Antiviral drugs that exclusively target viral factors rather than host cell processes offer the possibility of fewer side effects but at the cost of limiting possible targets and of providing selective pressure for the generation of drug-resistant viral variants. Recent advances in siRNA gene-targeting technologies provide a way of identifying host cell mechanisms that are essential for viral replication and researchers at the University of Texas Medical Branch at Galveston have used a siRNA screening approach to identify host gene products that play critical roles in Zaire Ebola virus infection. The team screened a siRNA library targeting cell kinases and phosphatases for inhibitors of cell penetration using a high-throughput assay based on pseudotyped virus which can be handled under biosafety level 2 conditions. Viral pseudotypes carry the glycoproteins of the virus of interest over the core of another virus and typically display the receptor specificity, cell tropism, and entry characteristics of the foreign glycoproteins. Ranking of siRNA efficacy followed by gene cluster analysis identified proteins in phosphatidylinositol-3-kinase and calcium/calmodulin kinase related pathways as important for Zaire Ebola virus infection and compounds targeting these pathways were prioritised for testing against both pseudotyped virus and wild type Ebola virus.
LY294002 was used to target the phosphatidylinositol-3-kinase (PI3K) pathway and KN-93 was chosen as an inhibitor of the calmodulin kinase 2 (CAMK2) pathway. LY294002, which directly inhibits the kinase activity of PI3K, had an IC50 of 7µM and KN-93, which prevents association of CAMK2 with calmodulin (required for kinase activity), had an IC50 of 21µM against infection by the pseudotyped virus. It was further shown that both KN-93 and LY294002 are able to reduce infection by wild type Ebola virus: at a concentration of 50µM, LY294002 reduced viral titres in cell culture experiments by 65% and KN-93 reduced viral titres by > 95%.
This is the first example of siRNA profiling coupled with network analysis being used to identify critical pathways for viral entry, together with lead compounds with confirmed antiviral activity. The study supports the use of pseudotyped viruses to facilitate lower containment level high-throughput screening and may lead to effective treatments for Ebola virus.
The study is published in the journal Drug Discovery Research.