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1.  An orally available, small-molecule interferon inhibits viral replication 
Scientific Reports  2012;2:259.
Most acute hepatitis C virus (HCV) infections become chronic and some progress to liver cirrhosis or hepatocellular carcinoma. Standard therapy involves an interferon (IFN)-α-based regimen, and efficacy of therapy has been significantly improved by the development of protease inhibitors. However, several issues remain concerning the injectable form and the side effects of IFN. Here, we report an orally available, small-molecule type I IFN receptor agonist that directly transduces the IFN signal cascade and stimulates antiviral gene expression. Like type I IFN, the small-molecule compound induces IFN-stimulated gene (ISG) expression for antiviral activity in vitro and in vivo in mice, and the ISG induction mechanism is attributed to a direct interaction between the compound and IFN-α receptor 2, a key molecule of IFN-signaling on the cell surface. Our study highlights the importance of an orally active IFN-like agent, both as a therapy for antiviral infections and as a potential IFN substitute.
doi:10.1038/srep00259
PMCID: PMC3277087  PMID: 22355771
2.  High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery 
PLoS ONE  2012;7(5):e36594.
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.
doi:10.1371/journal.pone.0036594
PMCID: PMC3344904  PMID: 22574190
3.  Identification of Small Molecules with Type I Interferon Inducing Properties by High-Throughput Screening 
PLoS ONE  2012;7(11):e49049.
The continuous emergence of virus that are resistant to current anti-viral drugs, combined with the introduction of new viral pathogens for which no therapeutics are available, creates an urgent need for the development of novel broad spectrum antivirals. Type I interferon (IFN) can, by modulating the cellular expression profile, stimulate a non-specific antiviral state. The antiviral and adjuvant properties of IFN have been extensively demonstrated; however, its clinical application has been so far limited. We have developed a human cell-based assay that monitors IFN-β production for use in a high throughput screen. Using this assay we screened 94,398 small molecules and identified 18 compounds with IFN-inducing properties. Among these, 3 small molecules (C3, E51 and L56) showed activity not only in human but also in murine and canine derived cells. We further characterized C3 and showed that this molecule is capable of stimulating an anti-viral state in human-derived lung epithelial cells. Furthermore, the IFN-induction by C3 is not diminished by the presence of influenza A virus NS1 protein or hepatitis C virus NS3/4A protease, which make this molecule an interesting candidate for the development of a new type of broad-spectrum antiviral. In addition, the IFN-inducing properties of C3 also suggest its potential use as vaccine adjuvant.
doi:10.1371/journal.pone.0049049
PMCID: PMC3492183  PMID: 23145065
4.  Inhibition of Interferon-Stimulated JAK-STAT Signaling by a Tick-Borne Flavivirus and Identification of NS5 as an Interferon Antagonist 
Journal of Virology  2005;79(20):12828-12839.
The tick-borne encephalitis (TBE) complex of viruses, genus Flavivirus, can cause severe encephalitis, meningitis, and/or hemorrhagic fevers. Effective interferon (IFN) responses are critical to recovery from infection with flaviviruses, and the mosquito-borne flaviviruses can inhibit this response. However, little is known about interactions between IFN signaling and TBE viruses. Langat virus (LGTV), a member of the TBE complex of viruses, was found to be highly sensitive to the antiviral effects of IFN. However, LGTV infection inhibited IFN-induced expression of a reporter gene driven by either IFN-α/β- or IFN-γ-responsive promoters. This indicated that LGTV can inhibit the IFN-mediated JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway of signal transduction. The mechanism of inhibition was due to blocks in the phosphorylation of both Janus kinases, Jak1 and Tyk2, during IFN-α signaling and at least a failure of Jak1 phosphorylation following IFN-γ stimulation. To determine the viral protein(s) responsible, we individually expressed all nonstructural (NS) proteins and examined their ability to inhibit signal transduction. Expression of NS5 alone inhibited STAT1 phosphorylation in response to IFN, thus identifying NS5 as a potential IFN antagonist. Examination of interactions between NS5 and cellular proteins revealed that NS5 associated with IFN-α/β and -γ receptor complexes. Importantly, inhibition of JAK-STAT signaling and NS5-IFN receptor interactions were demonstrated in LGTV-infected human monocyte-derived dendritic cells, important target cells for early virus replication. Because NS5 may interfere with both innate and acquired immune responses to virus infection, this protein may have a significant role in viral pathogenesis.
doi:10.1128/JVI.79.20.12828-12839.2005
PMCID: PMC1235813  PMID: 16188985
5.  A High Throughput Assay to Identify Small Molecule Modulators of Prostatic Acid Phosphatase 
Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. Injection of the secretory isoform of PAP has potent antinociceptive effects in mouse models of chronic pain. These data suggested that a small molecule activator of PAP may have utility as a novel therapeutic for chronic pain, while inhibitors could be used to acutely inhibit PAP in vitro and in vivo. To identify small molecule modulators of PAP activity, we validated a high throughput, fluorescence-based biochemical assay and then used this assay to screen a compound library. We decreased the frequency of false positive activators by subtracting compound fluorescence from the final assay fluorescence. This approach significantly reduced the number of false positive activators found in the screen. While no activators were confirmed, seven novel inhibitors of PAP were identified. Our results suggest this high throughput assay could be used to identify small molecule modulators of PAP activity.
doi:10.2174/1875397300903010042
PMCID: PMC2808025  PMID: 20161835
6.  miR-19a: An Effective Regulator of SOCS3 and Enhancer of JAK-STAT Signalling 
PLoS ONE  2013;8(7):e69090.
Suppressors of cytokine signalling (SOCS) proteins are classic inhibitors of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Many cytokines and pathogenic mediators induce expression of SOCS, which act in a negative feedback loop to inhibit further signal transduction. SOCS mRNA expression is regulated by DNA binding of STAT proteins, however, their post-transcriptional regulation is poorly understood. microRNAs (miRNAs) are small non-coding RNAs that bind to complementary sequences on target mRNAs, often silencing gene expression. miR-19a has been shown to regulate SOCS1 expression during mutiple myeloma and be induced by the anti-viral cytokine interferon-(IFN)-α, suggesting a role in the regulation of the JAK-STAT pathway. This study aimed to identify targets of miR-19a in the JAK-STAT pathway and elucidate the functional consequences. Bioinformatic analysis identified highly conserved 3’UTR miR-19a target sequences in several JAK-STAT associated genes, including SOCS1, SOCS3, SOCS5 and Cullin (Cul) 5. Functional studies revealed that miR-19a significantly decreased SOCS3 mRNA and protein, while a miR-19a antagomir specifically reversed its inhibitory effect. Furthermore, miR-19a-mediated reduction of SOCS3 enhanced IFN-α and interleukin (IL)-6 signal transduction through STAT3. These results reveal a novel mechanism by which miR-19a may augment JAK-STAT signal transduction via control of SOCS3 expression and are fundamental to the understanding of inflammatory regulation.
doi:10.1371/journal.pone.0069090
PMCID: PMC3718810  PMID: 23894411
7.  IRF9 is a key factor for eliciting the antiproliferative activity of IFN-α 
A number of tumors are still resistant to the antiproliferative activity of human interferon (IFN)-α. The Janus kinases/Signal Transducers and Activators of Transcription (JAK-STAT) pathway plays an important role in initial IFN signaling. In order to enhance the antiproliferative activity of IFN-α, it is important to elucidate which factors in the JAK-STAT pathway play a key role in eliciting this activity. In human ovarian adenocarcinoma OVCAR3 cells sensitive to both IFN-α and -γ, only IFN regulatory factor 9 (IRF9)-RNA interference (RNAi) completely inhibited the antiproliferative activity of IFN-α among the intracellular JAK-STAT pathway factors. Conversely, Stat1-RNAi did not inhibit the antiproliferative activity of IFN-α, while it partially inhibited that of IFN-γ. As a cell death pathway, it is reported that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via TRAIL-R (receptor) 1 and TRAIL-R2. In IFN-α-treated OVCAR3 cells, IRF9-RNAi inhibited transcription of TRAIL while Stat1-RNAi did not, suggesting that the transcription of TRAIL induced by IFN-α predominantly required IRF9. Furthermore, IFN-stimulated response element (ISRE)-like motifs of TRAIL bound to IFN-stimulated gene factor 3 (ISGF3) complex following IFN-α treatment. Subsequently, TRAIL-R2-RNAi inhibited both antiproliferative activities of IFN-α and TRAIL, suggesting that TRAIL-R2 mediated both IFN-α and TRAIL signals to elicit their antiproliferative activities. Finally, IRF9 overexpression facilitated IFN-α-induced apoptosis in T98G (human glioblastoma multiforme) cells, which were resistant to IFN-α. Thus, our present study suggests that IRF9 is the key factor for eliciting the antiproliferative activity of IFN-α and TRAIL may be one of the potential mediators.
doi:10.1097/CJI.0b013e3181ad4092
PMCID: PMC2796296  PMID: 19752753
IFN-α; JAK-STAT pathway; antiproliferative activity; IRF9; TRAIL
8.  Intracellular Delivery of a Cell-Penetrating SOCS1 that Targets IFN-γ Signaling 
Science signaling  2009;2(80):ra37.
Suppressor of cytokine signaling–1 (SOCS1) is an intracellular inhibitor of the Janus kinase–signal transducer and activator of transcription (JAK-STAT) pathway that couples interferon-γ (IFN-γ) signaling to the nucleus. Because several inflammatory diseases are associated with uncontrolled IFN-γ signaling, we engineered a recombinant cell-penetrating SOCS1 (CP-SOCS1) to target this pathway. Here, we show that CP-SOCS1, analogous to endogenous SOCS1, interacted with components of the IFN-γ signaling complex and functionally attenuated the phosphorylation of STAT1, which resulted in the subsequent inhibition of the production of proinflammatory chemokines and cytokines. Thus, controlled, intracellular delivery of recombinant CP-SOCS1 boosted the anti-inflammatory potential of the cell by restoring the homeostatic balance between pro- and anti-inflammatory signaling. This approach to controlling signal transduction has potential use for therapeutic targeting of signaling pathways associated with inflammatory diseases.
doi:10.1126/scisignal.1162191
PMCID: PMC2798805  PMID: 19622834
9.  Dysregulated Sonic hedgehog signaling and medulloblastoma consequent to IFN-α–stimulated STAT2-independent production of IFN-γ in the brain 
Journal of Clinical Investigation  2003;112(4):535-543.
The type I IFNs (IFN-α and IFN-β), which are crucial in antiviral defense and immune regulation, signal via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway with activation of STAT1 and STAT2. Here, the function of STAT2 was studied in transgenic mice (termed GIFN/STAT2–/–) with CNS production of IFN-α. Surprisingly, GIFN/STAT2–/–, but not GIFN/STAT1-null, transgenic mice, with CNS production of IFN-α, died prematurely with medulloblastoma. An immune response also induced in the brain of the GIFN/STAT2–/– mice was associated with IFN-γ gene expression by CD3+ T cells and the activation of the STAT1, STAT3, STAT4, and STAT5 molecules. Expression of the Sonic hedgehog (Shh) and the downstream transcriptional factor Gli-1 genes, implicated in the pathogenesis of medulloblastoma, was found to be significantly increased and cotranscribed in cerebellar granule neurons of the GIFN/STAT2–/– mice. IFN-γ, but not IFN-α, induced STAT1-dependent expression of the Shh gene in cultured cerebellar granule neurons. Thus, there is an unexpected and extraordinarily adverse biological potency of IFN-α in the CNS when the primary signal transduction molecule STAT2 is absent. Moreover, a hitherto unknown role is indicated for the immune system in the pathogenesis of developmental disorders and tumorigenesis of the CNS via dysregulated Shh signaling mediated by IFN-γ.
doi:10.1172/JCI200318637
PMCID: PMC171394  PMID: 12925694
10.  6-Bromoindirubin-3'-oxime inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma cells 
Cancer research  2011;71(11):3972-3979.
Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated and contributes to malignant progression in various cancers. Janus kinases (JAKs) phosphorylate STAT3 in response to stimulation with cytokines or growth factors. The STAT3 signaling pathway has been validated as a promising target for development of anti-cancer therapeutics. Small-molecule inhibitors of JAK/STAT3 signaling represent potential molecular-targeted cancer therapeutic agents. In this study, we investigated the role of JAK/STAT3 signaling in 6-bromoindirubin-3'-oxime (6BIO) mediated growth inhibition of human melanoma cells and assessed 6BIO as an anticancer drug candidate. We found that 6BIO is a pan-JAK inhibitor that induced apoptosis of human melanoma cells. 6BIO directly inhibited JAK family kinase activity both in vitro and in cancer cells. Apoptosis of human melanoma cells induced by 6BIO was associated with reduced phosphorylation of JAKs and STAT3 in both a dose- and time-dependent manners. Consistent with inhibition of STAT3 signaling, the anti-apoptotic protein Mcl-1 was down-regulated. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the AKT and MAPK signaling proteins were not inhibited in cells treated with 6BIO. Importantly, 6BIO suppressed tumor growth in vivo with low toxicity in a mouse xenograft model of melanoma. Taken together, these results demonstrate that 6BIO is a novel pan-JAK inhibitor that can selectively inhibit STAT3 signaling and induced tumor cell apoptosis. Our findings support further development of 6BIO as a potential anti-cancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.
doi:10.1158/0008-5472.CAN-10-3852
PMCID: PMC3107399  PMID: 21610112
bromoindirubin; JAK inhibitor; STAT3 signaling; apoptosis; melanoma
11.  Blocking of the Alpha Interferon-Induced Jak-Stat Signaling Pathway by Japanese Encephalitis Virus Infection 
Journal of Virology  2004;78(17):9285-9294.
The induction of alpha/beta interferon (IFN-α/β) is a powerful host defense mechanism against viral infection, and many viruses have evolved strategies to overcome the antiviral effects of IFN. In this study, we found that IFN-α had only some degree of antiviral activity against Japanese encephalitis virus (JEV) infection, in contrast to another flavivirus, dengue virus serotype 2, which was highly sensitive to IFN-α in the cultured cell system. JEV infection appeared to render cells resistant to IFN-α since the IFN-α-induced luciferase reporter activity driven by the IFN-stimulated response element (ISRE) was gradually reduced as the JEV infection progressed. Since the biological activities of IFNs are triggered by the Janus kinase (Jak) signal transducer and activation of transcription (Stat) signaling cascade, we then studied the activation of Jak-Stat pathway in the virus-infected cells. The IFN-α-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 was suppressed by JEV in a virus replication and de novo protein synthesis-dependent manner. Furthermore, JEV infection blocked the tyrosine phosphorylation of IFN receptor-associated Jak kinase, Tyk2, without affecting the expression of IFN-α/β receptor on the cell surface. Consequently, expression of several IFN-stimulated genes in response to IFN-α stimulation was also reduced in the JEV-infected cells. Overall, our findings suggest that JEV counteracts the effect of IFN-α/β by blocking Tyk2 activation, thereby resulting in inhibition of Jak-Stat signaling pathway.
doi:10.1128/JVI.78.17.9285-9294.2004
PMCID: PMC506928  PMID: 15308723
12.  A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon 
PLoS Pathogens  2012;8(4):e1002668.
Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.
Author Summary
Influenza viruses are rapidly developing resistance against available anti-influenza drugs and consequently there is an urgent demand for new treatment approaches. We identified compound ASN2 in a high-throughput screen for molecules that are capable of inducing the antiviral cytokine interferon (IFN) in the presence of influenza virus infection. Normally, influenza virus blocks IFN production, an activity that is dependent on the viral NS1 protein and contributes to the ability of the virus to cause disease in an infected host. We show that ASN2 is a potent inhibitor of influenza A virus and can partially protect infected animals from disease and death. ASN2 acts by targeting influenza virus polymerase function which results in inhibition of virus replication, and as a consequence, NS1 expression. Thus the ability of ASN2 to induce IFN is a “side-effect”, albeit a desirable one, of polymerase inhibition. This combination of directly inhibiting the virus while also stimulating the host immune response is a novel property for an antiviral compound.
doi:10.1371/journal.ppat.1002668
PMCID: PMC3343121  PMID: 22577360
13.  Gamma Interferon-Induced Interferon Regulatory Factor 1-Dependent Antiviral Response Inhibits Vaccinia Virus Replication in Mouse but Not Human Fibroblasts▿  
Journal of Virology  2009;83(8):3684-3695.
Vaccinia virus (VACV) replicates in mouse and human fibroblasts with comparable kinetics and efficiency, yielding similar titers of infectious progeny. Here we demonstrate that gamma interferon (IFN-γ) but not IFN-α or IFN-β pretreatment of mouse fibroblasts prior to VACV infection induces a long-lasting antiviral state blocking VACV replication. In contrast, high doses of IFN-γ failed to establish an antiviral state in human fibroblasts. In mouse fibroblasts, IFN-γ impeded the viral replication cycle at the level of late gene transcription and blocked the multiplication of VACV genomes. The IFN-γ-induced antiviral state invariably prevented the growth of different VACV strains but was not effective against the replication of ectromelia virus. The IFN-γ effect required intact IFN-γ receptor signaling prior to VACV infection through Janus kinase 2 (Jak2) and signal transducer and activator of transcription 1 (STAT1). The permissive state of IFN-γ-treated human cells was unrelated to the VACV-encoded IFN decoy receptors B8 and B18 and associated with a complete disruption of STAT1 homodimer formation and DNA binding. Unlike human fibroblasts, mouse cells responded with long-lasting STAT1 activation which was preserved after VACV infection. The deletion of the IFN regulatory factor 1 (IRF-1) gene from mouse cells rescued efficient VACV replication, demonstrating that IRF-1 target genes have a critical role in VACV control. These data have implications for the understanding of VACV pathogenesis and identify an incongruent IFN-γ response between the human host and the mouse model.
doi:10.1128/JVI.02042-08
PMCID: PMC2663247  PMID: 19211768
14.  Inhibition of Jak1-Dependent Signal Transduction in Airway Epithelial Cells Infected with Adenovirus 
Adenoviral evolution has generated mechanisms to resist host cell defense systems, but the biochemical basis for evasion of multiple antiviral pathways in the airway by adenoviruses is incompletely understood. We hypothesized that adenoviruses modulate airway epithelial responses to type I interferons by altering the levels and activation of specific Janus family kinase-signal transducer and activator of transcription (JAK-STAT) signaling components. In this study, specific effects of adenovirus type 5 (AdV) on selected JAK-STAT signal transduction pathways were identified in human tracheobronchial epithelial cells, with focus on type I interferon–dependent signaling and gene expression. We found that wild-type AdV infection inhibited IFN-α–induced expression of antiviral proteins in epithelial cells by blocking phosphorylation of the Stat1 and Stat2 transcription factors that are required for activation of type I interferon–dependent genes. These effects correlated with AdV-induced down-regulation of expression of the receptor-associated tyrosine kinase Jak1 through a decrease in Jak1 mRNA levels. Phosphorylation of Stat3 in response to IL-6 and oncostatin M was also lost in AdV-infected cells, indicating loss of epithelial cell responses to other cytokines that depend on Jak1. In contrast, IL-4– and IL-13–dependent phosphorylation of Stat6 was not affected during AdV infection, indicating that the virus modulates specific signaling pathways, as these Stat6-activating pathways can function independent of Jak1. Taken together, the results indicate that AdV down-regulates host epithelial cell Jak1 to assure inhibition of the antiviral effects of multiple mediators to subvert airway defense responses and establish a productive infection.
doi:10.1165/rcmb.2007-0158OC
PMCID: PMC2219548  PMID: 17641294
JAK-STAT signaling; interferon; interleukin
15.  JAK2 mutants (e.g., JAK2V617F) and their importance as drug targets in myeloproliferative neoplasms 
JAK-STAT  2013;2(3):e25025.
The Janus kinase 2 (JAK2) mutant V617F and other JAK mutants are found in patients with myeloproliferative neoplasms and leukemias. Due to their involvement in neoplasia and inflammatory disorders, Janus kinases are promising targets for kinase inhibitor therapy. Several small-molecule compounds are evaluated in clinical trials for myelofibrosis, and ruxolitinib (INCB018424, Jakafi®) was the first Janus kinase inhibitor to receive clinical approval. In this review we provide an overview of JAK2V617F signaling and its inhibition by small-molecule kinase inhibitors. In addition, myeloproliferative neoplasms are discussed regarding the role of JAK2V617F and other mutant proteins of possible relevance. We further give an overview about treatment options with special emphasis on possible combination therapies.
doi:10.4161/jkst.25025
PMCID: PMC3772115  PMID: 24069563
JAK2V617F; myeloproliferative neoplasms; polycythemia vera; essential thrombocythemia; primary myelofibrosis
16.  Identification of a Novel Sulfonamide Non-Nucleoside Reverse Transcriptase Inhibitor by a Phenotypic HIV-1 Full Replication Assay 
PLoS ONE  2013;8(7):e68767.
Classical target-based, high-throughput screening has been useful for the identification of inhibitors for known molecular mechanisms involved in the HIV life cycle. In this study, the development of a cell-based assay that uses a phenotypic drug discovery approach based on automated high-content screening is described. Using this screening approach, the antiviral activity of 26,500 small molecules from a relevant chemical scaffold library was evaluated. Among the selected hits, one sulfonamide compound showed strong anti-HIV activity against wild-type and clinically relevant multidrug resistant HIV strains. The biochemical inhibition, point resistance mutations and the activity of structural analogs allowed us to understand the mode of action and propose a binding model for this compound with HIV-1 reverse transcriptase.
doi:10.1371/journal.pone.0068767
PMCID: PMC3715523  PMID: 23874756
17.  A Quantitative High-Throughput Screen for Modulators of IL-6 Signaling: A Model for Interrogating Biological Networks using Chemical Libraries 
Molecular bioSystems  2009;5(9):1039-1050.
Small molecule modulators are critical for dissecting and understanding signaling pathways at the molecular level. Interleukin 6 (IL-6) is a cytokine that signals via the JAK/STAT pathway and is implicated in cancer and inflammation. To identify modulators of this pathway, we screened a chemical collection against an IL-6 responsive cell line stably expressing a beta-lactamase reporter gene fused to a sis-inducible element (SIE-bla cells). This assay was optimized for a 1536-well microplate format and screened against 11,693 small molecules using quantitative high-throughput screening (qHTS), a method that assays a chemical library at multiple concentrations to generate titration-response profiles for each compound. The qHTS recovered 564 actives with well-fit curves that clustered into 32 distinct chemical series of 13 activators and 19 inhibitors. A retrospective analysis of the qHTS data indicated that single concentration data at 1.5 and 7.7 uM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and nonselective series, we found four activator and one inhibitor series that modulated SIE-bla cells but did not show similar activity in reporter gene assays induced by EGF and hypoxia. Small molecules within these series will make useful tool compounds to investigate IL-6 signaling mediated by JAK/STAT activation.
doi:10.1039/b902021g
PMCID: PMC2747079  PMID: 19668870
IL-6; small molecule; HTS; STAT; assay
18.  Simulating Henipavirus Multicycle Replication in a Screening Assay Leads to Identification of a Promising Candidate for Therapy▿ †  
Journal of Virology  2009;83(10):5148-5155.
Nipah (NiV) and Hendra (HeV) viruses are emerging zoonotic paramyxoviruses that cause encephalitis in humans, with fatality rates of up to 75%. We designed a new high-throughput screening (HTS) assay for inhibitors of infection based on envelope glycoprotein pseudotypes. The assay simulates multicycle replication and thus identifies inhibitors that target several stages of the viral life cycle, but it still can be carried out under biosafety level 2 (BSL-2) conditions. These features permit a screen for antivirals for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. The screening of a small compound library identified several effective molecules, including the well-known compound chloroquine, as highly active inhibitors of pseudotyped virus infection. Chloroquine inhibited infection with live HeV and NiV at a concentration of 1 μM in vitro (50% inhibitory concentration, 2 μM), which is less than the plasma concentrations present in humans receiving chloroquine treatment for malaria. The mechanism for chloroquine's antiviral action likely is the inhibition of cathepsin L, a cellular enzyme that is essential for the processing of the viral fusion glycoprotein and the maturation of newly budding virions. Without this processing step, virions are not infectious. The identification of a compound that inhibits a known cellular target that is important for viral maturation but that had not previously been shown to have antiviral activity for henipaviruses highlights the validity of this new screening assay. Given the established safety profile and broad experience with chloroquine in humans, the results described here provide an option for treating individuals infected by these deadly viruses.
doi:10.1128/JVI.00164-09
PMCID: PMC2682105  PMID: 19264786
19.  High-Throughput Screening Identifies a Bisphenol Inhibitor of SV40 Large T Antigen ATPase Activity 
Journal of biomolecular screening  2011;17(2):194-203.
The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose–response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC50 of 41 μM in the primary assay and 6.2 μM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.
doi:10.1177/1087057111421630
PMCID: PMC3731742  PMID: 21948801
SV40; chemistry; large T antigen; HTS; antivirals
20.  AV-65, a novel Wnt/β-catenin signal inhibitor, successfully suppresses progression of multiple myeloma in a mouse model 
Blood Cancer Journal  2011;1(11):e43-.
Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Although new molecular targeting agents against MM have been developed based on the better understanding of the underlying pathogenesis, MM still remains an incurable disease. We previously demonstrated that β-catenin, a downstream effector in the Wnt pathway, is a potential target in MM using RNA interference in an in vivo experimental mouse model. In this study, we have screened a library of more than 100 000 small-molecule chemical compounds for novel Wnt/β-catenin signaling inhibitors using a high-throughput transcriptional screening technology. We identified AV-65, which diminished β-catenin protein levels and T-cell factor transcriptional activity. AV-65 then decreased c-myc, cyclin D1 and survivin expression, resulting in the inhibition of MM cell proliferation through the apoptotic pathway. AV-65 treatment prolonged the survival of MM-bearing mice. These findings indicate that this compound represents a novel and attractive therapeutic agent against MM. This study also illustrates the potential of high-throughput transcriptional screening to identify candidates for anticancer drug discovery.
doi:10.1038/bcj.2011.41
PMCID: PMC3256754  PMID: 22829079
multiple myeloma; Wnt; β-catenin; high-throughput screening
21.  JAK Inhibitors AG-490 and WHI-P154 Decrease IFN-γ-Induced iNOS Expression and NO Production in Macrophages 
Mediators of Inflammation  2006;2006(2):16161.
In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-γ (IFN-γ). JAK inhibitors AG-490 and WHI-P154 decreased IFN-γ-induced nuclear levels of signal transducer and activator of transcription 1α (STAT1α). JAK inhibitors AG-490 and WHI-P154 decreased also iNOS protein and mRNA expression and NO production in a concentration-dependent manner. Neither of the JAK inhibitors affected the decay of iNOS mRNA when determined by actinomycin D assay. Our results suggest that the inhibition of JAK-STAT1-pathway by AG-490 or WHI-P154 leads to the attenuation of iNOS expression and NO production in IFN-γ-stimulated macrophages.
doi:10.1155/MI/2006/16161
PMCID: PMC1592588  PMID: 16883061
22.  Reprogramming of the Macrophage Transcriptome in Response to Interferon-γ and Mycobacterium tuberculosis 
The Journal of Experimental Medicine  2001;194(8):1123-1140.
Macrophage activation determines the outcome of infection by Mycobacterium tuberculosis (Mtb). Interferon-γ (IFN-γ) activates macrophages by driving Janus tyrosine kinase (JAK)/signal transducer and activator of transcription–dependent induction of transcription and PKR-dependent suppression of translation. Microarray-based experiments reported here enlarge this picture. Exposure to IFN-γ and/or Mtb led to altered expression of 25% of the monitored genome in macrophages. The number of genes suppressed by IFN-γ exceeded the number of genes induced, and much of the suppression was transcriptional. Five times as many genes related to immunity and inflammation were induced than suppressed. Mtb mimicked or synergized with IFN-γ more than antagonized its actions. Phagocytosis of nonviable Mtb or polystyrene beads affected many genes, but the transcriptional signature of macrophages infected with viable Mtb was distinct. Studies involving macrophages deficient in inducible nitric oxide synthase and/or phagocyte oxidase revealed that these two antimicrobial enzymes help orchestrate the profound transcriptional remodeling that underlies macrophage activation.
PMCID: PMC2193509  PMID: 11602641
gene expression; microarray analysis; macrophage activation; innate immunity; phagocytosis
23.  Complementary Genomic Screens Identify SERCA as a Therapeutic Target in NOTCH1 Mutated Cancer 
Cancer cell  2013;23(3):390-405.
SUMMARY
Notch1 is a rational therapeutic target in several human cancers, but as a transcriptional regulator, it poses a drug discovery challenge. To identify Notch1 modulators, we performed two cell-based, high-throughput screens for small-molecule inhibitors and cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. SERCA calcium channels emerged at the intersection of these complementary screens. SERCA inhibition preferentially impairs the maturation and activity of mutated Notch1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human leukemia cells. A small-molecule SERCA inhibitor has on-target activity in two mouse models of human leukemia and interferes with Notch signaling in Drosophila. These studies “credential” SERCA as a therapeutic target in cancers associated with NOTCH1 mutations.
doi:10.1016/j.ccr.2013.01.015
PMCID: PMC3709972  PMID: 23434461
24.  Gene expression-based screening for inhibitors of PDGFR signaling 
Genome Biology  2008;9(3):R47.
Inhibitors of the platelet derived growth factor receptor (PDGFR) signaling pathway are isolated using gene expression-based high-throughput screening (GE-HTS), a method that is applicable to other pathways.
Here we describe a proof-of-concept experiment designed to explore the possibility of using gene expression-based high-throughput screening (GE-HTS) to find inhibitors of a signaling cascade, using platelet derived growth factor receptor (PDGFR) signaling as the example. The previously unrecognized ability of aurintricarboxylic acid to inhibit PDGFR signaling, discovered through a screen of 1,739 compounds, demonstrates the feasibility and generalizability of GE-HTS for the discovery of small molecule modulators of any signaling pathway of interest.
doi:10.1186/gb-2008-9-3-r47
PMCID: PMC2397499  PMID: 18312689
25.  Structurally Modified Curcumin Analogs Inhibit STAT3 Phosphorylation and Promote Apoptosis of Human Renal Cell Carcinoma and Melanoma Cell Lines 
PLoS ONE  2012;7(8):e40724.
The Janus kinase-2 (Jak2)-signal transducer and activator of transcription-3 (STAT3) pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC) and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3), and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma.
doi:10.1371/journal.pone.0040724
PMCID: PMC3416819  PMID: 22899991

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