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. 2012 Oct 12;287(42):35324-35332.
doi: 10.1074/jbc.M112.392142. Epub 2012 Aug 21.

Obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection

Oxana V Denisova  1 , Laura Kakkola  1 , Lin Feng  2 , Jakob Stenman  3 , Ashwini Nagaraj  1 , Johanna Lampe  1 , Bhagwan Yadav  1 , Tero Aittokallio  1 , Pasi Kaukinen  4 , Tero Ahola  4 , Suvi Kuivanen  5 , Olli Vapalahti  6 , Anu Kantele  7 , Janne Tynell  8 , Ilkka Julkunen  8 , Hannimari Kallio-Kokko  9 , Henrik Paavilainen  10 , Veijo Hukkanen  10 , Richard M Elliott  11 , Jef K De Brabander  12 , Xavier Saelens  13 , Denis E Kainov  14
Affiliations

Obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection

Oxana V Denisova et al. J Biol Chem. .

Abstract

Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

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Figures

FIGURE 1.
FIGURE 1.
8 of 201 potent host-directed IAV inhibitors attenuate infection of reporter IAV strain in human nonmalignant RPE cells at noncytotoxic concentrations. A, schematic represents the efficacy and cytotoxicity testing of 201 potential and known inhibitors of IAV infection in human RPE cells. B, 8 hit compounds targeting indicated host factors were validated against reporter PR8-GFP IAV (m.o.i. 3) in RPE cells (24 hpi). Green curves show the percent GFP fluorescence of cells with the increasing concentrations of compounds. Blue curves show percent viability of noninfected cells with increasing concentrations of compounds. The error bars represent the S.D. C, cytotoxicity (IC50), antiviral efficacy (EC50), and selectivity index (SI) of the hit compounds were calculated based on the data from B.
FIGURE 2.
FIGURE 2.
Obatoclax, gemcitabine, and SaliPhe efficiently attenuate replication of wild type IAV strains in human RPE cells and PBMC-derived macrophages at noncytotoxic concentrations. A, the antiviral efficacy and cytotoxicity of five representative inhibitors of Bcl2-like proteins, ribonucleotide reductase, vATPase, proteasome, or HSP90 were examined in noninfected (blue curves) and PR8- or WSN-infected (green and red curves) RPE cells and macrophages using a CTG assay. The error bars represent the S.D. The ATS are shown. B, PR8 IAVs produced in nontreated or obatoclax- (0.4 μm), gemcitabine- (1 μm), or SaliPhe- (3 μm) treated human macrophages and RPE cells were titered on MDCK cells. Representative plaque assays and degrees of inhibition of PR8 virus production are shown.
FIGURE 3.
FIGURE 3.
Obatoclax, SaliPhe, and gemcitabine inhibit IAV entry. A, PR8-infected (m.o.i. 3) PBMC-derived macrophages were treated with obatoclax (0.4 μm), SaliPhe (3 μm), or gemcitabine (1 μm) at the indicated time points. Control cells remained nontreated. Cell viability was measured at 24 hpi using a CTG assay. The error bars represent the S.D. B, RPE cells were treated with obatoclax (0.4 μm), SaliPhe (3 μm), gemcitabine (1 μm), or nontreated, infected with WSN IAV at m.o.i. 30, fixed at 4 hpi, and imaged for the viral NP. DAPI stains the nucleus. Scale bars, 10 μm. C, RPE cells were treated with obatoclax (0.4 μm), SaliPhe (3 μm), gemcitabine (1 μm), or nontreated followed by infection with WSN IAV at m.o.i. 3. Total RNA was extracted at the indicated time points and subjected to quantitative PCRs detecting viral-positive (+) and -negative (−) strands of Pb2 and cellular β-actin RNA. The error bars represent the S.D. D, cells were treated and infected as in A. Total cell lysates were obtained at the indicated time points and analyzed by immunoblotting. E, WSN IAVs produced in nontreated or obatoclax- (0.4 μm), gemcitabine- (1 μm), or SaliPhe- (3 μm) treated RPE cells were titered on MDCK cells. Representative plaque assays and degrees of inhibition of PR8 virus production are shown.
FIGURE 4.
FIGURE 4.
Mcl-1 is a target for obatoclax and an essential host factor for IAV infection. A, RPE cells were transfected with Mcl-1-specific or control siRNA, and Mcl-1 levels were analyzed 48 h later by immunoblotting. B, si-Mcl-1 and si-Ctrl RPE cells were prepared as in A and infected with PR8-GFP virus. 24 hpi representative fluorescent and bright-field images were taken, and GFP expression and cell viability were quantified. The error bars represent the S.D. C, obatoclax-treated or nontreated RPE cells were WSN- (m.o.i. 3) or MOCK-infected. Cells were harvested at indicated times, and Mcl-1, NP, and LRP130 (loading control) levels were analyzed by immunoblotting. D, RPE cells were treated with obatoclax (0.4 μm) or remained nontreated and infected with WSN IAV at m.o.i. 30, fixed at 1 and 4 hpi, and imaged for the viral M1 and Mcl-1. DAPI stains the nucleus. Scale bars, 20 μm. E, RPE cells were treated with obatoclax (0.4 μm), stained, and imaged 4 h later. Autofluorescent obatoclax localizes to the perinuclear region of the cells. Scale bars, 10 μm.
FIGURE 5.
FIGURE 5.
Obatoclax, by contrast to SaliPhe and gemcitabine, suppressed production of IFNs by IAV-infected cells. RPE cells were infected with WSN at m.o.i. 3. At 24 hpi the supernatants were collected and assayed for chemokine/cytokine levels.
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