# High throughput screening of orthosteric inhibitors of flavivirus protease

> **NIH NIH R01** · UNIVERSITY OF ARIZONA · 2022 · $550,021

## Abstract

Project Summary/Abstract:
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.

## Key facts

- **NIH application ID:** 10376239
- **Project number:** 5R01AI131669-05
- **Recipient organization:** UNIVERSITY OF ARIZONA
- **Principal Investigator:** HONGMIN LI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $550,021
- **Award type:** 5
- **Project period:** 2018-02-15 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10376239

## Citation

> US National Institutes of Health, RePORTER application 10376239, High throughput screening of orthosteric inhibitors of flavivirus protease (5R01AI131669-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10376239. Licensed CC0.

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