Cardiotonic Steroids May Provide Avenue for the Treatment of Human Adenovirus

In response to: Saha, B., Varette, O., Stanford, W. L., Diallo, J. S., &Parks, R. J. (2019). Development of a novel screening platform for the identification of small molecule inhibitors of human adenovirus. Virology.

           

            Human adenovirus (HAdV) includes a broad group of DNA viruses that can cause infected patients to develop symptoms ranging from respiratory infections or conjunctivitis¹. Over eighty strains of this extremely prevalent nuclear virus have been identified². While this virus is quite prevalent, current anti-viral medications prescribed to people infected with the disease have only limited effectiveness against the disease. Improved anti-viral drug treatments for HAdV are needed because this virus can be fatal in immunocompromised hosts such as patients with AIDS, the elderly or infants³.

           One avenue for developing an effective treatment for human adenovirus involves the identification of small molecules that may disrupt the organization pathways that HAdV uses to infect a cell. When infecting a human host, HAdV must rely on proteins and molecular pathways native to the host cell to generate more viruses that are capable of infecting more host cells. The HAdV hijacks host cell mechanism to multiply and infect other cells in the human host. Small molecules such as selective estrogen receptor modulators have previously been shown to successfully disrupt the propagation of viruses like Ebola by interfering with the viruses’ ability to manipulate host cell proteins and pathways⁴. Until recently, however, very few studies have investigated the effectiveness of small molecules in disrupting the progression of HAdV, and those studies that have investigated the impact of small molecules on the disruption of HAdV have failed to perform a large enough exploration of small molecules⁵. Thus, the Parks laboratory at the Ottawa Hospital designed a new experimental method for visualizing the interaction between HAdV and small molecules that has broad application for testing purposes.

           Researchers at the Parks laboratory ultimately developed a fluoresce tagging protocol that fused a red fluorescent protein (RFP) with the HAdV’s major late promoter (MLP). The MLP is active during the end of HAdV replication process and, consequently, is expressed in greater quantities when the virus is about to spread to other cells. Greater expression of MLP in a cell indicates that the virus is forming more viral particles as the MLP is involved in the construction of structural proteins used for the new viruses to exit the host cell. By fusing a RFP with the MLP, the researchers could determine whether or not specific small molecules were able to disrupt HAdV by visualizing a reduction in MLP activation. HAdV’s that are unable to express MLP would be unable to reproduce effectively and thus the small molecule will have affectively disrupt the virus. 

           After manipulating the HAdV’s genome to include RFP, the virus was incubated with a human lung-derived cell line that was tagged with a green fluorescent protein and infection was allowed to occur. The two different fluorescent colors enabled the researchers to optimize their study and identification technique. The relative amount of green fluorescence permitted the researches to assess whether or not the cells utilized in the experiment were damaged. If the cells were damaged, a decrease in red fluoresce could be the result of a decrease in viable cells instead of a suppression of HAdV replication. Thus, the multiple fluorescent tags improved the validity of the Parks’ study. Researchers quantified the level of RFP in the virally infected cell line after twenty-four hours to determine the base level of RFP in HAdV infected human lung cell lines. 

Figure 1: Imagining of Human Lung Cells Infected with RFP Tagged HAdV. Live-cell images of human lung cells following 18, 21, or 24 respective hours following infection with RFP tagged HAdV. Images display the effectiveness of the tag to increase fluorescence following longer incubation periods in cells and thus higher presence of the virus. 

           After testing the validity of their viral construct through imaging seen in Figure 1, the researchers then used their newly developed fluorescent method to examine the effectiveness of 1200 FDA approved small molecules to inhibit the spread of HAdV. From their screen assays, the Parks laboratory determined that digoxin, digitoxigenin, and lanatoside C were the drugs that yielded the greatest reduction in RFP. All three drugs are cardiotonic steroids that inhibit the activity of Na+/K+ ATPases which are critical for the maintenance of cell membrane potential⁶. Through plaque assays that measure virally induced cell death, the experimenters also showed that exposure to dioxin, digitoxigenin, and lanatoside C lead to a significant reduction in HAdV pathogenicity after twenty-four hours of exposure. Cardiotonic steroids were previously shown in another study to effectively repress the infectious capabilities of HAdV⁷. The combination of the experimenters’ RFP and viral plaque assay findings and the previous work of Grosso et al. provides substantial support for the experimenters’ conclusion that cardiotonic steroids have the potential to be used as effective anti-viral drugs against HAdV. 

Figure 2: Impact of Cardiotonic Steroids on Expression of MLP in HAdV Infected Cells. As the concentration of the cardiotonic steroids (dioxin, digitoxigenin, and lanatoside C) increases in the cell culture there is a decrease in the intensity of RFP in the culture. The decrease in RFP is far greater in the cardiotonic steroid molecules than in the control small molecule (SAHA).

            Ultimately, the Parks’ study is important to the field of virology and the general public for two key reasons. First, the research group was able to construct and execute a new fluorescent technique that could be used to determine the impact of small molecules like cardiotonic steroids on the infectiousness of HAdV. The development of valid visualization methods to monitor HAdV is critically important for the development of effective anti-virals. The Parks’ fluorescence method could potentially be used to monitor the impact of other anti-HAdV treatments beyond small molecules to inhibit the production of late viral proteins in infect cells and thus inhibit the spread of the disease. Additionally, the research group was able to add to the body of literature which has suggested the cardiotonic steroid molecules could be used as an effective anti-viral therapy against HAdV. More research on the impact of cardiotonic steroid molecules on cell cultures infected with HAdV should be performed to determine whether or not these molecules could be used as an effective treatment for people infected with HAdV. Developing an effective anti-viral to HAdV could significantly improve the survival chances and quality of life of immunocompromised people with HAdV. 

1 Lion T. (2014). Adenovirus infections in immunocompetent and immunocompromised patients. Clinical microbiology reviews, 27(3), 441–462. doi:10.1128/CMR.00116-13

2 Binder, A. M., Biggs, H. M., Haynes, A. K., Chommanard, C., Lu, X., Erdman, D. D., … Gerber, S. I. (2017). Human Adenovirus Surveillance - United States, 2003-2016. MMWR. Morbidity and mortality weekly report, 66(39), 1039–1042. doi:10.15585/mmwr.mm6639a2

3 Bhatti, Z., & Dhamoon, A. (2017). Fatal adenovirus infection in an immunocompetent host. The American journal of emergency medicine, 35(7), 1034-e1.

4 Johansen, L. M., Brannan, J. M., Delos, S. E., Shoemaker, C. J., Stossel, A., Lear, C., ... & Lehár, J. (2013). FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection. Science translational medicine, 5(190), 190ra79-190ra79.

5 Duffy, M. R., Parker, A. L., Kalkman, E. R., White, K., Kovalskyy, D., Kelly, S. M., & Baker, A. H. (2013). Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach. Journal of controlled release, 170(1), 132-140.

6 Prassas, I., & Diamandis, E. P. (2008). Novel therapeutic applications of cardiac glycosides. Nature reviews Drug discovery, 7(11), 926.

7 Grosso, F., Stoilov, P., Lingwood, C., Brown, M., & Cochrane, A. (2017). Suppression of adenovirus replication by cardiotonic steroids. Journal of virology, 91(3), e01623-16.