Date Published: June 26, 2014
Publisher: Public Library of Science
Author(s): Dong-Hoon Chung, Colleen B. Jonsson, Nichole A. Tower, Yong-Kyu Chu, Ergin Sahin, Jennifer E. Golden, James W. Noah, Chad E. Schroeder, Julie B. Sotsky, Melinda I. Sosa, Daniel E. Cramer, Sara N. McKellip, Lynn Rasmussen, E. Lucile White, Connie S. Schmaljohn, Justin G. Julander, Jeffrey M. Smith, Claire Marie Filone, John H. Connor, Yasuteru Sakurai, Robert A. Davey, Timothy L. Tellinghuisen.
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.
Emergence and re-emergence of arboviruses such as alphaviruses continue to present serious health and economic threats , . New World alphaviruses, family Togaviridae, including Venezuelan (VEEV), eastern (EEEV), and western (WEEV) equine encephalitis viruses, also represent significant biological defense threats, prompting these agents to be classified as Category B priority biodefense agents . Most VEEV infections in humans are non-lethal, however, about 14% of the cases show acute disease symptoms affecting the central nervous system, resulting in fatalities in a small percentage of cases (<1%) . Children are more susceptible to the neurological disease than adults. The lack of therapeutics for treatment, the possibility of accidental aerosol exposure of laboratory workers and its possible use as a bioterrorism agent highlight the importance of developing safe and effective anti-VEEV therapies. We report the discovery of a novel antiviral for VEEV with an excellent potency and safety profile from a large HTS. For HTS, we took advantage of the attenuated VEEV strain, TC-83 . We developed a series of assays to screen and further evaluated the hits from a >340,000-compound library. We selected CID15997213, a quinazolinone compound as our top hit and confirmed its potency and lack of toxicity in several assays. Further, because VEEV is neurotropic, we assessed the antiviral activity of the compound in neuronal cells and found that the antiviral activity in the neuronal cells is almost identical to that observed in Vero 76 cell culture (Table 1). We also tested the CID15997213 compound for its spectrum of antiviral activity against several additional alphaviruses and viruses from other families. Neither EEEV nor CHIKV were inhibited in vitro by CID15997213. Broader spectrum screening also did not show any additional antiviral activity against Ebola virus, vesicular stomatitis virus, vaccinia virus, or human respiratory syncytial virus, suggesting selectivity for VEEV and WEEV.