Development of a novel cell-based screening platform to identify inhibitors of viral interferon antagonists from clinically important viruses
Abstract
All viruses encode for at least one viral interferon (IFN) antagonist, which is
used to subvert the cellular IFN response, a powerful antiviral innate immune response.
Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial
for virus survival, suggesting that viral IFN antagonists could represent promising
therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an
important human pathogen for which there is no vaccine or virus-specific antiviral drug.
RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV
replication and pathogenicity. We developed a high-throughput screening (HTS) assay
to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP
gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN-
signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression
in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using
a HTS approach, we screened 16,000 compounds to identify small molecules that
inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling,
leading to restoration of GFP expression. A total of twenty-eight hits were identified;
elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the
most promising. These four hit compounds have EC₅₀ values in the single μM range and
three of them (AV-14, -16, -18) represent a chemically related series with an indole
structure. We demonstrated that the hit compounds specifically inhibit the STAT2
degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists.
At the current time, compounds do not restrict RSV replication in vitro, hence hit
optimization is required to improve their potency. Nonetheless, these compounds could
be used as chemical tools to determine the unknown mechanism by which NS2
mediates STAT2 degradation and tackle fundamental questions about RSV biology.
Type
Thesis, PhD Doctor of Philosophy
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