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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.  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
3.  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
4.  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
5.  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
6.  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
7.  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.
PMCID: PMC3344904  PMID: 22574190
8.  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
9.  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
10.  Cross-species chemogenomic profiling reveals evolutionarily conserved drug mode of action 
Chemogenomic screens were performed in both budding and fission yeasts, allowing for a cross-species comparison of drug–gene interaction networks.Drug–module interactions were more conserved than individual drug–gene interactions.Combination of data from both species can improve drug–module predictions and helps identify a compound's mode of action.
Understanding the molecular effects of chemical compounds in living cells is an important step toward rational therapeutics. Drug discovery aims to find compounds that will target a specific pathway or pathogen with minimal side effects. However, even when an effective drug is found, its mode of action (MoA) is typically not well understood. The lack of knowledge regarding a drug's MoA makes the drug discovery process slow and rational therapeutics incredibly difficult. More recently, different high-throughput methods have been developed that attempt to discern how a compound exerts its effects in cells. One of these methods relies on measuring the growth of cells carrying different mutations in the presence of the compounds of interest, commonly referred to as chemogenomics (Wuster and Babu, 2008). The differential growth of the different mutants provides clues as to what the compounds target in the cell (Figure 2). For example, if a drug inhibits a branch in a vital two-branch pathway, then mutations in the second branch might result in cell death if the mutants are grown in the presence of the drug (Figure 2C). As these compound–mutant functional interactions are expected to be relatively rare, one can assume that the growth rate of a mutant–drug combination should generally be equal to the product of the growth rate of the untreated mutant with the growth rate of the drug-treated wild type. This expectation is defined as the neutral model and deviations from this provide a quantitative score that allow us to make informed predictions regarding a drug's MoA (Figure 2B; Parsons et al, 2006).
The availability of these high-throughput approaches now allows us to perform cross-species studies of functional interactions between compounds and genes. In this study, we have performed a quantitative analysis of compound–gene interactions for two fungal species (budding yeast (S. cerevisiae) and fission yeast (S. pombe)) that diverged from each other approximately 500–700 million years ago. A collection of 2957 compounds from the National Cancer Institute (NCI) were screened in both species for inhibition of wild-type cell growth. A total of 132 were found to be bioactive in both fungi and 9, along with 12 additional well-characterized drugs, were selected for subsequent screening. Mutant libraries of 727 and 438 gene deletions were used for S. cerevisiae and S. pombe, respectively, and these were selected based on availability of genetic interaction data from previous studies (Collins et al, 2007; Roguev et al, 2008; Fiedler et al, 2009) and contain an overlap of 190 one-to-one orthologs that can be directly compared. Deviations from the neutral expectation were quantified as drug–gene interactions scores (D-scores) for the 21 compounds against the deletion libraries. Replicates of both screens showed very high correlations (S. cerevisiae r=0.72, S. pombe r=0.76) and reproduced well previously known compound–gene interactions (Supplementary information). We then compared the D-scores for the 190 one-to-one orthologs present in the data set of both species. Despite the high reproducibility, we observed a very poor conservation of these compound–gene interaction scores across these species (r=0.13, Figure 4A).
Previous work had shown that, across these same species, genetic interactions within protein complexes were much more conserved than average genetic interactions (Roguev et al, 2008). Similarly we observed a higher cross-species conservation of the compound–module (complex or pathway) interactions than the overall compound–gene interactions. Specifically, the data derived from fission yeast were a poor predictor of S. cerevisaie drug–gene interactions, but a good predictor of budding yeast compound–module connections (Figure 4B). Also, a combined score from both species improved the prediction of compound–module interactions, above the accuracy observed with the S. cerevisae information alone, but this improvement was not observed for the prediction of drug–gene interactions (Figure 4B). Data from both species were used to predict drug–module interactions, and one specific interaction (compound NSC-207895 interaction with DNA repair complexes) was experimentally verified by showing that the compound activates the DNA damage repair pathway in three species (S. cerevisiae, S. pombe and H. sapiens).
To understand why the combination of chemogenomic data from two species might improve drug–module interaction predictions, we also analyzed previously published cross-species genetic–interaction data. We observed a significant correlation between the conservation of drug–gene and gene–gene interactions among the one-to-one orthologs (r=0.28, P-value=0.0078). Additionally, the strongest interactions of benomyl (a microtubule inhibitor) were to complexes that also had strong and conserved genetic interactions with microtubules (Figure 4C). We hypothesize that a significant number of the compound–gene interactions obtained from chemogenomic studies are not direct interactions with the physical target of the compounds, but include many indirect interactions that genetically interact with the main target(s). This would explain why the compound interaction networks show similar evolutionary patterns as the genetic interactions networks.
In summary, these results shed some light on the interplay between the evolution of genetic networks and the evolution of drug response. Understanding how genetic variability across different species might result in different sensitivity to drugs should improve our capacity to design treatments. Concretely, we hope that this line of research might one day help us create drugs and drug combinations that specifically affect a pathogen or diseased tissue, but not the host.
We present a cross-species chemogenomic screening platform using libraries of haploid deletion mutants from two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We screened a set of compounds of known and unknown mode of action (MoA) and derived quantitative drug scores (or D-scores), identifying mutants that are either sensitive or resistant to particular compounds. We found that compound–functional module relationships are more conserved than individual compound–gene interactions between these two species. Furthermore, we observed that combining data from both species allows for more accurate prediction of MoA. Finally, using this platform, we identified a novel small molecule that acts as a DNA damaging agent and demonstrate that its MoA is conserved in human cells.
PMCID: PMC3018166  PMID: 21179023
chemogenomics; evolution; modularity
11.  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
12.  A Systematic Analysis of Host Factors Reveals a Med23-Interferon-λ Regulatory Axis against Herpes Simplex Virus Type 1 Replication 
PLoS Pathogens  2013;9(8):e1003514.
Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome.
Author Summary
Herpes simplex virus type 1 (HSV-1) infects the vast majority of the global population. Whilst most people experience the relatively mild symptoms of cold sores, some individuals suffer more serious diseases like viral meningitis and encephalitis. HSV-1 is also becoming more common as a cause of genital herpes, traditionally associated with HSV-2 infection. Co-infection with HSV-2 is a major contributor to HIV transmission, so a better understanding of HSV-1/HSV-2 disease has wide implications for global healthcare. After initial infection, all herpesviruses have the ability to remain dormant, and can awaken to cause a symptomatic infection at any stage. Whether the virus remains dormant or active is the result of a finely tuned balance between our immune system and evasion techniques developed by the virus. In this study we have found a new method by which the replication of the virus is counteracted. The cellular protein Med23 was found to actively induce an innate anti-viral immune response in the form of the Type III interferons (IFN-lambda), by binding IRF7, a key regulator of interferons, and modulating its activity. Interferon lambda is well known to be important in the control of Hepatitis C infection, and a genetic mutation correlating to an increase in interferon lambda levels is strongly linked to clearance of infection. Here we find the same association between this genetic mutation and the clinical severity of recurrent cases of HSV-1 infection (coldsores). These data identify a Med23-interferon lambda regulatory axis of innate immunity, show that interferon lambda plays a significant role in HSV-1 infection, and contribute to the expanding evidence for interferon lambda in disease control.
PMCID: PMC3738494  PMID: 23950709
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.  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
15.  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
16.  Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole 
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
Chemical screens with a library enriched for known drugs identified a diverse set of 148 compounds that potentiated the action of the antifungal drug fluconazole against the fungal pathogens Cryptococcus neoformans, Cryptococcus gattii and Candida albicans, and the model yeast Saccharomyces cerevisiae, often in a species-specific manner.Chemogenomic profiles of six confirmed hits in S. cerevisiae revealed different modes of action and enabled the prediction of additional synergistic combinations; three-way synergistic interactions exhibited even stronger synergies at low doses of fluconazole.The synergistic combination of fluconazole and the antidepressant sertraline was active against fluconazole-resistant clinical fungal isolates and in an in vivo model of Cryptococcal infection.
Rising fungal infection rates, especially among immune-suppressed individuals, represent a serious clinical challenge (Gullo, 2009). Cancer, organ transplant and HIV patients, for example, often succumb to opportunistic fungal pathogens. The limited repertoire of approved antifungal agents and emerging drug resistance in the clinic further complicate the effective treatment of systemic fungal infections. At the molecular level, the paucity of fungal-specific essential targets arises from the conserved nature of cellular functions from yeast to humans, as well as from the fact that many essential yeast genes can confer viability at a fraction of wild-type dosage (Yan et al, 2009). Although only ∼1100 of the ∼6000 genes in yeast are essential, almost all genes become essential in specific genetic backgrounds in which another non-essential gene has been deleted or otherwise attenuated, an effect termed synthetic lethality (Tong et al, 2001). Genome-scale surveys suggest that over 200 000 binary synthetic lethal gene combinations dominate the yeast genetic landscape (Costanzo et al, 2010). The genetic buffering phenomenon is also manifest as a plethora of differential chemical–genetic interactions in the presence of sublethal doses of bioactive compounds (Hillenmeyer et al, 2008). These observations frame the difficulty of interdicting network functions in eukaryotic pathogens with single agent therapeutics. At the same time, however, this genetic network organization suggests that judicious combinations of small molecule inhibitors of both essential and non-essential targets may elicit additive or synergistic effects on cell growth (Sharom et al, 2004; Lehar et al, 2008). Unbiased screens for drugs that synergistically enhance a specific bioactive effect, but which are not themselves individually active—termed a syncretic combination—are one means to substantially elaborate chemical space (Keith et al, 2005). Indeed, compounds that enhance the activity of known agents in model yeast and cancer cell line systems have been identified both by focused small molecule library screens and by computational methods (Borisy et al, 2003; Lehar et al, 2007; Nelander et al, 2008; Jansen et al, 2009; Zinner et al, 2009).
To extend the stratagem of chemical synthetic lethality to clinically relevant fungal pathogens, we screened a bioactive library of known drugs for synergistic enhancers of the widely used fungistatic drug fluconazole against the clinically relevant pathogens C. albicans, C. neoformans and C. gattii, as well as the genetically tractable budding yeast S. cerevisiae. Fluconazole is an azole drug that inhibits lanosterol 14α-demethylase, the gene product of ERG11, an essential cytochrome P450 enzyme in the ergosterol biosynthetic pathway (Groll et al, 1998). We identified 148 drugs that potentiate the antifungal action of fluconazole against the four species. These syncretic compounds had not been previously recognized in the clinic as antifungal agents, and many acted in a species-specific manner, often in a potent fungicidal manner.
To understand the mechanisms of synergism, we interrogated six syncretic drugs—trifluoperazine, tamoxifen, clomiphene, sertraline, suloctidil and L-cycloserine—in genome-wide chemogenomic profiles of the S. cerevisiae deletion strain collection (Giaever et al, 1999). These profiles revealed that membrane, vesicle trafficking and lipid biosynthesis pathways are targeted by five of the synergizers, whereas the sphingolipid biosynthesis pathway is targeted by L-cycloserine. Cell biological assays confirmed the predicted membrane disruption effects of the former group of compounds, which may perturb ergosterol metabolism, impair fluconazole export by drug efflux pumps and/or affect active import of fluconazole (Kuo et al, 2010; Mansfield et al, 2010). Based on the integration of chemical–genetic and genetic interaction space, a signature set of deletion strains that are sensitive to the membrane active synergizers correctly predicted additional drug synergies with fluconazole. Similarly, the L-cycloserine chemogenomic profile correctly predicted a synergistic interaction between fluconazole and myriocin, another inhibitor of sphingolipid biosynthesis. The structure of genetic networks suggests that it should be possible to devise higher order drug combinations with even greater selectivity and potency (Sharom et al, 2004). In an initial test of this concept, we found that the combination of a non-synergistic pair drawn from the membrane active and sphingolipid target classes exhibited potent three-way synergism with a low dose of fluconazole. Finally, the combination of sertraline and fluconazole was active in a G. mellonella model of Cryptococcal infection, and was also efficacious against fluconazole-resistant clinical isolates of C. albicans and C. glabrata.
Collectively, these results demonstrate that the combinatorial redeployment of known drugs defines a powerful antifungal strategy and establish a number of potential lead combinations for future clinical assessment.
Resistance to widely used fungistatic drugs, particularly to the ergosterol biosynthesis inhibitor fluconazole, threatens millions of immunocompromised patients susceptible to invasive fungal infections. The dense network structure of synthetic lethal genetic interactions in yeast suggests that combinatorial network inhibition may afford increased drug efficacy and specificity. We carried out systematic screens with a bioactive library enriched for off-patent drugs to identify compounds that potentiate fluconazole action in pathogenic Candida and Cryptococcus strains and the model yeast Saccharomyces. Many compounds exhibited species- or genus-specific synergism, and often improved fluconazole from fungistatic to fungicidal activity. Mode of action studies revealed two classes of synergistic compound, which either perturbed membrane permeability or inhibited sphingolipid biosynthesis. Synergistic drug interactions were rationalized by global genetic interaction networks and, notably, higher order drug combinations further potentiated the activity of fluconazole. Synergistic combinations were active against fluconazole-resistant clinical isolates and an in vivo model of Cryptococcus infection. The systematic repurposing of approved drugs against a spectrum of pathogens thus identifies network vulnerabilities that may be exploited to increase the activity and repertoire of antifungal agents.
PMCID: PMC3159983  PMID: 21694716
antifungal; combination; pathogen; resistance; synergism
17.  Interferon- α 2b reduces phosphorylation and activity of MEK and ERK through a Ras / Raf -independent mechanism 
British Journal of Cancer  2000;83(4):532-538.
Interferon (IFN)-α affects the growth, differentiation and function of various cell types by transducing regulatory signals through the Janus tyrosine kinase/signal transducers of activation and transcription (Jak/STAT) pathway. The signalling pathways employing the mitogen-activated ERK-activating kinase (MEK) and the extracellular-regulated kinase (ERK) are critical in growth factors signalling. Engagement of the receptors, and subsequent stimulation of Ras and Raf, initiates a phosphorylative cascade leading to activation of several proteins among which MEK and ERK play a central role in routing signals critical in controlling cell development, activation and proliferation. We demonstrate here that 24–48 h following treatment of transformed T- and monocytoid cell lines with recombinant human IFN-α2b both the phosphorylation and activity of MEK1 and its substrates ERK1/2 were reduced. In contrast, the activities of the upstream molecules Ras and Raf -1 were not affected. No effect on MEK/ERK activity was observed upon short-term exposure (1–30 min) to IFN. The anti-proliferative effect of IFN-α was increased by the addition in the culture medium of a specific inhibitor of MEK, namely PD98059. In conclusion, our results indicate that IFN-α regulates the activity of the MEK/ERK pathway and consequently modulates cellular proliferation through a Ras / Raf -independent mechanism. Targeting the MEK/ERK pathway may strengthen the IFN-mediated anti-cancer effect. © 2000 Cancer Research Campaign
PMCID: PMC2374650  PMID: 10945503
IFN-α; cellular proliferation; MEK/ERK pathway
18.  The anti-HBV effect mediated by a novel recombinant eukaryotic expression vector for IFN-α 
Virology Journal  2013;10:270.
Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-α) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-α at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-α) in vitro was carried out.
HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-α or empty plasmid by using Lipofectamine™ 2000 reagent. The expression levels of IFN-α were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-α on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-α on IFN-α-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-α and the combination with lamivudine on HBV were also examined.
pSecTagB-IFN-α could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-α-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-α, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-α vector revealed effectively anti-HBV effect than exogenously added IFN-α. Moreover, lamivudine combined with endogenously expressed IFN-α exhibited stronger anti-HBV effect than with exogenous IFN-α.
Our results showed that endogenously expressed IFN-α can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-α.
PMCID: PMC3766191  PMID: 23984795
HBV; IFN-α; Eukaryotic expression vector
19.  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
20.  Inhibitor of IκB kinase activity, BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells 
Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IκB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs.
We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-α production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-α production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U.
Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-α production, more strongly inhibited IFN-α production than TNF-α production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-α production by human PBMCs induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with poly U.
These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.
PMCID: PMC2911871  PMID: 20470398
21.  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.
PMCID: PMC3277087  PMID: 22355771
22.  Cooperative effects of Janus and Aurora kinase inhibition by CEP701 in cells expressing Jak2V617F 
The Janus kinase 2 mutant V617F occurs with high frequency in myeloproliferative neoplasms. Further mutations affecting the Janus kinase family have been discovered mostly in leukaemias and in myeloproliferative neoplasms. Owing to their involvement in neoplasia, inflammatory diseases and in the immune response, Janus kinases are promising targets for kinase inhibitor therapy in these disease settings. Various quantitative assays including two newly developed screening assays were used to characterize the function of different small-molecule compounds in cells expressing Jak2V617F. A detailed comparative analysis of different Janus kinase inhibitors in our quantitative assays and the subsequent characterization of additional activities demonstrated for the first time that the most potent Jak2 inhibitor in our study, CEP701, also targets Aurora kinases. CEP701 shows a unique combination of both activities which is not found in other compounds also targeting Jak2. Furthermore, colony forming cell assays showed that Janus kinase 2 inhibitors preferentially suppressed the growth of erythroid colonies, whereas inhibitors of Aurora kinases preferentially blocked myeloid colony growth. CEP701 demonstrated a combined suppression of both colony types. Moreover, we show that combined application of a Janus and an Aurora kinase inhibitor recapitulated the effect observed for CEP701 but might allow for more flexibility in combining both activities in clinical settings, e.g. in the treatment of myeloproliferative neoplasms. The newly developed screening assays are high throughput compatible and allow an easy detection of new compounds with Janus kinase 2 inhibitory activity.
PMCID: PMC3822589  PMID: 23301855
Janus kinases; Jak2V617F; Aurora kinases; kinase inhibitors; MPN; CEP701
23.  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
24.  Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library 
Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.
Author Summary
Recent outbreaks and expanding global distribution of Chikungunya virus (CHIKV) in different regions of Asia, Africa and Europe necessitates the development of effective therapeutic interventions. At present, only two antiviral compounds (chloroquine and ribavirin) that inhibit viral infection in vitro have been used in clinical cases of chikungunya infections. However, neither of these compounds have shown strong efficacy in vivo. Recent attempts to identify new antiviral candidates for CHIKV using cell-based phenotypic approach have been reported. In this study, we developed a simple cell-based high-throughput assay using resazurin to identify potential anti-CHIKV compounds. This high-throughput assay is based on the metabolic reduction of resazurin to the highly fluorescent resorufin by viable cells as an indicator of activity against CHIKV-induced CPE. We screened 4,000 small molecules belonging to the BioFocus kinase inhibitor chemical library and found a cluster of related molecules with antiviral activity against CHIKV. Finally, we characterized the putative mode of action of these active compounds using an image-based high content assay and conventional virological methods (i.e., virus yield reduction assay, microneutralization assay).
PMCID: PMC3814572  PMID: 24205414
25.  Ascorbic Acid Has Superior Ex Vivo Antiproliferative, Cell Death-Inducing and Immunomodulatory Effects over IFN-α in HTLV-1-Associated Myelopathy 
Clear therapeutic guidelines for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are missing due to the lack of randomized double-blind controlled clinical trials. Moderate yet similar clinical benefit has been demonstrated for IFN-α and high-dose ascorbic acid (AA) monotherapy in a large open clinical trial. However, there is a lack of in vivo and in vitro studies exploring and comparing the effects of high-dose AA and IFN-α treatment in the context of HAM/TSP. Therefore, we performed the first comparative analysis of the ex vivo and in vitro molecular and cellular mechanisms of action of IFN-α and high-dose AA in HAM/TSP.
Principal Findings
Through thymidine incorporation and quantification of Th1/Th2/Th17 cytokines, we demonstrate that high-dose AA displays differential and superior antiproliferative and immunomodulatory effects over IFN-α in HAM/TSP PBMCs ex vivo. In addition, high-dose AA, but not IFN-α, induced cell death in both HAM/TSP PBMCs and HTLV-1-infected T-cell lines MT-2 and MT-4. Microarray data combined with pathway analysis of MT-2 cells revealed AA-induced regulation of genes associated with cell death, including miR-155. Since miR-155 has recently been demonstrated to up-regulate IFN-γ, this microRNA might represent a novel therapeutic target in HAM/TSP, as recently demonstrated in multiple sclerosis, another neuroinflammatory disease. On the other hand, IFN-α selectively up-regulated antiviral and immune-related genes.
In comparison to IFN-α, high-dose AA treatment has superior ex vivo and in vitro cell death-inducing, antiproliferative and immunomodulatory anti-HTLV-1 effects. Differential pathway activation by both drugs opens up avenues for targeted treatment in specific patient subsets.
Author Summary
HAM/TSP is a chronic and disabling neuroinflammatory disease, for which clinical management is mostly empirical and symptomatic rather than evidence-based, due to the lack of biomarkers and controlled clinical trials. Although similar clinical benefit has been demonstrated for IFN-α and high-dose ascorbic acid (vitamin C) in one major open clinical trial with 200 patients, their cellular and molecular mechanisms of action remain unexplored in HAM/TSP. We demonstrate that high-dose ascorbic acid strongly inhibits lymphoproliferation of HAM/TSP mononuclear cells in ex vivo cultures, in contrast to IFN-α. Furthermore, high-dose ascorbic acid, but not IFN-α, significantly decreased ex vivo TNF-α and IFN-γ pro-inflammatory cytokine levels in supernatant of mononuclear cells from HAM/TSP patients. In addition, ascorbic acid, but not IFN-α, induced cell death in HTLV-1-infected T-cell lines, which was confirmed by gene expression profiling, revealing cell death-associated pathways activated by high-dose ascorbic acid, including miR-155. This microRNA has previously been shown up-regulated in HTLV-1-infected cells, as well as in blood and brain samples of multiple sclerosis patients, another neuroinflammatory disease. In addition, miR-155 has also been reported to up-regulate IFN-γ production in human natural killer cells, thus linking both cell death and cytokine signaling pathways, rendering it a potential therapeutic target in neuroinflammatory disorders. Thus, our findings reveal molecular mechanisms of action as well as candidate biomarkers for high-dose ascorbic acid therapy and provide a rational basis, rather than an empirical basis, for its use in HAM/TSP treatment.
PMCID: PMC3404116  PMID: 22848768

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