A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon

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

An Infant Mouse Model of Influenza Virus Transmission Demonstrates the Role of Virus-Specific Shedding, Humoral Immunity, and Sialidase Expression by Colonizing Streptococcus pneumoniae

This study provides insight into the role of the virus strain, age, immunity, and URT flora on IAV shedding and transmission efficiency. Using the infant mouse model, we found that (i) differences in viral shedding of various IAV strains are dependent on specific hemagglutinin (HA) and/or neuraminidase (NA) proteins, (ii) host age plays a key role in the efficiency of IAV transmission, (iii) levels of IAV-specific immunoglobulins are necessary to limit infectiousness, transmission, and susceptibility to IAV, and (iv) expression of sialidases by colonizing S. pneumoniae antagonizes transmission by limiting the acquisition of IAV in recipient hosts. Our findings highlight the need for strategies that limit IAV shedding and the importance of understanding the function of the URT bacterial composition in IAV transmission. This work reinforces the significance of a tractable animal model to study both viral and host traits affecting IAV contagion and its potential for optimizing vaccines and therapeutics that target disease spread.The pandemic potential of influenza A viruses (IAV) depends on the infectivity of the host, transmissibility of the virus, and susceptibility of the recipient. While virus traits supporting IAV transmission have been studied in detail using ferret and guinea pig models, there is limited understanding of host traits determining transmissibility and susceptibility because current animal models of transmission are not sufficiently tractable. Although mice remain the primary model to study IAV immunity and pathogenesis, the efficiency of IAV transmission in adult mice has been inconsistent. Here we describe an infant mouse model that supports efficient transmission of IAV. We demonstrate that transmission in this model requires young age, close contact, shedding of virus particles from the upper respiratory tract (URT) of infected pups, the use of a transmissible virus strain, and a susceptible recipient. We characterize shedding as a marker of infectiousness that predicts the efficiency of transmission among different influenza virus strains. We also demonstrate that transmissibility and susceptibility to IAV can be inhibited by humoral immunity via maternal-infant transfer of IAV-specific immunoglobulins and modifications to the URT milieu, via sialidase activity of colonizing Streptococcus pneumoniae. Due to its simplicity and efficiency, this model can be used to dissect the host’s contribution to IAV transmission and explore new methods to limit contagion

Identification of small molecules with type I interferon inducing properties by high-throughput screening.

The continuous emergence of virus that are resistant to current anti-viral drugs, combined with the introduction of new viral pathogens for which no therapeutics are available, creates an urgent need for the development of novel broad spectrum antivirals. Type I interferon (IFN) can, by modulating the cellular expression profile, stimulate a non-specific antiviral state. The antiviral and adjuvant properties of IFN have been extensively demonstrated; however, its clinical application has been so far limited. We have developed a human cell-based assay that monitors IFN-β production for use in a high throughput screen. Using this assay we screened 94,398 small molecules and identified 18 compounds with IFN-inducing properties. Among these, 3 small molecules (C3, E51 and L56) showed activity not only in human but also in murine and canine derived cells. We further characterized C3 and showed that this molecule is capable of stimulating an anti-viral state in human-derived lung epithelial cells. Furthermore, the IFN-induction by C3 is not diminished by the presence of influenza A virus NS1 protein or hepatitis C virus NS3/4A protease, which make this molecule an interesting candidate for the development of a new type of broad-spectrum antiviral. In addition, the IFN-inducing properties of C3 also suggest its potential use as vaccine adjuvant

Cytotoxicity and apoptosis induction by C3. A.

<p>Percentage of ATP (representative of cell viability) in 293T-FF (black dots) and A549 (white dots) cells after 24 hrs incubation with C3 at increasing concentrations. The amount of ATP in mock-treated cells was set to 100%. Error bars represent standard deviation of three replicates. <b>B. </b><i>In vitro</i> determination of caspase 3 activity by fluorimetric immunosorbent enzyme assay (Roche) after 24 hrs of C3 or Staurosporine treatment at the indicated concentrations.</p

Activation of the 293T IFN reporter cell line by small molecules.

<p><b>Top.</b> Luciferase fold induction of 293T-FF cells after 24 hrs treatment with C3 (<b>A</b>), L56 (<b>B</b>) or E51 (<b>C</b>) over the mock-treated cells. <b>Middle</b>. IFN-β (black bars) and ISG54 (white bars) mRNA induction in A549 cells after 24 hrs incubation with C3 (<b>A</b>), L56 (<b>B</b>) or E51 over the mock-treated cells. <b>Bottom</b>. Chemical structures of the small molecules, <b>A.</b> C3 3-[2-(diethylamino)ethyl]-2,3-diazatetracyclo[7.6.1.0?{5,16}.0?{10,15}]hexadeca-1,5(16),6,8,10,12,14-heptaen-4-one, <b>B.</b> L56 (N1,N1-dimethyl-N2-[2-(2-thienyl)-4-quinolyl]-1,2-ethanediamine) and <b>C.</b> E51 3,10-diazapentacyclo [10.7.1.0?{2,11}.0?{4,9}.0?{16,20}]icosa-1(19),2,4,6,8,10,12,14,16(20),17-decaene-6-sulfonamide.</p

Induction of type I IFN by C3.

<p>To confirm the production of type I IFN A549 (<b>A</b>) or VERO (<b>B</b>) cells were incubated with increasing concentrations of C3 for 24 hrs, the media with the compound was then removed, the cells washed with PBS (2×) and new media containing VSV-GFP was added. Twenty-four hrs later the GFP (as an indirect estimation of the viral replication, dashed gray line) and the ATP (as marker for the cell viability, solid line) were measured. ATP and GFP values in non-treated cells were set to 100%. Error bars represent standard deviation of three replicates. <b>C.</b> C3 induction of luciferase in the IFN-β MDCK reporter cell line. MDCK cells expressing firefly luciferase under the control of the IFN-β promoter were treated with C3 at the indicated concentration. After 24 hrs the luciferase signal was analyzed and standardized based on the values obtained in the mock treated cells.</p

IFN-β induction by C3 in the presence of viral antagonist, NS1 and NS3/4A.

<p><b>A</b> Induction of IFN by C3 in the absence (white bars) and presence (dark bars) of influenza A/PR/8/34 virus. Cells (either 293T-FF or MDCK expressing the firefly luciferase under the control of the IFN-β promoter) were infected with influenza A/PR/8/34 virus at an MOI of 10 and 2 hrs later treated with C3 at the indicated concentrations. Twenty hrs post-treatment the luciferase activity in the cells was measured as an indirect read-out of the IFN levels. The maximum luciferase value obtained from the mock infected cells for each cell line was set to 100%. <b>B.</b> and <b>C.</b> Induction of IFN in the presence of plasmid expressed NS1 from influenza A/PR/8/34 virus or NS3/4A from HCV. 293T-FF cells were transfected with either GFP (white bars), NS1 (dark bars) or NS3/4A (hatched bars), 24 hrs later the media was removed and fresh media containing C3 (C) or Sendai Virus (D) added. The luciferase levels were measured 24 hrs later and the relative luminescence units (RLU) of the GFP transfected cells set to 100%. Error bars represent standard deviation of three replicates. The significance of the differences was calculated using a t-test, ns indicates not significant, **pvalue<0.01 and ***pvalue<0.001.</p

Induction of IFN and ISGs by C3 in hTBE cells.

<p><b>A.</b> IFN-β (black bars) and Mx (white bars) mRNA induction in primary human tracheobronchial epithelial (hTBE) cells after 24 hrs incubation with C3. <b>B.</b> Percentage of ATP (representative of cell viability) in hTBE cells after 24 hrs incubation with C3 at increasing concentrations. The amount of ATP in mock-treated cells was set to 100%. Error bars represent standard deviation of three replicates.</p

Concept for high-throughput compound screen and identification of ASN2.

<p>(A) MDCK cells stably expressing IFNβ-luciferase reporter are not responsive to wild type (wt) influenza A virus infection due to the presence of a fully functional NS1 protein (left panel). Infection with a mutant virus expressing a truncated NS1 protein (rPR8 NS1-113), which is unable to antagonize the IFNβ production pathway, can induce the IFNβ-luciferase reporter (middle panel). The HTS assay consisted of infecting the reporter cells with wt influenza A virus in the presence of small molecular weight compounds. The aim was to identify compounds that are able to restore IFNβ in the presence of wt influenza A virus (right panel). (B) Chemical structure of ASN2 with its molecular weight (MW) and chemical formula. (C) Reporter assays of MDCK IFNβ-luciferase cells treated with increasing concentrations of ASN2 for 2 hours prior to mock infection or infection with influenza A/PR/8/34 virus and VSV-GFP. Luciferase activity was assayed 18 hours post infection. Curves represent the mean of triplicate values ± standard deviation.</p

ASN2 inhibits the replication of influenza A virus and displays <i>in vivo</i> antiviral activity.

<p>(A) Virus titers from A549 cells infected with influenza A/WSN/33 virus (MOI = 0.01) and treated with increasing concentrations of ASN2 for 48 hours (orange curve). Cell viability analysis of A549 cells treated with increasing concentrations of ASN2 for 48 hours (black curve). Curves represent means of triplicate values ± standard deviation. (B) Bodyweight and survival curves of BALB/c mice (groups of 9) infected with influenza A/WSN/33 virus (5LD₅₀) and treated with 100 mg/kg of ASN2 every 8 hours for 8 days. Compound was delivered intraperitoneally beginning 8 hr prior to infection. Three mice from each group were sacrificed on days 3 and 8 post infection to determine viral lung titers (data not shown). Curves represent means ± standard deviation, *p<0.001. Mice that fell below 75% of their initial weight were sacrificed in accordance with our animal protocol.</p