Conceived and designed the experiments: DAP ACP MJH. Performed the experiments: DAP WCN YZ. Analyzed the data: DAP ACP. Wrote the paper: DAP MJH.
DAP is the recipient of a Roche Postdoctoral Fellowship, and MJH is the principal investigator for research grants to Washington University from Hoffmann-La Roche and Forest Labs. These awards do not alter the authors' adherence to all of the PLoS ONE policies on sharing data and materials.
Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z′>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.
There has been significant progress in the development of vaccines and therapeutics against viruses, but there are still major gaps in medical therapy for some of the most common types of viral infections. For these types of infections, vaccines can still be ineffective due to new and emergent strains and can exhibit significant off-target effects
Type I IFN signaling starts by activation of the IFN-α/β receptor (IFNAR) and subsequent activation of the IFNAR1-associated TYK2 and IFNAR2-associated JAK1, with consequent recruitment of STAT2. Phosphorylation of STAT2 enables reruitment of STAT1 and release of the phosphorylated STAT1-STAT2 heterodimer bound to IRF-9. This complex binds to the IFN stimulated response element (ISRE) and in concert with recruited transcriptional co-activators such as p300/CBP then drives IFN-stimulated gene (ISG) transcription.
In the present study, we aimed to mimic the beneficial actions of STAT1 modification with a small molecule that also enhances the activity of the IFN signaling pathway. We describe here the development of a high-throughput screening (HTS) system for novel small molecular weight compounds (so-called “small molecules”) that might increase ISG expression and antiviral activity. To develop this screening system, we generated cell lines that stably express the human interferon-stimulated response element (ISRE) driving a luciferase reporter gene. The ISRE gene promoter element is responsible for type I IFN signaling that mediates host defense against a wide range of viruses
Based on the observation that STAT1-CC-expressing cells show increased activity of the endogenous ISRE promoter element
(A) Schematic representation of the vector construct (ISRE-CBG99) used to establish stable cell lines for monitoring ISRE activity driving a click beetle luciferase (CBL) reporter gene. (B) 2fTGH cells stably expressing the ISRE-CBG99 construct (2fTGH-ISRE-CBG99 cells) were treated with IFN-β (1000 U/ml for 0–14 h) and then monitored for luciferase-catalyzed luminescence over 0–4 h. Signal maximum was found at 50 min, and the optimal measurement window (OMW) with at least 90% preservation of signal) was found at 40–70 min. (C) Time course for effect of IFN-β (1000 U/ml) and IFN-γ (100 U/ml) on ISRE activity in 2fTGH-ISRE-CBG99 cells. (D) Corresponding time course for HEK293T-ISRE-CBG99 cells. (E) Ratio of ISRE activities when each cell line is treated with saturating concentrations of IFN-β versus IFN-γ for 0–24 h. * indicates significant differences between values for 2fTGH versus HEK293T cell lines. (F) Concentration-response curves for effect of IFN-β and IFN-γ on ISRE activity in 2fTGH-ISRE-CBG99 cells. (G) Corresponding concentration-response for HEK293T-ISRE-CBG99 cells.
To achieve assay automation and miniaturization, the ISRE activity assay was first automated in 96-well plates and then reformatted for 384-well plates. In the 384-well format, the assay exhibited a near-maximal signal at 8000 cells per well and consistent well-to-well and plate-to-plate reproducibility (
Format | S/B | CV (%) | Z′-factor |
96 well | 206 | 5.1 | 0.84 |
384 well | 112 | 10.9 | 0.70 |
Abbreviations: S/B, signal to background ratio; CV, coefficient of variation.
We used the automated ISRE-activity assay to perform a screen of a 2240 chemical compound library. This library consisted of 2160 compounds from the Johns Hopkins Clinical Compound Library (JHCCL) of FDA approved or approvable drugs
(A) Schematic of assay plate map used in screening the chemical compound library. (B) Reproducibility of raw data between duplicate assay plates for control wells containing IFN-β (0–200 U/ml) and test wells containing compound plus IFN-β (5 U/ml). Dashed lines indicate median ± SD of each data set from the test wells. (C) Signal consistency through a full screen of 56 assay plates. Values represent replicate A of assay plate 1 (the first plate) and plate 55 (the last plate). Box plots represent median and 25th and 75th percentile, and whiskers indicate minimum and maximum of data. Arrows indicate values from test wells.
After raw data were normalized, scaled to z-scores, and summarized, we found that 321 data points (out of a total of 8960 data points representing the 2240 compounds tested at 4 concentrations) had an ISRE activity z-score ≥2 (
(A) Plot of z-scores for each of the 2240 compounds subjected to the primary screen for ISRE activation. Values represent mean of two replicates per compound concentration. Compounds are ranked by highest z-score achieved at any of the four compound concentrations tested. Dashed lines demarcate the compounds with z-scores greater than 2 SD above the mean; and red color denotes the 64 hit compounds selected from this group for validation. (B) The distribution of the 64 hit compounds into various therapeutic classes. Values indicate the number of compounds in each class.
Each of the 64 primary hits was subjected to primary validation for ISRE activity over a broader range of concentrations of drug and IFN-β (0–15 U/ml). Among the primary and confirmed screening hits, idarubicin hydrochloride ranked highest in potency for enhancing ISRE activity (i.e., idarubicin exhibited a significant effect at a lower concentration than other compounds). During the ISRE validation, we found that idarubicin caused a concentration-dependent increase in ISRE activity over a range of IFN-β treatment concentrations, with highly significant effects as low as 25 nM idarubicin in combination with 15 U/ml IFN-β (
(A) Idarubicin concentration-response for ISRE activity without and with treatment with IFN-β (1, 5, and 15 U/ml). 2fTGH-ISRE-CBG99 cells were treated with idarubicin and IFN-β for 8 h. Overall significance for idarubicin dose P<0.0001, IFN-β dose p<0.0001, and Interaction p<0.0001. The p-values for individual comparisons (versus control without idarubicin) are from Bonferroni post-tests from two-way ANOVA (idarubicin x IFN dose). Symbols: * p<0.05, ** p<0.01, *** p<0.001. (B) Corresponding data for daunorubicin, doxorubicin, and epirubicin concentration-response for ISRE activity. (C) Corresponding data for DMXAA and Imiquimod concentration-response for ISRE activity. Overall significance for compound dose p = ns, IFN-β dose p<0.0001, interaction p = ns. (D) Idarubicin toxicity determined using Alamar Blue metabolism assay. Cells were treated with drug for 12 h. (E) Etoposide, Hu-0331, and ICRF-193 (0–100 µM) concentration-response for ISRE activity without and with IFN-β (1, 5, and15 U/ml). Overall significance for ICRF-193 dose p = ns, IFN-β dose p<0.0001, interaction p = ns; for Etoposide and HU-0331 dose, there is a significant inhibitory effect on ISRE activity, p<0.0001, IFN-β dose p<0.0001, interaction p<0.0001.
The structure for idarubicin shows characteristic features of an anthracycline antibiotic unrelated to any other antiviral compound in clinical use. To further validate the effect of idarubicin on ISRE activity, we tested three other anthracyclines (daunorubicin, doxorubicin, epirubicin) with very similar chemical structures to idarubicin. Each of these compounds also showed a capacity to significantly increase ISRE activity (
We also found a cytotoxic effect of idarubicin that is consistent with previous observations
We also observed that the effect of idarubin and the other anthracyclines on ISRE activity occurred at a lower concentration of drug when IFN-β was co-administered, particularly at the highest concentration of IFN-β (15 U/ml) (
Idarubicin concentration-response for ISRE activity without and with treatment with IFN-β (1, 5, and15 U/ml) in the absence or presence of anti-IFNAR2 blocking mAb. *,† = p<0.05 ; **,†† = p<0.01; ***,††† = p<0.001. p-values for individual comparisons are from Bonferroni post-tests from repeated measures two-way ANOVA, comparing the 0 nM Idarubicin condition to the other drug doses. For 15 U/ml IFN-β, overall significance: anti-IFNARII, p<0.0001, IDA dose, p<0.0001; interaction, p = 0.0023. For 5 U/ml IFN-β, overall significance: anti-IFNARII, p<0.0001, IDA dose, p<0.0001; interaction, p = 0.3294. For 1 U/ml IFN-β, overall significance: anti-IFNARII, p<0.0892, IDA dose, p<0.0001; interaction, p = 0.1542. For 0 U/ml IFN-β, overall significance: anti-IFNARII, p<0.4432, IDA dose, p<0.0001; interaction, p = 0.7677.
IFN-β (U/ml) | EC50 |
Confidence Interval | R2 |
0 | 394.7 | 378.7–411.2 | 0.93 |
1 | 361.9 | 321.1–408.0 | 0.92 |
5 | 224.7 | 149.2–338.5 | 0.69 |
15 | 15.97 | 11.75–21.70 | 0.68 |
Values for EC50 were calculated by fitting the data to a four-parameter concentration-response curve for idarubicin effect on ISRE activity as described in
We subjected idarubicin to further validation as an ISRE activator in assays of ISG expression and antiviral activity. For ISG expression, U3A (STAT1-null) and U3A-STAT1 cells were treated with a range of concentrations of idarubicin and IFN-β and then harvested for gene expression using quantitative real-time PCR assay. We found that idarubicin increased the expression of the antiviral gene 2′,5′-oligoadenylate synthetase 1 (
U3A-STAT1 and U3A (STAT1-null) cells were treated with idarubicin for 1.5 h followed by IFN-β for 12 h and then determination of
For antiviral activity, 2fTGH cells were treated with idarubicin along with or without IFN-β and then assessed for control of encephalomyocarditis virus (EMCV) levels and virus-induced cytopathic effect. We selected EMCV since it was previously found to be sensitive to STAT1-CC-dependent improvement in IFN signaling
(A) 2fTGH cells were treated with idarubicin or DMSO vehicle and with IFN-β for 6 h and then inoculated with EMCV (MOI 1) for 1 h. Viral titers in cell supernatant were measured at 28 h after inoculation using real-time quantitative PCR assay for EMCV RNA. Values represent mean ± SE (n = 3 biological replicates, n = 2 technical replicates). Overall significance for idarubicin dose p<0.0001, IFN dose p<0.0001, and Interaction p<0.05. (B) For the protocol used in (A), the corresponding levels of virus-induced cell toxicity based on cell viability from propidium iodide (PI) staining. Overall significance for idarubicin dose P<0.0001, IFN dose p<0.0001, and Interaction p = ns. The p-values for individual comparisons (vs 0 idarubicin control) are from Bonferroni post-tests from two-way ANOVA (idarubicin x IFN dose). Symbols: *p<0.05, **p<0.01, ***p<0.001.
The present study was undertaken to discover antiviral therapeutics that broadly increase host defense. We focused on the IFN system that is central to the antiviral response, although we recognized that other labs have pursued this target with limited success in the past. Some of these previous investigators have used administration of IFN itself to increase the antiviral response, but for this therapeutic goal and others, the side effects of IFN administration have proven to be rate-limiting
Our specific approach was based on previous success with the use of a modified STAT1 signaling pathway. In this work, we demonstrated that a designer form of STAT1 (designated STAT1-CC based on double-cysteine substitutions) was able to enhance IFN signaling and better control viral replication
Considering these factors and observations in the STAT1-CC model system, we designed a cell-based luciferase reporter assay for measuring type I-dependent ISRE activity. This assay proved to yield excellent signal-to-background and Ź factors, specificity for IFN-β treatment over IFN-γ treatment, and suitability for automation and screening. Furthermore, because the construct design uses the Click Beetle Green luciferase, the assay can be paired with other luciferase reporter genes to develop dual color assays to report activity of other signaling pathways, including the type II IFN-γ activated sequence (GAS) promoter activity that mediates defense against intracellular bacteria. A related approach was used to screen for small molecules that increase GAS activity for anti-proliferative and pro-apoptotic effects in cancer cells
Our primary screen identified idarubicin on the basis of its capacity to significantly increase ISRE activity. Subsequent validation assays demonstrated that idarubicin facilitates STAT1-dependent ISG expression and STAT1-directed control of viral replication and cytopathic effect. While others previously reported the antiviral properties of anthracyclines some time ago, no mechanism of their antiviral action was elucidated
In a further analysis of drug mechanism, our study demonstrates that the antiviral activity of idarubicin and other closely related anthracyclines is derived from enhancing the activity of the type I IFN signaling pathway. Our data further show that the enhancing effect of idarubicin is based on ISRE activation and ISG expression independent of IFN production or IFN–IFN-receptor interaction, since the effect of idarubicin is unchanged by IFN-receptor blockade. These findings suggest that idarubicin activation of the ISRE is due to an action in the IFN signaling pathway distal to ligand-receptor binding, e.g., at the level of receptor-associated JAK kinases or further downstream at the formation, transport, binding, and/or assembly of the ISRE transcriptional complex. In that regard, anthracyclines are known to inhibit DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand to prevent replication, but whether this mechanism can affect ISRE or other gene promoter elements still needs to be defined. The present screening approach overcomes the uncertainty in molecular mechanism by using a phenotypic (rather than a target-based) screening approach and thereby captures compounds that increase the activity of the IFN signaling pathway by either established or undefined mechanisms.
In sum, we describe and validate a phenotypic screening strategy to identify small molecules that enhance the activity of the type I IFN signaling pathway and consequently improve antiviral host defense. This approach is designed to lead to discovery of drugs with activity against a broad range of viruses for clinical application as well as experimental tool compounds to further understand IFN-dependent immune mechanisms. Current approaches to defining the basis for IFN signal transduction, particularly in vivo, often rely on complex transgenic and gene targeting approaches. Thus, the use of small molecule enhancers (SMEs) of the IFN signaling pathway may provide much greater flexibility and ease of application to achieve transient adjustment of IFN-related actions and consequent scientific and clinical benefit. Our approach should thereby prove useful to discover drugs with activity against a broad range of viruses as well as effectiveness in other conditions (e.g., multiple sclerosis and melanoma) where the efficacy of IFN treatment might benefit from enhancing the IFN signaling pathway.
IFN-β and IFN-γ were obtained from PBL Interferon source (Piscataway, NJ), diluted and aliquoted according of manufacturer's recommendation, and stored at −80°C. The Johns Hopkins Clinical Compound Library (JHCCL) was obtained from Dr. David Sullivan at the Johns Hopkins University
To construct the pISRE-CBG99 vector, we first generated a 5x-repeat of the ISRE sequence and a TATAA box (5xISRE-TATAA) in the pUCMinusMCS vector from Blue Heron Biotechnology (Bothell, WA) and then cloned this sequence into the Chroma-Luc pCBG99-Basic reporter vector from Promega (Madison, WI) and ligated into Xma1 and Nco1 sites using T4 DNA ligase from Life Technologies (Carlsbad, CA). The DNA sequence of the resultant pISRE-CBG99 vector was confirmed by carrying out BigDye Terminator v3.1 sequencing reactions (Life Technologies) on an ABI capillary sequencer. This vector and the pPUR selection vector from Clontech (Mountain View, CA) were co-transfected at a 9∶1 ratio into 2fTGH or HEK293T cells to increase the likelihood that cells tolerating puromycin selection (0.5 μg/ml) contained one or more copies of pISRE-CBG99 in addition to pPUR. The 2fTGH cells
To optimize the luciferase light reaction, we tested a series of flash and glow luminescent substrate systems in both lysed and live 2fTGH-ISRE-CBG99 and HEK293T-ISRE-CBG99 cells, including D-luciferin (Fisher Scientific, Pittsburgh, PA), the Chroma-Glo Luciferase assay system from Promega and the steadylite plus reporter gene assay system from Perkin Elmer (Waltham, MA) under a range of incubation conditions. The steadylite plus system was selected based on kinetic profile. Effects of IFN-β concentration and treatment time were assessed with all other variables constant.
The assay was automated in a 96-well format with a customized and fully integrated robotic system. The system equipment included: a Caliper Sciclone ALH 3000 workstation (Perkin Elmer) and a EL406 washer (BioTek) for liquid handling, an automated Liconic incubator (Thermo Scientific) for cold storage of plates, an automated Cytomat incubator (Thermo Scientific) for cell culture environment, a separate hotel for storage of plates at room temperature, a Synergy 4 plate reader, a Flexiseal plate heat sealer (K Biosciences, Beverly, MA), a Caliper Twister II, and a Beckman Sagian Orca robotic arm on a linear rail (Beckman Coulter, Fullerton, CA). Construction allowed for transfer of plates, reagents, and plasticware between all instruments, so that there was no need for any manual interference during screening assays. This entire system was enclosed in a custom-made laminar flow hood to allow for HTS screening capability under BSL2 sterile conditions. After the system demonstrated satisfactory performance in a 96-well format, the assay was miniaturized to a 384-well format and re-tested for reproducibility and stability under IFN-β and vehicle (1% DMSO) treatment conditions.
To achieve simultaneous treatment of cells with IFN-β and various compound concentrations and to avoid reagent degradation over time, the screen was run in a modular manner with a precise timeline (Figure S2). The first step included production of plates with appropriate concentrations of compound and IFN-β and then storage at 4°C. A separate plate was made for each of the four compound concentrations (0.24, 1.2, 6 and 30 μM). The Twister II, Sciclone, Orca, and Liconic cold storage incubator handled this step. For the second step, cells were plated at 8000 cells per well in 384-well assay plates (n = 56). This step was accomplished in seven batches (8 assay plates per batch) using the Sciclone. A uniform suspension of cells was maintained by intermittent mixing on the Sciclone deck between cell plating. Simultaneously, the compound stock plates were sealed using the Flexiseal and stacked back into a Twister II rack for storage. For the third step, cells were allowed to grow for 11 h and then were treated with compound and IFN solutions. This step required that a plate containing cells be brought from the Cytomat incubator to the Sciclone deck in concert with a set of compound/IFN dilutions plates from the cold storage incubator. Cell treatments were timed so that each assay plate would be incubated for 10.3 h before the final step of performing the luciferase assay. For this last step, robotics were programmed so that each assay plate developed the luciferase light reaction for 40 min at 25°C in the plate hotel and then was delivered to the Synergy 4 plate reader for determination of luminescence. For this assay, the BioTek EL406 washer was used for aspiration of media and dispersion of substrate. In entirety, the screen took 41.6 h to complete.
The raw data from the HTS assay were subjected to statistical analysis using cellHTS2
Hits from the primary screen were validated using the ISRE activity-luciferase reporter assay over a broad range of compound concentrations (0.01–25 µM) in the absence or presence of IFN-β (1, 5, and 15 U/ml). To determine drug potency, as defined by half-maximal effective concentration (EC50), this data was fit to a four-parameter concentration-response curve as described previously
A resazurin (Alamar Blue) metabolism assay was used to assess cell toxicity during compound treatment
Expression of ISG's was assessed with real-time quantitative PCR assay for the corresponding mRNA level. For these experiments, U3A and U3A-STAT1 cells were first treated with the programmed combination of compound and IFN-β for 12 h and then were washed twice with cold Dulbecco's PBS followed by lysis with Cells-to-cDNA II lysis buffer (Life Technologies) and treatment with DNase according to the manufacturer's instructions. The U3A cells were obtained from G. Stark (Cleveland Clinic) and complemented with STAT1 to generate U3A-STAT1 cells as described previously
Cells were cultured overnight and then treated with compound and IFN-β for 6 h. Thereafter, cells were washed and then were inoculated with EMCV (strain VR-129B, ATCC, Manassas, VA) for 1 h at MOI 1 as described previously
(EPS)
(EPS)
We thank Dr. Yael Alevy and Ms. Cathy Huang for excellent technical advice and assistance.