Antiviral Therapeutics Discovery for Enterovirus D68.

Abstract

Introduction

Enterovirus D68 (EV-D68) can cause a spectrum of clinical symptoms from mild respiratory symptoms to severe disease which can lead to hospitalization and even fatality in the immunocompromised, the elderly, and young children. To date, there are no effective antiviral successfully developed for enterovirus infection and the current understanding on pathogenesis is limited, which has resulted in the persistent circulation and outbreaks across the globe.

Methods

An immunofluorescence-based phenotypic high-throughput antiviral screen was performed on both RD and H1299 cells, where 7,987 compounds across nine commercially-available drug libraries were screened for antiviral activity against Enterovirus D68 (EV-D68) strain US/KY/14-18953. Using a hit threshold of 50% inhibition and a toxicity threshold of nuclei count > 1000, 178 hits were identified. These hits were further filtered for novelty and potency, which led to the validation of 12 compounds. MARVAS DF01 was selected for subsequent experiments due to its high efficacy (IC50: 1.804 µM).

Results

MARVAS DF01 treatment of EV-D68 infected cells potently reduced viral protein and viral genomic RNA levels. The time-of-addition, time-of-removal, and entry bypass studies showed that MARVAS DF01 acts in the post-entry stages of EV-D68 replication, specifically between 4 to 6 hours post-infection. Transfecting a Nanoluciferase viral replicon containing a truncated viral 3D protein still resulted in a luminescence reduction, indicating that MARVAS DF01 affects viral protein translation. Following serial passaging of EV-D68 in the presence of MARVAS DF01, no resistant mutants were found after 18 passages, suggesting the target of MARVAS DF01 to be a host factor. To elucidate the specific target, siRNA knockdown of the four currently known host targets of MARVAS DF01 was performed, from which ULK1 and ULK2 were identified to be involved in EV-D68 replication. As these two proteins are canonically known as part of the autophagy pathway, MARVAS DF01 was investigated for its role in EV-D68-induced autophagy. Through western blot and immunofluorescence staining, the LC3-II/LC3-I ratio and number of LC3 puncta-positive cells increased upon EV-D68 infection, indicating autophagy induction in infected cells. MARVAS DF01 treatment reversed this phenomenon, suggesting its mechanism of inhibition to be through the autophagy pathway. Additionally, MARVAS DF01 is a broad-spectrum enterovirus antiviral, inhibiting the replication of representative viruses from Enterovirus A-D and Rhinovirus A. MARVAS DF01 also reduced EV-D68 replication primary human nasal epithelial cells.

Discussion

Our research has taken a leap into the identification of novel antivirals for EV-D68 and highlight the gap that necessitates further research to devise novel broad spectrum treatment approaches and modalities to thwart human enterovirus infection.

Conclusion

MARVAS DF01 is a potent antiviral for EV-D68 and can serve as a broad spectrum antiviral for human enteroviruses by targeting the host autophagy processes that is essential for human enterovirus replication.