Many flaviviruses such as Dengue virus, Zika virus, West Nile virus, and Yellow Fever virus cause significant human diseases. However, no clinically approved antiviral therapy is available for treatment of flavivirus infections. Therefore, the development of vaccines and antiviral agents for prevention and treatment of flavivirus infections is a clear public health priority. The long- range goal of this proposal is to address this need by developing a new system to identify, screen and validate a class of potential flavivirus inhibitors, and successfully identify and analyze candidate compounds that show promise in disrupting viral survival. During flaviviral infection of the host cell, a single viral polyprotein is synthesized from the viral genome. This polyprotein is cleaved into its component proteins by a combination of host cell proteases and a two-component viral protease, encoded in genes NS2B and NS3. The function of this viral protease is indispensable for virus assembly and replication. The objectives of this project are to develop innovative high throughput screening strategies to identify and characterize compounds that orthosterically inhibit the function of the critical protease NS2B- NS3. To achieve project objectives, in Specific Aim 1, we will develop and perform novel high- throughput screening primary, secondary, and tertiary assays capable of screening large chemical libraries to identify orthosteric inhibitors.
In Specific Aim 2, after optimizing these assays, we will perform large-scale HTS to screen a compound library of >400,000 compounds for potential protease inhibitor candidates, employing five levels of screening to fully eliminate false positives.
In Specific Aim 3, we will evaluate the efficacy of the resulting candidate compounds for their capacity to block protease activity, and determined their modes of actions. Lead compounds against the protease will be tested for cellular toxicity, reduction of flavivirus titer in cell culture, resistant variants, and limited structure-activity relationship. We will ultimately test the toxicity, pharmacokinetics, and efficacy in live animals for the most potent compounds. We anticipate the outcome of this study will be the generation of crucial new information on flavivirus inhibitor-enzyme interaction at structurally significant sites. This creates the potential for identifying novel classes of flaviviral inhibitors, suitable for addressing the global public health need for new antiviral agents to fight flaviviruses.

Public Health Relevance

Flaviviruses such as West Nile virus, Dengue virus, and Zika virus are worldwide endemic arthropod-borne human pathogens that infect over 200 million and kill over 100,000 people per year. Despite effective vaccines for JEV, YFV, and TBEV, difficulty in vaccinating large at risk populations and a lack of safe vaccines for devastating viruses such as Dengue and Zika necessitate development of antiviral therapeutics to combat severe flavivirus infections. The proposed work will develop specific inhibitors against an essential flavivirus enzyme using Dengue virus as a model system.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Davis, Mindy I
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Wadsworth Center
United States
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Li, Zhong; Sakamuru, Srilatha; Huang, Ruili et al. (2018) Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease. Antiviral Res 150:217-225
Li, Zhong; Brecher, Matthew; Deng, Yong-Qiang et al. (2017) Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction. Cell Res 27:1046-1064