Abstract
Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.
Highlights
Emergence and re-emergence of arboviruses such as alphaviruses continue to present serious health and economic threats [1,2]
While most previously reported antialphavirus compounds inhibit host proteins, evidence supported that this scaffold targeted the Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) protein, and that inhibition was associated with viral replication
Molecular Libraries Small Molecule Repository (MLSMR) library at a concentration of 20 mM with a Vero 76-based assay that measures cytopathic effect (CPE) from VEEV TC-83 infection
Summary
Emergence and re-emergence of arboviruses such as alphaviruses continue to present serious health and economic threats [1,2]. The TC-83 vaccine is an attenuated virus derived from wild-type Trinidad donkey (TrD) strain (subtype IAB) by Author Summary Alphaviruses occur worldwide, causing significant diseases such as encephalitis or arthritis in humans and animals. Some alphaviruses, such as VEEV, pose a biothreat due to their high infectivity and lack of available treatments. Compound resistance studies with VEEV mapped activity to the N-terminal domain of nsP2, to which no known function has been attributed. This discovery has delivered a small moleculederived class of potent VEEV inhibitors whose activity is coupled to the nsP2 viral protein, a novel target with a previously unestablished biological role that is implicated in viral replication
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