# High Throughput Screening of Inhibitors Targeting the Enterovirus A71 and D68 2A Proteases

> **NIH NIH R01** · UNIVERSITY OF ARIZONA · 2021 · $27,733

## Abstract

PROJECT SUMMARY
 The enterovirus genus includes many medically and socioeconomically important human pathogens such as
poliovirus, coxsackievirus, EV-A71, EV-D68, and rhinoviruses. Non-polio enteroviruses (NPEV) such as EV-A71
and EV-D68 are of particular concern as they are causative pathogens for hand, foot, and mouth disease
(HFMD), moderate to severe respiratory illness and, in rare cases, central nervous system (CNS) infections in
humans. Both EV-A71 and EV-D68 are listed among the NIH NIAID priority pathogens. Despite a significant
disease burden, no approved antiviral drugs are currently available for the prevention and treatment of EV-A71
and EV-D68 infection. Accordingly, the goal of the proposed research is to identify viral 2A protease inhibitors
as potent EV-A71 and EV-D68 antivirals through high-throughput screening (HTS).
 The EV-A71 and EV-D68 2A proteases play an essential role in viral replication by cleaving the viral
polyprotein after viral transcription, initiating viral replication. The EV-A71 and EV-D68 2A proteases represent
unexplored novel antiviral drug targets—only three weak or promiscuous EV-A71 2Apro inhibitors have been
reported and there is no EV-D68 2Apro inhibitor in the literature. To identify EV-A71 and EV-D68 2Apro inhibitors
through HTS, we developed fluorescence resonance energy transfer (FRET)-based enzymatic assays for both
the EV-A71 and EV–D68 2Apro proteins. The EV-D68 2Apro FRET assay was subsequently used to conduct a
pilot screening against the Selleckchem Bioactive compound library (1902 compounds), leading to the
identification of telaprevir (VX-950) as the first EV-D68 2Apro inhibitor with an IC50 of 0.2 µM. In secondary cell
culture assays, telaprevir showed sub- to low micromolar potency against several contemporary human EV-D68
strains in different human cell lines. The success of the pilot screening suggests that it is feasible to identify 2Apro
inhibitors as EV antivirals through FRET-based HTS.
 As EV-A71 and EV-D68 cause both respiratory infection and, in rare cases, neurological infection, there is a
need for both blood-brain barrier (BBB)-impermeable and BBB-permeable 2Apro inhibitors. BBB-impermeable
2Apro inhibitors are the most needed for the treatment of respiratory infection as well as the prevention of viral
spread to the CNS. In contrast, BBB-permeable 2Apro inhibitors are needed for the treatment of neurological
infection when virus has spread to the spinal cord through viremia. In addition, as the EV-A71 and EV-D68 2A
proteases share ~60% sequence similarity, we expect to identify inhibitors that target either EV-A71 2Apro or EV-
D68 2Apro or both by conducting two FRET-based enzymatic HTS assays—one for EV-A71 2Apro and one for EV-
D68 2Apro.
 The expected outcome of this project is the identification of first-in-class EV-A71 and EV-D68 2Apro inhibitors
with validated mechanisms of action and drug-like properties. The proposed study is significant because it
represents a step fo...

## Key facts

- **NIH application ID:** 10149927
- **Project number:** 5R01AI147325-03
- **Recipient organization:** UNIVERSITY OF ARIZONA
- **Principal Investigator:** Jun Wang
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $27,733
- **Award type:** 5
- **Project period:** 2019-06-07 → 2022-01-03

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10149927

## Citation

> US National Institutes of Health, RePORTER application 10149927, High Throughput Screening of Inhibitors Targeting the Enterovirus A71 and D68 2A Proteases (5R01AI147325-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10149927. Licensed CC0.

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