RNAi-based small molecule repositioning reveals clinically approved urea-based kinase inhibitors as broadly active antivirals

Influenza viruses (IVs) tend to rapidly develop resistance to virus-directed vaccines and common antivirals targeting pathogen determinants, but novel host-directed approaches might preclude resistance development. To identify the most promising cellular targets for a host-directed approach against influenza, we performed a comparative small interfering RNA (siRNA) loss-of-function screen of IV replication in A549 cells. Analysis of four different IV strains including a highly pathogenic avian H5N1 strain, an influenza B virus (IBV) and two human influenza A viruses (IAVs) revealed 133 genes required by all four IV strains. According to gene enrichment analyses, these strain-independent host genes were particularly enriched for nucleocytoplasmic trafficking. In addition, 360 strain-specific genes were identified with distinct patterns of usage for IAVs versus IBV and human versus avian IVs. The strain-independent host genes served to define 43 experimental and otherwise clinically approved drugs, targeting reportedly fourteen of the encoded host factors. Amongst the approved drugs, the urea-based kinase inhibitors (UBKIs) regorafenib and sorafenib exhibited a superior therapeutic window of high IV antiviral activity and low cytotoxicity. Both UBKIs appeared to block a cell signaling pathway involved in IV replication after internalization, yet prior to vRNP uncoating. Interestingly, both compounds were active also against unrelated viruses including cowpox virus (CPXV), hantavirus (HTV), herpes simplex virus 1 (HSV1) and vesicular stomatitis virus (VSV) and showed antiviral efficacy in human primary respiratory cells. An in vitro resistance development analysis for regorafenib failed to detect IV resistance development against this drug. Taken together, the otherwise clinically approved UBKIs regorafenib and sorafenib possess high and broad-spectrum antiviral activity along with substantial robustness against resistance development and thus constitute attractive host-directed drug candidates against a range of viral infections including influenza. Author summary Conventional medications against influenza infections, including vaccination and antiviral drug therapy, are targeted against viral determinants-an approach collectively referred to as pathogen-directed. However, influenza viruses mutate fast and quickly develop resistance to these pathogen-directed treatments. An alternative, yet not well established, is to block host cellular molecules required by the virus to successfully multiply. Such a host-directed approach is anticipated to be more robust against the development of drug resistance. This notion is founded on the different modes of action of the two principal approaches: Virus-directed therapeutics target the virus itself. Thus, just a single mutation could abrogate sensitivity to a virus-directed therapeutic. In contrast, it is unlikely that viruses can easily circumvent a pharmacological blockage of a cellular factor by means of just a few mutations. Instead, the virus needs to either exploit an immediate parallel cellular pathway or adjust its replication cycle to a different cellular factor-the latter being a process likely to require multiple mutations, if possible at all. To identify the most promising targets for a host-directed therapy, we performed a small interfering RNA (siRNA) screen with four different influenza virus strains using a lung epithelial cell line. Subsequently, we tested a series of drugs, specific for the products of the genes that are required for replication of all four influenza virus strains tested. Regorafenib and sorafenib, two chemically related urea-based kinase inhibitors already clinically approved for cancer treatment, turned out to be effective inhibitors of all influenza viruses and displayed low cytotoxicity. These drugs blocked viral replication at an early stage of the life cycle not only in cell lines but also in human primary respiratory cells. Moreover, these drugs exhibited high efficacy even against unrelated viruses. In addition, no development of resistance was observed against regorafenib, which was used in an in vitro assay representatively of urea-based kinase inhibitors. Our results suggest that regorafenib and sorafenib are promising drug candidates for a host-directed therapy of influenza and other viral infections.