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1.  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.
PMCID: PMC3343121  PMID: 22577360
2.  Safety, Tolerability, and Immunogenicity of Interferons 
Pharmaceuticals  2010;3(4):1162-1186.
Interferons (IFNs) are class II cytokines that are key components of the innate immune response to virus infection. Three IFN sub-families, type I, II, and III IFNs have been identified in man, Recombinant analogues of type I IFNs, in particular IFNα2 and IFNβ1, have found wide application for the treatment of chronic viral hepatitis and remitting relapsing multiple sclerosis respectively. Type II IFN, or IFN gamma, is used principally for the treatment of chronic granulomatous disease, while the recently discovered type III IFNs, also known as IFN lambda or IL-28/29, are currently being evaluated for the treatment of chronic viral hepatitis. IFNs are in general well tolerated and the most common adverse events observed with IFNα or IFNβ therapy are “flu-like” symptoms such as fever, headache, chills, and myalgia. Prolonged treatment is associated with more serious adverse events including leucopenia, thrombocytopenia, increased hepatic transaminases, and neuropsychiatric effects. Type I IFNs bind to high-affinity cell surface receptors, composed of two transmembrane polypeptides IFNAR1 and IFNAR2, resulting in activation of the Janus kinases Jak1 and Tyk2, phosphorylation and activation of the latent cytoplasmic signal transducers and activators of transcription (STAT1) and STAT2, formation of a transcription complex together with IRF9, and activation of a specific set of genes that encode the effector molecules responsible for mediating the biological activities of type I IFNs. Systemic administration of type I IFN results in activation of IFN receptors present on essentially all types of nucleated cells, including neurons and hematopoietic stem cells, in addition to target cells. This may well explain the wide spectrum of IFN associated toxicities. Recent reports suggest that certain polymorphisms in type I IFN signaling molecules are associated with IFN-induced neutropenia and thrombocytopenia in patients with chronic hepatitis C. IFNγ binds to a cell-surface receptor composed of two transmembrane polypeptides IFGR1 and IFGR2 resulting in activation of the Janus kinases Jak1 and Jak2, phosphorylation of STAT1, formation of STAT1 homodimers, and activation of a specific set of genes that encode the effector molecules responsible for mediating its biological activity. In common with type I IFNs, IFNγ receptors are ubiquitous and a number of the genes activated by IFNγ are also activated by type I IFNs that may well account for a spectrum of toxicities similar to that associated with type I IFNs including “flu-like” symptoms, neutropenia, thrombocytopenia, and increased hepatic transaminases. Although type III IFNs share the major components of the signal transduction pathway and activate a similar set of IFN-stimulated genes (ISGs) as type I IFNs, distribution of the IFNλ receptor is restricted to certain cell types suggesting that IFNλ therapy may be associated with a reduced spectrum of toxicities relative to type I or type II IFNs. Repeated administration of recombinant IFNs can cause in a break in immune tolerance to self-antigens in some patients resulting in the production of neutralizing antibodies (NABs) to the recombinant protein homologue. Appearance of NABs is associated with reduced pharmacokinetics, pharmacodynamics, and a reduced clinical response. The lack of cross-neutralization of IFNβ by anti-IFNα NABs and vice versa, undoubtedly accounts for the apparent lack of toxicity associated with the presence of anti-IFN NABs with the exception of relatively mild infusion/injection reactions.
PMCID: PMC4034027
cytokines; interferons; interleukins; innate immunity; Toll-like receptors
3.  Dual Modulation of Type I Interferon Response by Bluetongue Virus 
Journal of Virology  2014;88(18):10792-10802.
Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus that causes an economically important disease in ruminants. BTV infection is a strong inducer of type I interferon (IFN-I) in multiple cell types. It has been shown recently that BTV and, more specifically, the nonstructural protein NS3 of BTV are able to modulate the IFN-I synthesis pathway. However, nothing is known about the ability of BTV to counteract IFN-I signaling. Here, we investigated the effect of BTV on the IFN-I response pathway and, more particularly, the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. We found that BTV infection triggered the expression of IFN-stimulated genes (ISGs) in A549 cells. However, when BTV-infected cells were stimulated with external IFN-I, we showed that activation of the IFN-stimulated response element (ISRE) promoter and expression of ISGs were inhibited. We found that this inhibition involved two different mechanisms that were dependent on the time of infection. After overnight infection, BTV blocked specifically the phosphorylation and nuclear translocation of STAT1. This inhibition correlated with the redistribution of STAT1 in regions adjacent to the nucleus. At a later time point of infection, BTV was found to interfere with the activation of other key components of the JAK/STAT pathway and to induce the downregulation of JAK1 and TYK2 protein expression. Overall, our study indicates for the first time that BTV is able to interfere with the JAK/STAT pathway to modulate the IFN-I response.
IMPORTANCE Bluetongue virus (BTV) causes a severe disease in ruminants and has an important impact on the livestock economy in areas of endemicity such as Africa. The emergence of strains, such as serotype 8 in Europe in 2006, can lead to important economic losses due to commercial restrictions and prophylactic measures. It has been known for many years that BTV is a strong inducer of type I interferon (IFN-I) in vitro and in vivo in multiple cell types. However, the ability of BTV to interact with the IFN-I system remains unclear. Here, we report that BTV is able to modulate the IFN-I response by interfering with the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. These findings contribute to knowledge of how BTV infection interferes with the host's innate immune response and becomes pathogenic. This will also be important for the design of efficacious vaccine candidates.
PMCID: PMC4178850  PMID: 25008919
4.  Influenza Virus Non-Structural Protein 1 (NS1) Disrupts Interferon Signaling 
PLoS ONE  2010;5(11):e13927.
Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.
PMCID: PMC2978095  PMID: 21085662
5.  An interferon-beta promoter reporter assay for high throughput identification of compounds against multiple RNA viruses 
Antiviral research  2014;107:56-65.
Virus infection of host cells is sensed by innate pattern recognition receptors (PRRs) and induces production of type I interferons (IFNs) and other inflammatory cytokines. These cytokines orchestrate the elimination of the viruses but are occasionally detrimental to the hosts. The outcomes and pathogenesis of viral infection are largely determined by the specific interaction between the viruses and their host cells. Therefore, compounds that either inhibit viral infection or modulate virus-induced cytokine response should be considered as candidates for managing virus infection. The aim of the study was to identify compounds in both categories, using a single cell-based assay. Our screening platform is a HEK293 cell-based reporter assay where the expression of a firefly luciferase is under the control of a human IFN-β promoter. We have demonstrated that infection of the reporter cell line with a panel of RNA viruses activated the reporter gene expression that correlates quantitatively with the levels of virus replication and progeny virus production, and could be inhibited in a dose-dependent manner by known antiviral compound or inhibitors of PRR signal transduction pathways. Using Dengue virus as an example, a pilot screening of a small molecule library consisting of 26,900 compounds proved the concept that the IFN-β promoter reporter assay can serve as a convenient high throughput screening platform for simultaneous discovery of antiviral and innate immune response modulating compounds. A representative antiviral compound from the pilot screening, 1-(6-ethoxybenzo[d]thiazol-2-yl)-3-(3-methoxyphenyl) urea, was demonstrated to specifically inhibit several viruses belonging to the family of flaviviridae.
PMCID: PMC4143146  PMID: 24792753
high throughput assay; antiviral; innate immune modulator; dengue virus
6.  Type III Interferon Induces Distinct SOCS1 Expression Pattern that Contributes to Delayed but Prolonged Activation of Jak/STAT Signaling Pathway: Implications for Treatment Non-Response in HCV Patients 
PLoS ONE  2015;10(7):e0133800.
Suppressor of cytokine signaling 1 (SOCS1) has long been thought to block type I interferon signaling. However, IFN-λ, a type III IFN with limited receptor expression in hepatic cells, efficiently inhibits HCV (Hepatitis C virus) replication in vivo with potentially less side effects than IFN-α. Previous studies demonstrated that type I and type III activated Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway differently, with delayed but prolonged activation by IFN-λ stimulation compared to IFNα/β. However, the molecular mechanisms underlying this observation is not well understood. Here, we found that there are distinct differences in SOCS1 expression patterns in Huh-7.5.1 cells following stimulation with IFN-α and IFN-λ. IFN-λ induced a faster but shorter expression of SOCS1. Furthermore, we confirmed that SOCS1 over-expression abrogates anti-HCV effect of both IFN-α and IFN-λ, leading to increased HCV RNA replication in both HCV replicon cells and JFH1 HCV culture system. In line with this, SOCS1 over-expression inhibited STAT1 phosphorylation, attenuated IFN-stimulated response elements (ISRE) reporter activity, and blocked IFN-stimulated genes (ISGs) expression. Finally, we measured SOCS1 mRNA expression levels in peripheral blood mononuclear cells (PBMCs) with or without IFN-α treatment from 48 chronic hepatitis C patients and we found the baseline SOCS1 expression levels are higher in treatment non-responders than in responders before IFN-α treatment. Taken together, SOCS1 acts as a suppressor for both type I and type III IFNs and is negatively associated with sustained virological response (SVR) to IFN-based therapy in patients with HCV. More importantly, faster but shorter induction of SOCS1 by IFN-λ may contribute to delayed but prolonged activation of IFN signaling and ISG expression kinetics by type III IFN.
PMCID: PMC4508043  PMID: 26193702
7.  Alpha/Beta Interferon and Gamma Interferon Synergize To Inhibit the Replication of Herpes Simplex Virus Type 1 
Journal of Virology  2002;76(22):11541-11550.
In vivo evidence suggests that T-cell-derived gamma interferon (IFN-γ) can directly inhibit the replication of herpes simplex virus type 1 (HSV-1). However, IFN-γ is a weak inhibitor of HSV-1 replication in vitro. We have found that IFN-γ synergizes with the innate IFNs (IFN-α and -β) to potently inhibit HSV-1 replication in vitro and in vivo. Treatment of Vero cells with either IFN-β or IFN-γ inhibits HSV-1 replication by <20-fold, whereas treatment with both IFN-β and IFN-γ inhibits HSV-1 replication by ∼1,000-fold. Treatment with IFN-β and IFN-γ does not prevent HSV-1 entry into Vero cells, and the inhibitory effect can be overcome by increasing the multiplicity of HSV-1 infection. The capacity of IFN-β and IFN-γ to synergistically inhibit HSV-1 replication is not virus strain specific and has been observed in three different cell types. For two of the three virus strains tested, IFN-β and IFN-γ inhibit HSV-1 replication with a potency that approaches that achieved by a high dose of acyclovir. Pretreatment of mouse eyes with IFN-β and IFN-γ reduces HSV-1 replication to nearly undetectable levels, prevents the development of disease, and reduces the latent HSV-1 genome load per trigeminal ganglion by ∼200-fold. Thus, simultaneous activation of IFN-α/β receptors and IFN-γ receptors appears to render cells highly resistant to the replication of HSV-1. Because IFN-α or IFN-β is produced by most cells as an innate response to virus infection, the results imply that IFN-γ secreted by T cells may provide a critical second signal that potently inhibits HSV-1 replication in vivo.
PMCID: PMC136787  PMID: 12388715
8.  Effect of ethanol on innate antiviral pathways and HCV replication in human liver cells 
Virology Journal  2005;2:89.
Alcohol abuse reduces response rates to IFN therapy in patients with chronic hepatitis C. To model the molecular mechanisms behind this phenotype, we characterized the effects of ethanol on Jak-Stat and MAPK pathways in Huh7 human hepatoma cells, in HCV replicon cell lines, and in primary human hepatocytes. High physiological concentrations of acute ethanol activated the Jak-Stat and p38 MAPK pathways and inhibited HCV replication in several independent replicon cell lines. Moreover, acute ethanol induced Stat1 serine phosphorylation, which was partially mediated by the p38 MAPK pathway. In contrast, when combined with exogenously applied IFN-α, ethanol inhibited the antiviral actions of IFN against HCV replication, involving inhibition of IFN-induced Stat1 tyrosine phosphorylation. These effects of alcohol occurred independently of i) alcohol metabolism via ADH and CYP2E1, and ii) cytotoxic or cytostatic effects of ethanol. In this model system, ethanol directly perturbs the Jak-Stat pathway, and HCV replication.
Infection with Hepatitis C virus is a significant cause of morbidity and mortality throughout the world. With a propensity to progress to chronic infection, approximately 70% of patients with chronic viremia develop histological evidence of chronic liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The situation is even more dire for patients who abuse ethanol, where the risk of developing end stage liver disease is significantly higher as compared to HCV patients who do not drink [1,2].
Recombinant interferon alpha (IFN-α) therapy produces sustained responses (ie clearance of viremia) in 8–12% of patients with chronic hepatitis C [3]. Significant improvements in response rates can be achieved with IFN plus ribavirin combination [4-6] and pegylated IFN plus ribavirin [7,8] therapies. However, over 50% of chronically infected patients still do not clear viremia. Moreover, HCV-infected patients who abuse alcohol have extremely low response rates to IFN therapy [9], but the mechanisms involved have not been clarified.
MAPKs play essential roles in regulation of differentiation, cell growth, and responses to cytokines, chemokines and stress. The core element in MAPK signaling consists of a module of 3 kinases, named MKKK, MKK, and MAPK, which sequentially phosphorylate each other [10]. Currently, four MAPK modules have been characterized in mammalian cells: Extracellular Regulated Kinases (ERK1 and 2), Stress activated/c-Jun N terminal kinase (SAPK/JNK), p38 MAP kinases, and ERK5 [11]. Interestingly, ethanol modulates MAPKs [12]. However, information on how ethanol affects MAPKs in the context of innate antiviral pathways such as the Jak-Stat pathway in human cells is extremely limited.
When IFN-α binds its receptor, two receptor associated tyrosine kinases, Tyk2 and Jak1 become activated by phosphorylation, and phosphorylate Stat1 and Stat2 on conserved tyrosine residues [13]. Stat1 and Stat2 combine with the IRF-9 protein to form the transcription factor interferon stimulated gene factor 3 (ISGF-3), which binds to the interferon stimulated response element (ISRE), and induces transcription of IFN-α-induced genes (ISG). The ISGs mediate the antiviral effects of IFN. The transcriptional activities of Stats 1, 3, 4, 5a, and 5b are also regulated by serine phosphorylation [14]. Phosphorylation of Stat1 on a conserved serine amino acid at position 727 (S727), results in maximal transcriptional activity of the ISGF-3 transcription factor complex [15]. Although cross-talk between p38 MAPK and the Jak-Stat pathway is essential for IFN-induced ISRE transcription, p38 does not participate in IFN induction of Stat1 serine phosphorylation [14,16-19]. However, cellular stress responses induced by stimuli such as ultraviolet light do induce p38 MAPK mediated Stat1 S727 phosphorylation [18].
In the current report, we postulated that alcohol and HCV proteins modulate MAPK and Jak-Stat pathways in human liver cells. To begin to address these issues, we characterized the interaction of acute ethanol on Jak-Stat and MAPK pathways in Huh7 cells, HCV replicon cells lines, and primary human hepatocytes.
PMCID: PMC1318489  PMID: 16324217
HCV; IFN; virus-host interactions; signal transduction; alcohol
9.  Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen 
ACS Chemical Biology  2012;7(4):715-722.
TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.
PMCID: PMC3331904  PMID: 22260433
10.  Modulation of Gamma Interferon-Induced Major Histocompatibility Complex Class II Gene Expression by Porphyromonas gingivalis Membrane Vesicles 
Infection and Immunity  2002;70(3):1185-1192.
Gamma interferon (IFN-γ)-induced endothelial cells actively participate in initiating immune responses by interacting with CD4+ T cells via class II major histocompatibility complex (MHC) surface glycoproteins. Previously, Porphyromonas gingivalis membrane vesicles were shown to selectively inhibit IFN-γ-induced surface expression of HLA-DR molecules by human umbilical cord vascular endothelial cells. In this study, we demonstrated an absence of HLA-DRα mRNA from IFN-γ-induced cells in the presence of P. gingivalis membrane vesicles by using reverse transcriptase-PCR and Southern blotting. Vesicles also prevented transcription of the gene encoding class II transactivator, a transactivator protein required for IFN-γ-induced expression of MHC class II genes. In addition, the effects of vesicles on IFN-γ signal transduction involving Jak and Stat proteins were characterized by using immunoprecipitation and Western blot analyses. Jak1 and Jak2 proteins could not be detected in endothelial cells treated with membrane vesicles. Consequently, IFN-γ-induced phosphorylation of Jak1, Jak2, and Stat1α proteins was prevented. The class II-inhibitory effect of the membrane vesicles could be eliminated by heating vesicles at 100°C for 30 min or by treating them with a cysteine proteinase inhibitor. This indicates that the cysteine proteinases were most likely responsible for the absence of Jak proteins observed in vesicle-treated cells. The observed increased binding of radiolabeled IFN-γ to vesicle-treated cells suggests that vesicles may also modulate the IFN-γ interactions with the cell surface. However, no evidence was obtained demonstrating that vesicles affected the expression of IFN-γ receptors. Thus, P. gingivalis membrane vesicles apparently inhibited IFN-γ-induced MHC class II by disrupting the IFN-γ signaling transduction pathway. Vesicle-inhibited class II expression also occurred in other IFN-γ-inducible cells. This suggested that the ability of P. gingivalis membrane vesicles to modulate antigen presentation by key cells may be an important mechanism used by this particular bacterium to escape immunosurveillance, thereby favoring its colonization and invasion of host tissues.
PMCID: PMC127778  PMID: 11854199
11.  Inhibition of the signalling kinase JAK3 alleviates inflammation in monoarthritic rats 
British Journal of Pharmacology  2011;164(1):106-118.
Many cytokines associated with autoimmune disorders and inflammation have been shown to activate the signalling kinase JAK3, implying that JAK3 plays key roles in the pathogenesis of these diseases. Therefore, investigating the alterations of JAK3 activity and the efficacy of selective JAK3 antagonists in animal models of such disorders is essential to a better understanding of the biology of JAK3 and to assess the potential clinical benefits of JAK3 inhibitors.
Through high-throughput cell-based screening using the NCI compound library, we identified NSC163088 (berberine chloride) as a novel inhibitor of JAK3. Specificity and efficacy of this compound were investigated in both cellular and animal models.
We show that berberine chloride has selectivity for JAK3 over other JAK kinase members, as well as over other oncogenic kinases such as Src, in various cellular assays. Biochemical and modelling studies strongly suggested that berberine chloride bound directly to the kinase domain of JAK3. Also phospho-JAK3 levels were significantly increased in the synovial tissues of rat joints with acute inflammation, and the treatment of these rats with berberine chloride decreased JAK3 phosphorylation and suppressed the inflammatory responses.
The up-regulation of JAK3/STATs was closely correlated with acute arthritic inflammation and that inhibition of JAK3 activity by JAK3 antagonists, such as berberine chloride, alleviated the inflammation in vivo.
PMCID: PMC3171864  PMID: 21434883
JAK; STAT; inflammation; small molecule inhibitor, berberine, IL-2, IL-3
12.  Signature-Based Small Molecule Screening Identifies Cytosine Arabinoside as an EWS/FLI Modulator in Ewing Sarcoma 
PLoS Medicine  2007;4(4):e122.
The presence of tumor-specific mutations in the cancer genome represents a potential opportunity for pharmacologic intervention to therapeutic benefit. Unfortunately, many classes of oncoproteins (e.g., transcription factors) are not amenable to conventional small-molecule screening. Despite the identification of tumor-specific somatic mutations, most cancer therapy still utilizes nonspecific, cytotoxic drugs. One illustrative example is the treatment of Ewing sarcoma. Although the EWS/FLI oncoprotein, present in the vast majority of Ewing tumors, was characterized over ten years ago, it has never been exploited as a target of therapy. Previously, this target has been intractable to modulation with traditional small-molecule library screening approaches. Here we describe a gene expression–based approach to identify compounds that induce a signature of EWS/FLI attenuation. We hypothesize that screening small-molecule libraries highly enriched for FDA-approved drugs will provide a more rapid path to clinical application.
Methods and Findings
A gene expression signature for the EWS/FLI off state was determined with microarray expression profiling of Ewing sarcoma cell lines with EWS/FLI-directed RNA interference. A small-molecule library enriched for FDA-approved drugs was screened with a high-throughput, ligation-mediated amplification assay with a fluorescent, bead-based detection. Screening identified cytosine arabinoside (ARA-C) as a modulator of EWS/FLI. ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model. Given the poor outcomes of many patients with Ewing sarcoma and the well-established ARA-C safety profile, clinical trials testing ARA-C are warranted.
We demonstrate that a gene expression–based approach to small-molecule library screening can identify, for rapid clinical testing, candidate drugs that modulate previously intractable targets. Furthermore, this is a generic approach that can, in principle, be applied to the identification of modulators of any tumor-associated oncoprotein in the rare pediatric malignancies, but also in the more common adult cancers.
Todd Golub and colleagues show that a gene expression-based screen of small-molecule libraries can identify candidate drugs that modulate cancer-associated oncoproteins.
Editors' Summary
Cancer occurs when cells accumulate genetic changes (mutations) that allow them to divide uncontrollably and to travel throughout the body (metastasize). Chemotherapy, a mainstay of cancer treatments, works by killing rapidly dividing cells. Because some normal tissues also contain dividing cells and are therefore sensitive to chemotherapy drugs, it is hard to treat cancer without causing serious side effects. In recent years, however, researchers have identified some of the mutations that drive the growth of cancer cells. This raises the possibility of designing drugs that kill only cancer cells by specifically targeting “oncoproteins” (the abnormal proteins generated by mutations that transform normal cells into cancer cells). Some “targeted” drugs have already reached the clinic, but unfortunately medicinal chemists do not know how to inhibit the function of many classes of oncoproteins with the small organic molecules that make the best medicines. One oncoprotein in this category is EWS/FLI. This contains part of a protein called EWS fused to part of a transcription factor (a protein that controls cell behavior by telling the cell which proteins to make) called FLI. About 80% of patients with Ewing sarcoma (the second commonest childhood cancer of bone and soft tissue) have the mutation responsible for EWS/FLI expression. Localized Ewing sarcoma can be treated with nontargeted chemotherapy (often in combination with surgery and radiotherapy), but treatment for recurrent or metastatic disease remains very poor.
Why Was This Study Done?
Researchers have known for years that EWS/FLI expression drives the development of Ewing sarcoma by activating the expression of target genes needed for tumor formation. However, EWS/FLI has never been exploited as a target for therapy of this cancer—mainly because traditional approaches used to screen libraries of small molecules do not identify compounds that modulate the activity of transcription factors. In this study, the researchers have used a new gene expression–based, high-throughput screening (GE-HTS) approach to identify compounds that modulate the activity of EWS/FLI.
What Did the Researchers Do and Find?
The researchers used a molecular biology technique called microarray expression profiling to define a 14-gene expression signature that differentiates between Ewing sarcoma cells in which the EWS/FLI fusion protein is active and those in which it is inactive. They then used this signature to screen a library of about 1,000 chemicals (many already approved for other clinical uses) in a “ligation-mediated amplification assay.” For this, the researchers grew Ewing sarcoma cells with the test chemicals, extracted RNA from the cells, and generated a DNA copy of the RNA. They then added two short pieces of DNA (probes) specific for each signature gene to the samples. In samples that expressed a given signature gene, both probes bound and were then ligated (joined together) and amplified. Because one of each probe pair also contained a unique “capture sequence,” the signature genes expressed in each sample were finally identified by adding colored fluorescent beads, each linked to DNA complementary to a different capture sequence. The most active modulator of EWS/FLI activity identified by this GE-HTS approach was cytosine arabinoside (ARA-C). At levels achievable in people, this compound reduced the abundance of EWS/FLI protein in and the viability and cancer-like behavior of Ewing sarcoma cells growing in test tubes. ARA-C treatment also slowed the growth of Ewing sarcoma cells transplanted into mice.
What Do These Findings Mean?
These findings identify ARA-C, which is already used to treat children with some forms of leukemia, as a potent modulator of EWS/FLI activity. More laboratory experiments are needed to discover how ARA-C changes the behavior of Ewing sarcoma cells. Nevertheless, given the poor outcomes currently seen in many patients with Ewing sarcoma and the historical reluctance to test new drugs in children, these findings strongly support the initiation of clinical trials of ARA-C in children with Ewing sarcoma. These results also show that the GE-HTS approach is a powerful way to identify candidate drugs able to modulate the activity of some of the oncoproteins (including transcription factors and other previously intractable targets) that drive cancer development.
Additional Information.
Please access these Web sites via the online version of this summary at
Cancerquest from Emory University, provides information on cancer biology (also includes information in Spanish, Chinese and Russian)
The MedlinePlus encyclopedia has pages on Ewing sarcoma
Information for patients and health professionals on Ewing sarcoma is available from the US National Cancer Institute
Cancerbackup offers information for patients and their parents on Ewing sarcoma
Wikipedia has pages on DNA microarrays and expression profiling (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1851624  PMID: 17425403
13.  IFN-λ3 Inhibits HIV Infection of Macrophages through the JAK-STAT Pathway 
PLoS ONE  2012;7(4):e35902.
Interferon lambda 3 (IFN-λ3) is a newly identified cytokine with antiviral activity, and its single nucleotide polymorphisms are strongly associated with the treatment effectiveness and development of chronic hepatitis C virus infection. We thus examined the potential of IFN-λ3 to inhibit HIV replication and the possible mechanisms of the anti-HIV action by IFN-λ3 in human macrophages.
Principal Findings
Under different conditions (before, during, and after HIV infection), IFN-λ3 significantly inhibited viral replication in macrophages, which was associated with the induction of multiple antiviral cellular factors (ISG56, MxA, OAS-1, A3G/F and tetherin) and IFN regulatory factors (IRF-1, 3, 5, 7 and 9). This anti-HIV action of IFN-λ3 could be compromised by the JAK-STAT inhibitor. In addition, IFN-λ3 treatment of macrophages induced the expression of toll-like receptor 3 (TLR3) and two key adaptors (MyD88 and TRIF) in type I IFN pathway activation. However, HIV infection compromised IFN-λ3-mediated induction of the key elements in JAK-STAT signaling pathway.
These data indicate that IFN-λ3 exerts its anti-HIV function by activating JAK-STAT pathway-mediated innate immunity in macrophages. Future in vivo studies are necessary in order to explore the potential for developing IFN-λ3-based therapy for HIV disease.
PMCID: PMC3338759  PMID: 22558263
14.  Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway 
PLoS Genetics  2013;9(5):e1003487.
Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10−9). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10−9), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.
Author Summary
A current challenge in human genetics is to follow-up “hits” from genome-wide association studies (GWAS) to guide drug discovery for complex traits. Previously, we identified a common variant in the CD40 locus as associated with risk of rheumatoid arthritis (RA). Here, we fine-map the CD40 signal of association through a combination of dense genotyping and exonic sequencing in large patient collections. Further, we demonstrate that the RA risk allele is a gain-of-function allele that increases the amount of CD40 on the surface of primary human B lymphocyte cells from healthy control individuals. Based on these observations, we develop a high-throughput assay to recapitulate the biology of the RA risk allele in a system suitable for a small molecule drug screen. After a series of primary screens and counter screens, we identify small molecules that inhibit CD40-mediated NF-kB signaling in human B cells. While this is only the first step towards a more comprehensive effort to identify CD40-specific inhibitors that may be used to treat RA, our study demonstrates a successful strategy to progress from a GWAS to a drug screen for complex traits such as RA.
PMCID: PMC3656093  PMID: 23696745
15.  Inhibition of TYK2 and JAK1 Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis by Inhibiting IL-22 and the IL-23/IL-17 axis 
Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making Janus kinase (JAK) inhibition an appealing strategy for the treatment of psoriasis. Here we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and Tyrosine Kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose-dependently (1 nM-10 μM) inhibited JAK1 and/or TYK2 dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild type mice with SAR-20347 significantly reduced IL-12 induced IFN-γ production and IL-22-dependent Serum Amyloid A (SAA) to similar extents, indicating that in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes, and proinflammatory cytokine levels compared to both TYK2 mutant mice and wild type controls. Taken together, these data indicate that targeting both JAK1 and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis.
PMCID: PMC4170002  PMID: 25156366
T cells; autoimmunity; psoriasis; cytokines; protein kinases; skin; JAK
16.  Influenza A Virus Inhibits Type I IFN Signaling via NF-κB-Dependent Induction of SOCS-3 Expression 
PLoS Pathogens  2008;4(11):e1000196.
The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNβ gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNβ gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5′ triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-κB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.
Author Summary
The type I interferon (IFN) system is one of the most powerful innate defenses against viral pathogens. Most RNA viruses are sensitive to the action of type I IFN. Therefore, these pathogens have evolved strategies to evade this response. For example, influenza viruses express a viral protein, the non-structural protein 1 (NS1), that suppresses production of IFNβ by lowering cellular sensitivity to viral nucleic acid as a pathogen pattern. Here we present data indicating that influenza A viruses are not only capable of suppressing production of the IFNβ gene but also inhibit action of this antiviral cytokine on cells. This occurs by viral induction of a cellular protein, the suppressor of cytokine signaling (SOCS)-3, a potent endogenous inhibitor of IFN signaling. This is a novel mechanism by which influenza viruses inhibit the antiviral response of the host and paves the path to efficient virus replication. This may be especially relevant for influenza viruses that induce high cytokine responses (cytokine burst), such as highly pathogenic avian influenza viruses of the H5N1 subtype. Induction of SOCS-3 expression would allow efficient replication despite high IFN and cytokine levels.
PMCID: PMC2572141  PMID: 18989459
17.  Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus 
PLoS Pathogens  2010;6(1):e1000721.
Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.
Author Summary
The closely related members of the filovirus family, Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic disease in humans with high fatality rates. Infected individuals exhibit dysregulated immune responses which appear to result from several factors, including virus-mediated impairment of innate immune responses. Previous studies demonstrated that both MARV and EBOV block the type I interferon-induced Jak-STAT signaling pathway. For EBOV, the viral protein VP24 mediates the inhibitory effects by interfering with the nuclear translocation of activated STAT proteins. Here, we show that MARV uses a distinct mechanism to block IFN signaling pathways. Our data revealed that MARV blocks the phosphorylation of Janus kinases and their target STAT proteins in response to type I and type II interferon and interleukin 6. Surprisingly, the observed inhibition is not achieved by the MARV VP24 protein, but by the matrix protein VP40 which also mediates viral budding. Over-expression studies indicate that MARV VP40 globally antagonizes Jak1-dependent signaling. Further, we show that a MARV VP40 mutant defective for budding retains interferon antagonist function. Our results highlight a basic difference between EBOV and MARV, define a new function for MARV VP40 and reveal new targets for the development of anti-MARV therapies.
PMCID: PMC2799553  PMID: 20084112
18.  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.
PMCID: PMC1235813  PMID: 16188985
19.  Thioredoxin Reductase Mediates Cell Death Effects of the Combination of Beta Interferon and Retinoic Acid 
Molecular and Cellular Biology  1998;18(11):6493-6504.
Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN-β–all-trans-retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.
PMCID: PMC109235  PMID: 9774665
20.  Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ 
PLoS Pathogens  2012;8(1):e1002483.
Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors.
Author Summary
Toxoplasma gondii is a common unicellular parasite of humans and other vertebrates and can lead to overt disease mostly in immune-suppressed patients or in fetuses. Since IFN-γ is the major mediator of resistance against T. gondii, inhibition of IFN-γ-mediated gene expression may be a crucial mechanism to allow parasite survival in the immune-competent hosts. Here, we used genome-wide expression profiling to show that parasite infection renders murine macrophages globally unresponsive to stimulation with IFN-γ. This results in severe defects of infected macrophages to regulate a variety of immune-related, but also immune-unrelated biological pathways. By analysing the underlying mechanisms, we provide substantial evidence that Toxoplasma interferes with the assembly of chromatin remodelling complexes at IFN-γ-responsive DNA sequences. Furthermore, binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to IFN-γ-regulated promoters, but not its nuclear import is disturbed in infected cells. The acetylation of histones at IFN-γ-regulated promoters was found to be severely impaired. Importantly, treatment with histone deacetylase inhibitors rescues Toxoplasma-infected macrophages from the inability to respond to IFN-γ. Our study reveals new insights into the evasion of IFN-γ-mediated host immunity by T. gondii, and opens the possibility of a novel intervention strategy against T. gondii by modulating this parasite-host interaction.
PMCID: PMC3262016  PMID: 22275866
21.  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.
PMCID: PMC3416819  PMID: 22899991
22.  Identification of signalling cascades involved in red blood cell shrinkage and vesiculation 
Bioscience Reports  2015;35(2):e00187.
Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca2+ ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)–Akt (protein kinase B) pathway, the Jak–STAT (Janus kinase–signal transducer and activator of transcription) pathway and the Raf–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.
After screening two libraries of small bioactive molecules and kinase inhibitors, we identified several signalling pathways to be involved in red blood cell (RBC) shrinkage and vesiculation. These include the Jak (Janus kinase)–STAT (signal transducer and activator of transcription) pathway, phosphoinositide 3-kinase (PI3K)–Akt pathway, the Raf–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal-regulated kinase) pathway and GPCR (G protein-coupled receptor) signalling.
PMCID: PMC4400636  PMID: 25757360
bioactive small molecule; compound screen; kinase inhibitor; red blood cell; vesiculation; AMPK, AMP-activated kinase; ATA, aurintricarboxylic acid; BCR-ABL, breakpoint cluster region protein–Abelson murine leukaemia viral oncogene homologue 1; CaM, calmodulin; CK2, casein kinase 2; Epo, erythropoietin; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; Jak, Janus kinase; LOPAC, Library of Pharmacologically Active Compounds; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NO, nitric oxide; nRTK, non-receptor tyrosine kinase; PC, phosphatidylcholine; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PS, phosphatidylserine; RBC, red blood cell; RTK, receptor tyrosine kinase; SAGM, saline-adenine-glucose-mannitol; SCD, sickle cell disease; SMase, acid sphingomyelinase; STAT, signal transducer and activator of transcription; VEGFR, vascular endothelial growth factor receptor; β-AR, β-adrenergic receptor
23.  The benzoxathiolone LYR-71 down-regulates interferon-γ-inducible pro-inflammatory genes by uncoupling tyrosine phosphorylation of STAT-1 in macrophages 
British Journal of Pharmacology  2009;158(8):1971-1981.
Background and purpose:
Benzoxathiolone derivatives have shown anti-inflammatory and immunomodulatory potential in acne and psoriatic disorders. However, little is known about the molecular basis for these pharmacological effects. In this study, we decided to investigate the anti-inflammatory actions of a benzoxathiolone derivative LYR-71, 6-methyl-2-propylimino-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one, in interferon (IFN)-γ-activated macrophages.
Experimental approach:
RAW 264.7 macrophages or primary macrophages, derived from bone marrow of C3H/HeJ mice, were stimulated with IFN-γ in the presence of LYR-71. Nitric oxide (NO) or chemokine production was measured by Griess reaction or enzyme-linked immunosorbent assay. RAW 264.7 cells were used to examine the molecular mechanisms of LYR-71 in modulating IFN-γ-induced inflammatory responses.
Key results:
LYR-71 down-regulated IFN-γ-induced transcription of inducible NO synthase, IFN-γ-inducible protein-10 and the monokine induced by IFN-γ genes in macrophages. This effect was mediated by uncoupling tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)-1 in response to IFN-γ. LYR-71 directly inhibited the in vitro catalytic activity of Janus kinase (JAK)-2. Further, the inhibitory actions of LYR-71 on IFN-γ-induced STAT-1 phosphorylation and NO production were consistently abolished in the presence of peroxyvanadate, implying another target dependent on protein tyrosine phosphatase.
Conclusions and implications:
Taken together, LYR-71 could restrain IFN-γ-induced inflammatory responses through uncoupling the tyrosine phosphorylation of STAT-1, an activation index of JAK–STAT-1 signalling, in macrophages. These results may provide a molecular mechanism underlying anti-inflammatory actions shown by benzoxathiolone derivatives.
PMCID: PMC2807659  PMID: 19922538
benzoxathiolone derivative; anti-inflammatory action; nitric oxide; chemokine; Janus kinase; STAT-1; IFN-γ; macrophages
24.  The antihypertension drug doxazosin suppresses JAK/STATs phosphorylation and enhances the effects of IFN-α/γ-induced apoptosis 
Genes & Cancer  2014;5(11-12):470-479.
Doxazosin, a commonly prescribed treatment for patients with benign prostatic hyperplasia, serves as an α1-blocker of the adrenergic receptors. In this study, we calculated its effect on the ovarian carcinoma cells. Doxazosin induces dose-dependent growth suppression and is additively activated through IFN-α or IFN-γ stimulation. They both enhanced G1 phase arrest, as well as the activity of caspase-3, and the reduction of cyclin D1 and CDK4 protein levels. Doxazosin growth suppression was abolished either by the Janus family of tyrosine kinase (JAK) or the signal transducer and activator of transcription (STAT) inhibitor treatment. The activity of JAK/STAT was dependent on the level of doxazosin, suggesting a requirement of doxazosin for the activation of JAK/STAT. Furthermore, doxazosin plus IFN-α or doxazosin plus IFN-γ additively suppressed the activation of the JAK/STAT signals through phosphorylation of JAK and STAT, thus affecting the activation of subsequent downstream signaling components PI3K, mTOR, 70S6K, and PKCδ. In vivo study demonstrated that doxazosin significantly suppressed tumor growth in an ovarian cancer cell xenograft mouse model, inducing apoptotic cell death by up-regulating the expression of p53, whereas c-Myc expression was markedly reduced. Our data indicate that doxazosin can modulate the apoptotic effects of IFN-α- and IFN-γ through the JAK/STAT signaling pathways. Collectively, we indicate that this action may be a potent chemotherapeutic property against ovarian carcinoma.
PMCID: PMC4279443  PMID: 25568671
doxazosin; interferon-α/γ; apoptotic cell death; JAK/STAT activation; cell cycle progression
25.  Noncytolytic Clearance of Sindbis Virus Infection from Neurons by Gamma Interferon Is Dependent on Jak/Stat Signaling▿  
Journal of Virology  2009;83(8):3429-3435.
The alphavirus Sindbis virus (SINV) causes encephalomyelitis in mice by infecting neurons of the brain and spinal cord. The outcome is age dependent. Young animals develop fatal disease, while older animals recover from infection. Recovery requires noncytolytic clearance of SINV from neurons, and gamma interferon (IFN-γ) is an important contributor to clearance in vivo. IFN-γ-dependent clearance has been studied using immortalized CSM14.1 rat neuronal cells that can be differentiated in vitro. Previous studies have shown that differentiated, but not undifferentiated, cells develop prolonged SINV replication and respond to IFN-γ treatment with noncytolytic clearance of virus preceded by suppression of genomic viral RNA synthesis and reactivation of cellular protein synthesis. To determine the signaling mechanisms responsible for clearance, the responses of SINV-infected differentiated neurons to IFN-γ were examined. IFN-γ treatment of SINV-infected differentiated CSM14.1 cells, AP-7 olfactory neuronal cells, and primary dorsal root ganglia neurons triggered prolonged Stat-1 Tyr701 phosphorylation, Stat-1 Ser727 phosphorylation, and transient Stat-5 phosphorylation. Inhibition of Jak kinase activity with Jak inhibitor I completely reversed the neuroprotective and antiviral activities of IFN-γ in differentiated cells. We conclude that activation of the Jak/Stat pathway is the primary mechanism for IFN-γ-mediated clearance of SINV infection from mature neurons.
PMCID: PMC2663278  PMID: 19176616

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