetylated p53 protein in samples was calculated by fitting the data into a standard curve of acetylated p53 protein on the same ELISA plate. To compare the levels of acetylated p53 among samples, the quantity of acetylated p53 was normalized against the amount of total protein in the lysates that had been transferred onto the ELISA plate.
HEK 293T/17 cells in 10 cm dishes were transfected with p300-HAT plasmid by FuGENE 6 transfection reagent (Roche, 11814443001). 18 hours after transfection, cells were pretreated with compounds for 6 hours and then stimulated with 20 ng/ml recombinant human TNFα for 20 minutes (Invitrogen, PHC3016). Cells were then lysed on plate and the supernatants after centrifugation were subject to immunoprecipitation by an anti-acetylated K310-p65 antibody (Abcam, ab19870) overnight at 4°C with rotating. On the next day, 50 µl Protein A Dynabeads were added to the immunoprecipitation samples and rotated for 3 hours at 4°C. After washing, the immunoprecipitated acetylated p65 protein was extracted by 2X SDS sample buffer and probed for p65 (anti-p65 antibody, Santa Cruz, sc-8008) by western blotting. A phospho-NF-κB (Ser536) antibody (Cell Signaling, 3031) and a phospho-IκBα (Ser32/36) antibody (Cell Signaling, 9246) were used to probe for phosphorylated p65 and phosphorylated IκBα in cell lysates. Densitometry quantitation of acetylated p65 protein on western blots was conducted by using Odyssey software. For the experiments to assess SIRT1 overexpression on TNFα-induced p65 acetylation, pcDNA-hSIRT1 plasmid or empty vector was co-transfected with p300-HAT plasmid at the same time into cells by FuGENE 6 transfection reagent.
HEK 293 cells stably expressing a luciferase reporter driven by a tandem of 3×κB DNA element were plated onto 384-well plates. On the day of experiment, cells were pretreated with compounds for 1 hour and then stimulated with 50 ng/ml of recombinant TNFα for 3 hours. Luciferase activity was measured by Steady-Glo Luciferase Assay System (Promega, E2550) according to the manufacturer’s protocol. For the experiments to examine SIRT1 overexpression on NF-κB transcriptional activity, SIRT1 plasmid or empty vector was transfected into cells 24 hours prior to TNFα treatment. Cell viability in the sister plate was measured by ATPlite according to the manufacturer’s protocol (PerkinElmer, 6016739).
RAW 264.7 macrophages were seeded at 4×104 cells per well in 96-well plates. 16 hours after seeding, cells were pretreated with compounds for 1 hour, followed by stimulation with 100 ng/ml LPS (
11 weeks old male BALB/c mice purchased from Jackson Laboratory were acclimated with minimum 4 days under the same conditions as for the actual test. Mice weighted at 25–28 grams were randomized into 6 groups with 8 mice each and were orally dosed with vehicle, 10% PEG, 10% VitE-TPGS, or SRTCX1003 at 3 mg/kg, 10 mg/kg, 30 mg/kg, 100 mg/kg, or dexamethasone at 1 mg/kg (Sigma, MO part#D1756, lot 096K1805), all diluted in vehicle of 10% PEG, 10% VitE-TPGS. One hour after oral dose, animals were administered 0.25 mg/kg LPS in PBS (Sigma, MO part# L2630, lot 128K4054) through intravenous injection. Ninety minutes later, animals were sacrificed by CO2 asphyxiation, and the blood was collected by cardiac puncture. Plasma was thereafter separated from blood cells by centrifugation for 8 minutes at 13,200 rpm in an Eppendorf using a 5214R centrifuge. A 10-fold dilution of plasma was performed prior to the measurement of TNFα (Invitrogen, CA, part# KMC3011) and IL-12p40 (Invitrogen, CA, part# KMC0121) by ELISA. In parallel, 30 µl of undiluted plasma was submitted for drug exposure analysis by mass spectrometry.
All animal studies were conducted at Sirtris, a GSK Company (Cambridge, MA) following the guidelines of the institutional animal use and care committee (IACUC). All protocols and animal ethics were approved by the Sirtris IACUC. Appropriate measures were taken to minimize distress to the animals, following the guidelines of Sirtris IACUC and GSK institutional animal use policies. The animals were anesthetized using isoflurane prior to intravenous injection of LPS and were allowed to recover for several minutes prior to returning to the cage. Mice were sacrificed by asphyxiation using carbon dioxide followed by cardiac puncture.
We developed a high throughput cellular assay to examine the ability of compounds to promote SIRT1-mediated deacetylation of p65 protein in cells. This assay system uses BacMam virus transduction in U2OS or HEK 293 cells to co-express p300 HAT and an HA-tagged p65 NF-κB subunit to enable detection of acetylated p65. SIRT1 has been shown to specifically deacetylate p65 at K310
Flow diagram depicts the assay procedure, including BacMam p65 and BacMam p300 viral transduction, plating cells, compound treatment, cell lysis and detection of acetylated p65 protein by AlphaScreen format.
(A) Levels of acetylated p65 in BacMam SIRT1 virus or BacMam GFP virus transduced cells. Western blot in the right upper corner shows the level of SIRT1 expression upon BacMam SIRT1 virus transduction. (B–C) Levels of acetylated p65 protein in cells transfected with SIRT1-siRNA or NT-siRNA as measured by AlphaScreen assay in (B), or demonstrated by immunoblotting in (C). (D) Dose-response effect of EX-527 on levels of acetylated p65 protein. All error bars represent s.d. of at least 4 replicates. *
STACs that were identified from biochemical assays (
(A) Levels of acetylated p65 in U2OS cells treated with varied concentrations of SRTCX1002 with or without 10 µM EX-527. (B) Levels of acetylated p65 in vehicle or 10 µM SRTCX1002 treated U2OS cells transfected with either SIRT1-siRNA or NT-siRNA. Western blots in the right upper corner indicate that SIRT1 was knocked down by 70% by SIRT1-siRNA. All error bars represent s.d. of at least 4 replicates. *
To see whether the reduction of acetylated p65 protein by STACs is mediated through SIRT1, levels of acetylated p65 protein in vehicle or STAC-treated cells with or without 10 µM EX-527 were measured. Our data showed that inhibition of SIRT1 by EX-527 significantly reversed STAC-mediated reduction of acetylated p65 (
We wanted to investigate the effect of STACs on a physiological proinflammatory stimulus since the assay described above relies on the overexpression of p65 and p300. It has been shown that TNFα stimulation can induce p65 acetylation, which can in turn be reduced by SIRT1 deacetylase activity
(A) Western blots of immunoprecipitated acetylated K310-p65 protein, p65 and actin protein in whole cell lysates from vector or p300 transfected cells with or without TNFα stimulation. (B) Left panel, western blots of immunoprecipitated acetylated K310-p65 protein, SIRT1, p65 and tubulin in whole cell lysates from TNFα-stimulated cells transfected with p300 plus empty vector or p300 plus SIRT1. Right panel, western blots of immunoprecipitated acetylated K310-p65 protein, p65 and tubulin in whole cell lysates from TNFα-stimulated p300 overexpressing cells that were pretreated with EX-527 for 6 hours. (C) Upper panels, western blots of immunoprecipitated acetylated K310-p65 protein, p65, phospho-NF-κB (Ser536), phospho-IκBα (Ser32/Ser36) and tubulin in whole cell lysates from cells pretreated with compounds for 6 hours followed by 20 minutes TNFα stimulation. Lower panels, densitometry quantitation of the western blot for immunoprecipitated acetylated p65 protein. The level of acetylated p65 in TNFα-stimulated and vehicle treated sample was set as 1. Experiments were repeated at least 2 times and western blots from one experiment were shown as representative. Error bars present s.d. of densitometry quantitation of western blots from at least two experiments. *
Next we tested whether STACs could affect TNFα-induced p65 acetylation. We pretreated HEK 293T/17 cells with each of the STACs for 6 hours and then stimulated them with TNFα. The western blot of the immunoprecipitated acetylated p65 protein from these samples showed that STACs significantly reduced TNFα-stimulated p65 acetylation, while the two biochemically inactive compounds showed no effect (
It has been shown previously that acetylation of p65 at lysine 310 is required for full activation of NF-κB function
(A) NF-κB luciferase reporter counts in HEK 293 cells transfected with SIRT1 or empty vector with or without TNFα stimulation. Western blot in the right upper corner indicated SIRT1 expression induced by SIRT1 plasmid transfection. (B) Dose-response effect of SRTCX1002 on NF-κB luciferase reporter activity in HEK293 cells stimulated with TNFα. Error bars represent s.d. of at least four replicates. (C) Dose-response effect of SRTCX1003 on LPS-induced TNFα secretion from RAW cells. *
Compound ID | NF-êB luciferase reporter assay IC50 (µM) | LPS-induced TNFá secretion assay IC50 (µM) |
SRTCX1002 | 0.71 | 7.58 |
SRTCX1003 | 0.95 | 12.52 |
SRTCZ1001 | >20 | >40 |
SRTCD1023 | 3.30 | 3.16 |
SRTCL1015 | 1.24 | 3.41 |
SRTCE1022 | >20 | >40 |
Table shows the IC50 values of the STACs in NF-κB luciferase reporter assay and LPS-induced TNFα secretion assay.
As NF-κB plays a significant role in regulating inflammatory cytokine production, we next determined whether STACs could block LPS-induced TNFα secretion from RAW 264.7 murine macrophage cells. As shown in
Previous studies on SRT1720, SRT2530 and SRT2379 have demonstrated that these earlier generation STACs can improve insulin sensitivity in mice fed with high fat diet in a SIRT1-dependent manner, which is at least partially due to their anti-inflammatory effects
(A) Schematic illustration of the acute LPS-induced inflammation mouse model. (B) Compound plasma concentration of mice dosed with SRTCX1003 for 2.5 hours. (C–D) Dose-response effect of SRTCX1003 and the effect of 1 mg/kg dexamethasone on LPS-induced production of TNFα in (C) and IL-12p40 in (D). Error bars represent s.d. of eight mice in each group. Please note that reduction of TNFα and IL-12p40 by SRTCX1003 at 100 mg/kg was comparable to that induced by 1 mg/kg dexamethasone. *
Inflammation has been shown to play a major role in contributing to the pathology of many chronic diseases, including COPD, rheumatoid arthritis, T2D, cancer, Alzheimer’s disease, cardiovascular, and renal diseases among others
The NAD+-dependent deacetylase SIRT1 has been shown to regulate the inflammatory response to multiple stimuli, as well as improve metabolic, neuronal, cardiovascular and renal functions in diseases of aging, thus validating this deacetylase as a potential therapeutic target for drug discovery and development
Mutational analyses of p65 have revealed the importance of lysine 310 acetylation on NF-κB activation
It is possible that other sirtuins could affect p65 acetylation. SIRT2 has also been shown to deacetylate p65 and regulate NF-κB
Previous studies have already demonstrated the effects of STACs on regulating the inflammation in liver and adipose that is associated with high fat diet and obesity
The efficacy seen in the LPS model prompts the question of whether STACs would show therapeutic benefit in inflammatory disease models. Preliminary studies of STACs on disease models, such as dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) show that STAC treatment reduces colonic inflammation in the IBD model (JL Ellis, unpublished data). Safety assessment studies on STACs are ongoing to assess whether STACs can be tested in chronic inflammation disease models, and to support progression of STACs into clinical trials. Collectively, these findings underscore the promise of SIRT1 modulators as a novel therapeutic approach for inflammatory diseases.
STACs promote SIRT1-mediated deacetylation of p65 protein. (A–C) Treatment of SRTCX1003, SRTCD1023 or SRTCL1015 at varied doses with or without 10 µM EX-527 on levels of acetylated p65 protein in U2OS cells. All error bars represent s.d. of at least 4 replicates. *
(TIF)
SRTCX1002 enhances SIRT1-mediated deacetylation of p53 protein.
(TIF)
SRTCX1003 reduces LPS-induced TNFα secretion from RAW cells via SIRT1. Effects of SRTCX1003 on LPS-induced TNFα secretion from RAW cells transfected with either SIRT1-siRNA or NT-siRNA. Western blots in the right upper corner indicate that SIRT1 was knocked down by 70% by SIRT1-siRNA. All error bars represent s.d. of at least 3 replicates. *
(TIF)
Materials and Methods.
(DOCX)
STACs activate SIRT1 deacetylase activity
(DOCX)
We thank Dr. Siva Lavu and Dr. Andrew C. Lake for critical reading of the manuscript.