Identification and in vivo Efficacy Assessment of Approved Orally Bioavailable Human Host Protein-Targeting Drugs With Broad Anti-influenza A Activity

Abstract

The high genetic variability of influenza A viruses poses a continual challenge to seasonal and pandemic vaccine development, leaving antiviral drugs as the first line of defense against antigenically different strains or new subtypes. As resistance against drugs targeting viral proteins emerges rapidly, we assessed the antiviral activity of already approved drugs that target cellular proteins involved in the viral life cycle and were orally bioavailable. Out of 15 candidate compounds, four were able to inhibit infection by 10- to 100-fold without causing toxicity, in vitro. Two of the drugs, dextromethorphan and ketotifen, displayed a 50% effective dose between 5 and 50 μM, not only for the classic H1N1 PR8 strain, but also for a pandemic H1N1 and a seasonal H3N2 strain. Efficacy assessment in mice revealed that dextromethorphan consistently resulted in a significant reduction of viral lung titers and also enhanced the efficacy of oseltamivir. Dextromethorphan treatment of ferrets infected with a pandemic H1N1 strain led to a reduction in clinical disease severity, but no effect on viral titer was observed. In addition to identifying dextromethorphan as a potential influenza treatment option, our study illustrates the feasibility of a bioinformatics-driven rational approach for repurposing approved drugs against infectious diseases.

Keywords: animal models; antiviral (H1N1 and H3N2) activity; drug repurposing; host protein-targeting drugs; influenza A virus.

Figure 1

Identification of candidate drugs. Network of influenza host factors with drug targets highlighted. From the network of 128 consensus human host genes (22), 319 virus and gene/protein interactions were retained. Among the 128 nodes, 14 gene targets of FDA-approved drugs were identified by a custom Perl script that identifies direct as well as alternative gene name matches in the DrugBank flat file version 3.0 (27).

Figure 2

Validation and inhibitory concentration determination of candidate drugs. Replication inhibition of the different drugs. MDCK cells were pretreated for 8 h and infected with the influenza strains A/Puerto Rico/8/34 (PR8) (A), A/Mexico/InDRE4487/2009 (H1N1pdm09) (B), or A/Victoria/36/2011 (H3N2) (C) at a MOI of 0.01 in the presence of 10-fold serial drug dilutions. After 16 h, the supernatant was harvested and the amount of virus quantified by limited dilution method. Bars represent the average of three independent duplicate experiments, and error bars indicate the standard deviation.

Figure 3

Inhibitory effect of drug candidates in mice. C57BL/6 mice were treated orally once daily starting 1 day before infection and continuing until sacrifice with the respective drug at the maximum tolerated dose individually (A) or in combination (B). The animals were infected intranasally with 10³ TCID₅₀ of PR8. Three days post-infection, the animals were sacrificed and the lung titers were quantified by limited dilution method and expressed as TCID₅₀/g tissue. Bars indicate group means, and the dotted line reflects the detection limit of the assay. One-way analysis of variance (ANOVA) with Dunnett's Multiple Comparison Test was used for statistical analyses (no significance (n.s.), ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 4

Dextromethorphan efficacy assessment in ferrets. Ferrets (n = 9) were treated orally every 8 h for 5 days or until sacrifice on day 3 (n = 4), starting 1 day before infection with 2 mg/kg dextromethorphan or 2.5 mg/kg oseltamivir, corresponding to recommended human doses. The animals were infected intranasally with 10⁵ TCID₅₀ of A/Mexico/InDRE4487/2009. (A) Nasal washes were collected daily, and titers were quantified by limited dilution method. For the first 4 days, animals were monitored twice daily and daily thereafter for weight (B) and clinical signs (C). The clinical score represents a composite score of body temperature, activity, nasal exudate, and respiratory signs scored on a 0-1-2 scale with 0 representing body temperatures ≤39.3°C and the physiologic state, 1 temperatures 39.4–39.9°C, calm behavior, low amounts of clear exudate, and occasional sneezing or congestion, and 2 temperatures ≥40.0°C, depressed behavior, larger amounts of yellow or brown exudate, and frequent sneezing, coughing and/or dyspnea. Symbols represent group averages, and error bars indicate the standard error mean. One-way analysis of variance (ANOVA) with Tukey's Multiple Comparison Test was used for statistical analyses (^***p < 0.001).